key: cord-0031724-e6t2f68x
authors: nan
title: ISEV2022 Abstract Book
date: 2022-05-17
journal: J Extracell Vesicles
DOI: 10.1002/jev2.12224
sha: 1b7bfa274dc2be0870f9a2c98d0e16b51a0a418f
doc_id: 31724
cord_uid: e6t2f68x

nan

Introduction: Anaplastic thyroid carcinoma (ATC) is a rare but deadly thyroid cancer. Hypoxia is frequent in aggressive solid tumors like ATC and can promote resistance to therapy via the hypoxia-inducible factor (HIF) pathway. Identifying and targeting hypoxic cells is an attractive approach to treating ATC. Methods: Ligation-based and -independent small RNA-seq (sRNA-seq) methods were used to compare miRNA expression in ATC lines (SW1736 and C643) vs. papillary thyroid cancer (PTC) lines (TPC-1 and BCPAP) at 2% (hypoxia) or atmospheric (normoxia) O2 levels. Differentially expressed miRNA was validated by Stem-Loop RT-qPCR. Extracellular vesicles (EVs) and ribonucleoproteins (RNPs) were isolated from cell-and debris-pre-cleared conditioned media by size-exclusion chromatography (SEC). EVs were characterized by electron microscopy (EM), resistive pulse sensing (RPS), and Western blot (WB). Results: sRNA-seq and qPCR showed 2x lower normoxic expression of miR-210 in ATC vs. PTC lines. In a 0, 2, 4, 8, 24, & 48hr time-course, miR-210 was >10x induced in hypoxic vs normoxic ATC cells but only 2-4-x in PTC. Both sRNA-seq and qPCR showed that miR-210 was the only miRNA significantly up-regulated (>2x) in hypoxic SW1736 (ATC) cells. In SW1736, miR-210 levels declined after 72hr of hypoxia, consistent with lower HIF-1alpha protein levels, but remained elevated (>2x) relative to normoxia. However, miR-210 increased (>2x) in media collected at 72hr, suggesting extracellular release. miR-210 in hypoxia relative to normoxia was enriched ∼3x in EV and RNP fractions separated by SEC. Lastly, similar particle counts per cell were observed for EVs released during hypoxia vs. normoxia (RPS), but there was an enrichment of CD63 and CD9 after 72hr hypoxia (WB). Summary/Conclusion: mir-210, a HIF target, indicates hypoxia in ATC lines and associates with EVs and extracellular RNPs. Therefore, miR-210 is a potential cellular and extracellular marker for hypoxia within ATC tumors. Further analysis of miR-210 levels in ATC tissues and plasma is warranted for the potential use of drugs that target hypoxia effectors such as HIF-1 and HIF-2.

Introduction: Cystic fibrosis (CF) is characterized by recruitment and hyperexocytosis of granules from polymorphonuclear neutrophils (PMNs) yet paradoxical failure to kill pathogens in the airways -a phenotype termed granule releasing, immunomodulatory and metabolically active (GRIM). Newly arrived, naïve PMNs are exposed to EVs from other cells including earlier arrived PMNs. We utilized a model in which blood PMNs can be conditioned into GRIM cells by transmigration toward CF sputum. We sought to determine what component of the sputum caused PMNs to become GRIM. Methods: EVs were purified by centrifugation at 800, 3000, 20000xg then by 300kDa MWCO column considered an "intermediate recovery, intermediate specificity" EV enrichment method by MISEV2018. EV integrity, purity and concentration were determined by EM and NTA and western blot of TSG101, CD63 and calnexin. Cell-specific EVs were validated by immunoprecipitation and nanoflow cytometry with antibodies against CD66b (PMN), CD326 (epithelial) or CD115 (macrophage). Results: EVs from CF sputum were necessary and sufficient to generate GRIM PMNs. Removal of CD66b+, but not CD326+ or CD115+EVs prevented the GRIM phenotype. EVs from GRIM PMNs were generated and applied to naïve neutrophils, which became GRIM. RNA-seq of GRIM and non-GRIM PMN EVs identified lncRNA MALAT1 as differentially expressed. Naïve PMNs became GRIM when transfected with MALAT1, which localized to EVs, and caused other naïve PMNs to become GRIM. HDAC11 was differentially expressed in GRIM vs non-GRIM PMNs. Expression of HDAC11 in GRIM PMNs caused upregulation and packaging of MALAT1 into EVs which could convert naïve PMNs to GRIM PMNs. Summary/Conclusion: PMNs become GRIM due to HDAC11 which caused expression and packaging of MALAT1 into EVs. MALAT1+ CD66b+ EVs caused naïve PMNs to express HDAC11 and become GRIM. This suggests that MALAT1 and HDAC11 are targets to lessen inflammation in CF and other airway diseases with PMNs such as COPD, asthma and COVID19. Funding: CF Foundation (TIROUV15A0), Emory Pediatrics Flow Core. further investigate the uptake mechanisms, experiments of EV co-localization with lysosomes and endoplasmic reticulum, are ongoing in the lab, together with treatments with inhibitors of distinct uptake pathways. Summary/Conclusion: Deciphering the neuronal uptake mechanisms of glial EVs in a TDP-43Q331K model uncover novel pathogenic mechanisms of ALS and FTD. Funding: Supported by the MSCA-IF ExItALS (752470), the Italian Ministry of Health (GR-2016-02361552) and 1 • Bando Eccellenze 2021 -Fondazione VRT.

Xin SU 1 ; Ariane Brassard 1 ; Ramin Rohanizadeh 2 ; Iqraa Dhoparee-Doomah 1 ; Betty Giannias 3 ; France Bourdeau 3 ; Roni Rayes 3 ; Julia Burnier 2 ; Jonathan Spicer 2 ; Veena Sangwan 2 ; Lorenzo Ferri 2 ; Jonathan Cools-Lartigue 2  RI-MUHC/ McGill University, MONTREAL, Canada;  RI-MUHC/McGill University, Montreal, Canada;  McGill University, Montreal, Canada Introduction: Lymph nodes (LNs) are the first sites of metastasis for most of solid cancers. The initiation of LN metastasis is mediated by immune cells including polymorphonuclear neutrophils (PMNs), and tumor derived factors, such as tumour extracellular vesicles (tEV). Our earlier work showed that PMNs and neutrophil extracellular traps (NETs) can capture circulating tumour cells and promote metastasis in distant sites. Thus, one potential mechanism of increased LN metastasis is that tEV recruit PMNs and induce NETs formation. Methods: Human tissue micro-arrays (TMAs) of gastroesophageal (GEA) cancer patients were stained with PMN and NETs markers and quantified by HALO software. C57BL/6 or pad4-/-mice were injected with B16F10 or H59 cells alone or treated with neutrophil elastase inhibitor (NEi) or neutrophil depletion antibody. LN sections were stained with NETs markers and quantified by ImageJ (NIH). EV were isolated from cell culture media by ultracentrifugation. Results: In the study of 175 GEA cancer patients, lymphatic NET deposition was observed. This was associated with reduced survival even in the absence of overt metastasis(p=0.03). Next, we demonstrated the lymphatic accumulation of tEVs both in vitro and in vivo. In both settings this was associated with PMN recruitment and NETs deposition. In vivo, NETs deposition was associated with increased development of lymphatic metastasis, and LN metastasis was abrogated through different kinds of NETs inhibition (neutrophil depletion, pad4 knockout and NEi treatment, n=10, p< 0.001), demonstrating the consequences of LN NETs deposition and its potential as a treatment target. Finally, we showed that tEV induced PMN recruitment and NETs formation was mediated by increased production of IL-8 by Lymphatic Endothelial Cells (LEC). Summary/Conclusion: Together, we demonstrated that tEVs can contribute to LN metastasis as PMN recruiter and NETs inducer, as well as a potential therapeutic targets. By further investigating the detailed mechanism, this project will lead to major advances in the management of cancer patients. Funding: The Thoracic Surgery Foundation and MGH start-up fund

Syrine Arif 1 ; Sébastien Larochelle 2 ; Véronique J. J. Moulin 3  Centre de recherche en organogénèse expérimentale de l'Université Laval/LOEX -Centre de recherche du CHU de Québec-Université Laval, Québec, QC, Canada, Québec, Canada;  Centre de recherche en organogénèse expérimentale de l'Université Laval/LOEX -Centre de recherche du CHU de Québec-Université Laval, Québec, QC, Canada, Quebec, Canada;  Centre de recherche en organogénèse expérimentale de l'Université Laval/LOEX -Centre de recherche du CHU de Québec-Université Laval, Québec, QC, Canada; Département de chirurgie, Faculté de médecine, Université Laval, Québec, QC, Canada, Quebec, Canada Introduction: The intercellular communication through the extracellular matrix (ECM) of a tissue is essential for skin wound healing. Myofibroblasts, specialized cells of this process, produce extracellular vesicles (EVs) that stimulate angiogenesis and tissue remodeling in vitro, two key steps in wound healing. EVs can target local or distal cells based on their free diffusion or binding to their environment. However, the diffusion mechanisms of EVs through solid tissues remain largely unexplored. Binding of EVs to the ECM could modulate EVs diffusion and therefore modify their action during healing. Methods: EVs were extracted from myofibroblast culture medium by differential centrifugation. To evaluate EVs binding to ECM molecules, we mixed fluorescent EVs, pretreated or not with proteinase K, with solutions of type I collagen or type III collagen. Following hydrogel polymerization, a buffer was added allowing a passive exchange between the two compartments. Petra Ilvonen; Ulla Impola; Saara Laitinen Finnish Red Cross Blood Service, Helsinki, Finland Introduction: Blood-derived extracellular vesicles (EVs) are shown to have great therapeutic potential. Until recently, characterizing the composition of clinically used platelet concentrate (PC) and red blood cell (RBC) product-derived EVs in modern technology has been possible. We have previously shown that PCs used for transfusions contain an increasing amount of EVs after a longer storage period and the number of EVs and platelet activation is also affected by platelet additive solutions (PASs). Few recent publications show that platelet EVs from plasma can target specifically certain mononuclear cell populations but little is known about PC-derived EVs and their immunological function as varying manufacturing and purification methods also yield different EV populations. Methods: Different isolation methods were tested and ultracentrifugation (UC) was selected to collect a pure population of PC-derived EVs. EVs were further analyzed for surface proteins and contaminating lipoprotein particles using Imaging Flow Cytometer and western blot analysis. Targeting into different mononuclear cells as well as their immunological relevance was investigated in vitro using imaging flow cytometry and IIF microscopy. Results: With UC we get a population of EVs with low contamination of plasma protein impurities. The main population of the PC-derived EVs are CD41 positive, whereas the population of EVs derived from RBC product contain several CD235 negative EVs. We show that the origin of EVs affects their targeting and uptake kinetics into mononuclear cells affecting their immunophenotype. Summary/Conclusion: In this study, we identified differences in the immunological function of PC and RBC-product EVs. Different EV populations isolated from blood products might be utilized either as therapeutic components or as drug delivery vehicles. This would give new applications for donated blood components. Funding: EV Ecosystem for Theranostic Platforms, funded by Business Finland University of Michigan Medical School, Ann Arbor, Michigan, USA., Ann Arbor, USA Introduction: Cancer cells use extracellular vesicles (EVs) as a mean to exchange information with its close-contact microenvironment and at distance, coordinating a myriad of biological functions. Still, how endogenously produced cancer EVs spontaneously flow in a multicellular organism, and with which organs/cells do they communicate remains highly unexplored.

We developed a CD63 multireporter transgenic mouse model in which tissue-specific derived EVs are fluorescently labelled, the ExoBow. Together with transgenic mice that faithfully recapitulate the pathogenesis of pancreatic ductal adenocarcinoma (PDAC) we have mapped the network of communication mediated by pancreatic cancer EVs within its microenvironment, and systemically, with other organs. Results: Notably, the observed network of communication mediated by pancreas cells is plastic and varies according to the biological context. Prior to disease onset, pancreas EVs are scarcely exchanged within the normal pancreas microenvironment, and we found significant inter-organ communication with the thymus and stomach. Upon cancer establishment, the level of communication increases both locally and systemically and, the biodistribution of cancer EVs changes along disease progression. At early PDAC stages, communication occurs with intestines and mesenteric lymph nodes, while at late stages occurs with thymus and kidneys. Within the tumor, communication from cancer cells occurs significantly with cancer associated fibroblasts, endothelial and distinct subsets of immune cells. Summary/Conclusion: Our work shows for the first time that the communication routes originated from cancer cells are not preferentially related with organs of metastasis. Rather, it suggests that cancer EVs are more actively involved in other processes of disease progression. Also, the dynamic nature of this communication network potentially contributes to the plastic capacity of the tumor.

Tatjana Vujic University of Geneva, Genève, Switzerland Introduction: Morphine, a member of opioid family, is commonly used as a gold standard for pain medication. This antinociceptive drug is transported to the Central Nervous System (CNS) by crossing the blood-brain barrier. The latter ensures the CNS homeostasis with brain endothelial cells, which are at the interface between the blood and the CNS. Moreover, one relatively unexplored form of cell-cell communication associated with brain in response to morphine exposure is extracellular vesicles (EVs). EVs are a nanosized particle released from the cells into the circulation and are a promising cargo reflecting the state of the cell of origin. Some studies suggested that morphine is involved in oxidative stress as well as inflammation and affects BBB permeability, but the biological processes are still not well understood. The purpose of this research was to characterize EVs derived from morphine-exposed brain endothelial cells. EVs were then analyzed with a combination of mass spectrometry-based proteomics and pathway enrichment strategies. Methods: Human Brain Microvascular Endothelial Cells (HBMECs) were exposed to morphine (0, 1, 10, 25, 50 and 100 μM) for 24h. EVs were isolated using EVtrap beads (Tymora Analytical Operations) and characterized by their size distribution (NTA), their concentration (NTA) and the presence of well-known EV markers (western blots). High-throughput mass spectrometrybased quantitative proteomics by Data Independent Acquisition was then applied on whole HBMECs and HBMECs-derived EVs. Signature pathways of morphine-exposed HBMECs and EVs were analyzed by gene ontology terms and pathway enrichment (MetaCoreTM). Results: EVs isolation was successful as the size of these particles was within the range (50-150nm) and that they possess common EV markers. The main results highlighted that HBMECs and HBMECs-derived EVs exposed to morphine have common modulated pathways, namely cell adhesion, oxidative stress and immune system. Summary/Conclusion: In conclusion, our data confirmed that morphine has a detrimental effect on HBMECs-derived EVs by modulating proteins involved in the same three main biological processes (cell adhesion, oxidative stress and immunity). These biological pathways might be associated. Indeed, a high level of oxidative stress may lead to a lack of cell adhesion triggering the immune system defense. This effect is also observable on the whole cell. As EVs mirror their cell of origin, their common biological pathways may potentially be molecular signatures of the morphine-induced damaged in the circulation. However, more studies have to be done to validate these results.

Introduction: Many extracellular vesicle (EV) studies rely on cell cultures for EV production, but the exogenous EVs from fetal bovine serum (FBS) or other sera supplements are difficult to remove and may cause confounding effects on downstream experiments. However, there are currently no adequate methods for routinely determining the relative amounts of FBS EVs within a given EV preparation. One potential solution involves the use of surface-enhanced Raman spectroscopy (SERS) to biochemically fingerprinting EVs in combination with machine learning algorithms to classify the otherwise convoluted spectra.

In this study, we combined EV SERS and a custom Autoencoder algorithm to quantitatively determine the relative amounts of FBS EVs to EVs from cultured cells within a mixture. Methods: We first tested the feasibility of this approach using known ratios of SERS chemical standards Rhodamine B and Rhodamine 6G, which have similar but distinguishable spectra, much like EVs. We then isolated both commercial FBS EVs (Sigma) and bioreactor-produced MDAMB231 breast cancer EVs using differential ultracentrifugation and size exclusion chromatography. Following MISEV validation, including western blotting, transmission electron microscopy, and nanoparticle tracking analysis, we performed EV SERS on known mixtures of the two EV populations and trained the autoencoder to find a linear function that describes their relative amounts within any mixture. Results: Our results show that EV SERS can be used to effectively fingerprint fetal bovine serum EVs and EVs from cell cultures, and that by applying a customized Autoencoder algorithm, their relative amounts within an EV mixture can be quantitatively determined. This approach is now being applied to EVs from mesenchymal stromal cell cultures, which often require serum supplementation, and which will require this type of quality control measure for future clinical translation. Summary/Conclusion: We demonstrate the non-destructive, label-free, ultrasensitive, and quantitative detection of the relative amounts of FBS EVs in mixtures with EVs from cell cultures. This approach should provide several useful applications in diverse EV studies, such as monitoring the integrity of semipermeable membranes within EV bioreactors, ensuring the quality or potency of therapeutic EV preparations, or even determining relative amounts of EVs produced in cocultures. University of Coimbra, Coimbra, Portugal, Coimbra, Portugal Introduction: Plastic antibodies (PAs) are biomimetic polymer materials with high affinity and selectivity to a target biomolecule. PAs act as synthetic receptors because they retain an imprinted molecular memory obtained during polymer formation in the presence of the target. After template removal, these binding sites are selective and mimic specific biological recognition mechanisms with higher stability and robustness. Thus, PAs have been extensively applied in biosensors. Novel technologies for separation of extracellular vesicles (EVs) are urged to surpass the existing limitations, owing to the intrinsic complexity of EVs. Simultaneously, tailoring PAs as photonic materials enables a label-free optical detection upon recognition and binding of the EVs. Methods: Various strategies of preparing PAs as structured hydrogels or as core-shell imprinted materials were pursued. Surface proteins on EVs, namely CD9 and CD81, were chosen as target molecules, and the polymer networks were combined with selfassembled colloids having photonic properties. Results: The photonic PAs-based biosensors demonstrated a selective and sensitive recognition of the target proteins, as observed by changes in the reflectance spectra of the sensing materials, while the non-imprinted controls had random variations. Also, the developed sensing materials responded to the presence of EVs. Summary/Conclusion: PAs can be tailored with different formats and conjugated with a label-free detection approach are very promising biomimetic materials to improve the sensing of EVs. Moreover, the selectivity presented by these materials, allied to their low cost of production and stability, are relevant features considering the capture of EVs by specific surface markers, and thus can be useful to analyse subpopulations of EVs. Funding: The authors gratefully acknowledge funding from the European Commission trough the project MindGAP (FET-Open/H2020/GA829040).

Introduction: Exosomes are small Extracellular Vesicles (sEV) formed by an endosomal route by inward budding of the late endosome/multivesicular body (MVB) membrane. Despite in recent years much progress has been made to better define sEV composition and biogenesis pathways, their small size and heterogeneity pose challenges to find new reliable labelling strategies to identify specific exosome populations. We developed an innovative methodology to metabolically label fluorescent sEV through the use of a fluorescent lipid (BODIPY C16) that is readily internalized by cells and is transformed into phospholipids which will form part of the lipid bilayer of the secreted vesicles. Methods: Fluorescent sEV secreted in the conditioned media of melanoma cells pulsed with BODIPY FL C16 were purified by differential ultracentrifugation, quantified by Flow Cytometry (FC) and Nanoparticle Tracking Analysis (NTA), sorted by Fluorescence Activated Cell Sorting (FACS) and further characterized by density gradient separation and Western Blot analysis for typical sEVs markers. Colocalization studies were performed by confocal microscopy and electron microscopy. Results: Confocal images showed colocalization of BODIPY lipids with lipid transformation sites such as ER and mitochondria and with specific markers of late endosomes/MVB or other organelles (tetraspanins, Golgi markers, lysosomes) but not with the Introduction: Extracellular vesicles (EVs) are important mediators of intercellular communication and are candidates for cancer immunotherapy. Immune checkpoint blockade, specifically of the PD-1/PD-L1 axis, mitigates T cell exhaustion but is only effective in a subset of cancer patients. Therefore, combination strategies are extensively explored. Reasons for therapy resistance can be low primary T cell activation to cancer antigens, poor antigen presentation and reduced T cell infiltration into the tumor. We here investigated if EV therapy could induce susceptibility to anti-PD-1 or -PD-L1 therapy in a checkpoint refractory B16 melanoma model. Methods: EVs carrying the model antigen ovalbumin (OVA) and adjuvant α-galactosylceramide were isolated from murine bone marrow-derived dendritic cells by ultracentrifugation and characterized by nanoparticle tracking analysis, transmission electron microscopy, western blot and bead-based flow cytometry. Mice were inoculated with B16 melanoma cells and treated with anti-PD-1 or -PD-L1. EVs were administered therapeutically or prophylactically. Tumor growth was monitored and immune cells from the tumor and spleen were analyzed by flow cytometry and ELISPOT. Results: Injection of EVs but not checkpoint blockade induced a potent antigen-specific T cell response and reduced tumor growth. The combination of EVs and anti-PD-1 or -PD-L1 in a therapeutic model caused a trend towards higher numbers of OVA-specific CD8+ T cells. Moreover, the combination therapy prolonged survival in a prophylactic model. Summary/Conclusion: EVs induced potent anti-tumor immune responses and furthermore induced anti-PD-1 or anti-PD-L1 sensitivity in a checkpoint-refractory mouse tumor model. This shows that EVs have potential as a treatment for patients not responding to checkpoint blockade.

Wei Guo 1 ; Wei Zhang 2 ; Wenqun Zhong 2 ; Beike Wang 2 ; Jiegang Yang 2 ; Jingbo Yang 2 ; Ziyan Yu 2 ; Xiaowei Xu 3  University of Pennsylvania, Philadelphia, USA;  University of Pennsylvania Department of Biology, Philadelphia, USA;  University of Pennsylvania Department of Pathology, Philadelphia, USA Introduction: Exosomes play a key role in the interaction between the immune system and cancer. Immune cell-derived, particularly T cell exosomes are known for their anti-cancer effect. However, to enhance the clinical applicability of T cell exosomes, it is essential to reinforce their anti-cancer potential. Methods: First, we engineered T cell exosomes by attaching cytokine to the cell surface using a flexible linker. Then, physical characteristics of engineered exosomes were verified by using NTA and TEM analysis. Protein and mRNA levels of T cell activation and proliferation markers were determined by western blot and quantitative real-time PCR. Cancer cell viability was measured by in vitro bioluminescent or MTS assay. To identify the altered payload of EXO-cyts causative for their anti-cancer activity, we conducted miRNA-sequencing analysis of EXO-cyts to profile miRNAs, respectively. To investigate in vivo efficacy of EXO-cyts, tumor growth and metastasis were analyzed after exosome injection in melanoma mouse models. In addition, to test the combination effect, EXO-cyts and existing anti-cancer drugs were administered together. Results: EXO-cyt increased the anti-cancer ability of CD8+ T cells, but interestingly, it did not affect the activity of regulatory T cells. Expression levels of cellular programmed cell death ligand-1 on melanoma cells and their exosomal PD-L1 were downregulated by EXO-cyt treatment, resulting in increase of CD8+ T cell-mediated cytotoxicity. The respective effects of EXO-cyt on CD8+ T cells and melanoma cells were reproduced by several miRNAs upregulated by the cytokine autocrine effect present inside EXO-cyt. In mouse models, EXO-cyt treatment inhibited tumor progression in melanoma-bearing immunocompetent mice but not in nude mice. The combination therapy of EXO-cyt and existing anti-cancer drugs significantly improved anti-tumor efficacy with decreasing PD-L1 in tumor tissues and plasma. Summary/Conclusion: In summary, the expression of cytokine through a flexible linker on the surface of T cells induces dramatic changes in anti-cancer miRNA contents of exosomes in addition to the direct immune stimulation by cytokine. Remarkably, these EXO-cyts preferentially increased the proliferation and activity of CD8+ T cells without affecting Treg cells. Furthermore, EXO-cyts inhibited cPD-L1 and ePD-L1 levels both in vitro and in vivo melanoma models. Here, we propose that reprogrammed EXO-cyt is a potent cancer immunotherapy that works by modulating both immune cells and cancer cells. We anticipate that the use of EXO-cyts alone or in combination with conventional immunotherapy could lead to significant advances in cancer therapy. In addition, our method to engineer exosome shows considerable potential in terms of robust reprogramming for exosome-based therapy beyond artificial payloads. Introduction: Current therapies do not improve brain function in Alzheimer's disease (AD). Hence, there is a need for new approaches capable of restraining disease progression and improving brain function. In this regard, extracellular vesicles (EVs) from human induced pluripotent stem cell-derived neural stem cells (hiPSC-NSCs) exhibiting the therapeutic effects of NSCs are of great interest. Methods: We investigated whether hiPSC-NSC-EV treatment in the early stage of AD would maintain better brain function. EVs from hiPSC-NSC cultures were purified through chromatography and then administered intranasally to 3-months old 5XFAD mice (∼200 billion/week for two weeks). A month later, the efficacy of EV treatment on cognitive and mood function was probed via several behavioral tests, following which brain tissues were examined for neuropathologies and hippocampal neurogenesis. Results: Both male and female AD mice receiving the vehicle exhibited impaired object location memory and pattern separation function, and anhedonia. Intranasally administered hiPSC-NSC-EVs incorporated into neurons and microglia in virtually all regions of the AD brain. Furthermore, both genders of AD mice receiving EVs displayed improved proficiency for object location memory and pattern separation with no anhedonia. Such functional benefits in EV-treated AD mice were associated with reductions in astrocyte hypertrophy, the density of microglia, oxidative stress markers malondialdehyde and protein carbonyls, and proinflammatory cytokines TNF-a, IL-1b, and MIP-1a in the hippocampus. EV-treatment also reduced amyloid-beta load and maintained a higher level of neurogenesis in AD mice. Summary/Conclusion: The results imply that intranasal administration of hiPSC-NSC-EVs in the early stage of AD can maintain better cognitive and mood function by restraining the progression of neuroinflammatory changes and amyloid-beta accumulation and facilitating higher levels of hippocampal neurogenesis. Funding: Supported by a grant from the National Institute of Neurological Disorders and Stroke (1R01NS106907 to A.K.S.) The long-term health effects of methamphetamine including cognitive impairments, anxiety, and depression, which can persist during recovery and are associated with poor treatment outcomes. Thus, biosignatures based on objectively quantifiable blood biomarkers that relate to neuropsychiatric data could be used clinically to monitor recovery. We previously identified seven plasma extracellular vesicle (EV) miRNAs that are differentially expressed in humans with active methamphetamine disorder (MA-ACT) vs. controls (CTL). Here we related the expression of plasma EVs and their miRNAs to measures of neuropsychiatric function. Methods: We measured the concentration of tetraspanin+ (CD9, CD63, CD81) and platelet EVs (CD41) in MA-ACT and CTL participants by vesicle flow cytometry. MiRNA expression in plasma EVs isolated by size exclusion chromatography were assayed by TaqMan arrays. Plasma EV concentrations and miRNA expression levels were related to measures of anxiety (GAD), depression (PHQ-9), and memory (PRMQ). Introduction: Age related macular degeneration (AMD) is a leading cause of legal blindness. Vision loss is caused by the progressive loss of retinal pigment epithelium (RPE) and photoreceptors, and/or retinal and choroidal angiogenesis. It is unclear how disease features spread in the outer retina and whether extracellular vesicles (EVs) play a role in this process. We used AMD patient specific RPE with the high-risk Y402H polymorphism in the complement factor H (CFH) gene to perform a comprehensive analysis of EVs, their cargo and role in AMD pathology. Methods: EVs were purified from cell conditioned media using size exclusion chromatography and characterised using transmission electron microscopy and western blot for EV markers (CD63, CD81 and Alix). RPE EV secretion was assessed using Tunable Resistive Pulse Sensing. Transcriptomic, proteomic and lipidomic analyses were used to identify the disease specific contents of EVs. Functional assays were employed to investigate the AMD RPE EV signaling in the outer retina. Results: Analyses showed enhanced and polarised EV secretion in AMD RPE cells. Transcriptomic, proteomic and lipidomic analyses demonstrated that AMD RPE EVs carry a repertoire of RNA, proteins and lipids that reflect disease changes in the RPE cells of origin and mediate key AMD pathological processes including oxidative stress, cytoskeletal dysfunction, angiogenesis and drusen accumulation. We demonstrated that exposure of control RPE to AMD RPE apical EVs leads to the formation of cytoplasmic stress vacuoles, cytoskeletal destabilization, abnormalities in the morphology of the nucleus in the recipient cells, and protein and oxidative stress, these being key pathological features in AMD. Treatment of laminated retinal organoids with apical AMD RPE EVs led to disrupted neuroepithelium and appearance of enlarged cytoprotective alpha B crystallin immunopositive cells, some of which co-expressed retinal progenitor cell markers PAX6 or VSX2, consistent with the activation of regenerative pathways upon injury. Summary/Conclusion: Our findings indicate that AMD RPE EVs act as signaling messengers in the outer retina with an important role in disease progression. Funding: This work was funded by the Macular Society UK and MRC Confidence in Concept. Introduction: Extracellular vesicles (EVs) have emerged as important mediators of cell-to-cell communication in brain development and they have been associated with neurodegenerative disease (ND) processes. However, the effects of ND insults on specific EV sub-populations remain poorly characterized. Here, we investigated the features of small EVs (SEVs) in cellular Introduction: Extracellular vesicles (EVs) play an emerging role in progression of neurodegenerative disorders, notably Alzheimer's disease (AD). We have recently reported that brain-derived extracellular vesicles (BDEVs) isolated from AD patients' brains efficiently spread misfolded tau protein in mouse brains. To uncover the specific cell type of BDEVs facilitating this phenomenon, we performed quantitative proteomic profiling of BDEVs isolated from brain tissues of AD patients, mild cognitive impairment (MCI) and control human subjects as well as APP/PS1 rodent AD model and control mice. We further examined EV secretory function of plaque associated microglia in APP knock-in mice. Methods: BDEVs were separated from human brain samples of 11 AD, 8 MCI and 11 control age-matched subjects as well as 6 brain samples of 8-month-old CAST.APP/PS1 and control female mice, using discontinuous sucrose gradient ultracentrifugation and subjected to tandem mass spectrometry for differentially expressed protein and pathway analysis. To monitor EV secretion specifically from microglia in vivo, we developed and injected a novel lentivirus expressing mEmerald-conjugated CD9 specifically in microglia with APPNL-G-F knock-in mice. Mice were euthanized 10 days after the lentivirus injection in the medial entorhinal cortex of mouse brains and brain tissues were subjected to immunohistochemistry to detect mEmerald+ EVs using GFP antibody and disease associated microglia using Mac2 antibody. Results: Cell type specific molecular analysis of BDEVs showed gradual shift from neuronal to glial origins between control, MCI and AD patients. Disease-associated microglia proteomic signature was significantly upregulated in BDEVs in AD patients (APOE, ITGAX, and CD63) compared to the control and/ or MCI. ITGAX was also enriched in BDEVs isolated from CAST.APP/PS1 mice, further supporting the involvement of microglial EVs in disease progression. Interestingly, Mac2+ plaque associated microglia secrete significantly more mEmerald-CD9+ EVs compared to Mac2-microglia in AD mouse models. Summary/Conclusion: We discovered an enrichment of glia-derived EVs in AD brains compared to the control or MCI cases, which was reproduced in APP/PS1 mouse model. Further, microglia appear as potent contributor of disease progression via hyper-secretion of EVs. Funding: Abbvie Inc. Cure Alzheimer's Fund, NIH RF1 AG054199, NIH R01 AG054672, NIH R01 AG066429, NIH R01 AG072719, NIH R01 AG067763, and NIH R21 NS104609.

Introduction: Bacterial flora in the body has clinical significance for the host. The metabolic environment created by this flora influences immunotherapy in urothelial carcinoma. However, there have been no report on the clinical significance of bacterial flora in the host bloodstream. Here, we aimed to clarify the clinical significance of blood flora in urothelial carcinoma patients. Methods: Clinical specimens: Serum were obtained from 50 patients with localized urothelial cancer (UC) who had undergone TURBT and from 31 metastatic UC patients who had undergone treatment with pembrolizumab, and 20 healthy donors. Written informed consent was obtained from each patient, and the study was approved by the ethics review board of the Osaka University Medical Hospital and was conducted according to the principles of the Declaration of Helsinki. Serum EVs isolation: Serum samples (200 ul) were centrifuged at 2,000 g for 30 minutes and filtered with 0.2 um syringe filter before applying into the following extracellular vesicles (EVs) isolation. Serum EVs were isolated using ultracentrifugation method or qEV columns, Exosome Isolation Kit and MagCapture Exosome Isolation Kit. DNA was isolated from EVs samples using the QIAamp Circulating Nucleic Acid Kit. 16S metagenomic sequencing: 16S rRNA metagenomic sequencing was performed on a MiSeq platform. QIIME was used to process all raw sequencing data. Linear discriminant analysis effect size (LEfSe) was used to elucidate bacterial genus classification taxa that were associated with malignancy of UC and immunological classification of T cells. Results: The expression of peripheral EVs carrying DNA of Firmicutes phylum was significantly correlated with the number and activation surface marker expression of infiltrating T cells within tumor tissues. In addition, Firmicutes abundance could predict the response and prognosis of patients with metastatic urothelial carcinoma treated with immunotherapy. Summary/Conclusion: Information of Firmicutes abundance derived from EVs in the blood can reflect the local immune status of the tumor microenvironment and could be used to predict the efficacy and prognosis of cancer immunotherapy.

Soumyalekshmi Nair 1 ; Nanthini Jayabalan 1 ; Andrew Lai 2 ; Dominic Guanzon 1 ; Flavio Carrion 3 ; Katherin Scholz-Romero 1 ; Martha Lappas 4 ; Carlos Salomon Gallo 1  The University of Queensland, Brisbane, Australia;  Exosome Biology Laboratory, University of Queensland Centre for Clinical Research, The University of Queensland, Brisbane, Australia;  Universidad del Alba, Santiago, Chile;  University of Melbourne, Melbourne, Australia Introduction: Gestational Diabetes Mellitus (GDM) is the glucose intolerance in pregnancy and leads to pregnancy complications and metabolic disorders in mother and offspring. Placenta-derived small extracellular vesicles (sEVs) play key roles in the regulation of maternal metabolism by transfer of miRNAs mediating cell communication. The aim of the present study is to identify the molecular mechanisms associated with selective packaging of miRNAs sEVs in placental cells in normal glucose tolerant (NGT) and GDM pregnancies. Methods: sEVs were isolated from cell-conditioned media of primary human trophoblast cultures from NGT and GDM patients. The miRNA profile in trophoblast cells and sEVs were analyzed using next generation sequencing and the proteins associated with the sEV enriched miRNAs were identified by biotin pull down and mass spectrometry. Further, siRNA mediated knock down of proteins and analysis of miRNA expression in sEVs using real time PCR were performed. Results: GDM alters the miRNA content of placental cells and sEVs. A specific set of miRNAs were highly enriched in sEVs compared to their cells of origin in NGT and GDM. We classified these miRNAs into three groups as miRNAs enriched in sEVs (1) only in NGT (2) only in GDM and (3) in both NGT and GDM. Candidate miRNAs were chosen for each group (miR-150-5p and miR-1246 for NGT; miR-1285-5p for GDM; and miR-486-5p for shared) based on their abundance and enrichment in sEVs. Further, we identified a repertoire of unique proteins interacting with miRNAs within each group. We found that RNA binding protein YBX3 is required for the sorting of miR-1246 in sEVs in NGT and proteins CWF19L1 and DISC3 for the sorting of miR-486 in NGT and GDM condition. Furthermore, proteins FASTKD2 and HDLBP inhibits the sorting and release of miR-1285 in sEVs from GDM trophoblast cells. Summary/Conclusion: These findings provide insights into the mechanisms by which miRNA-protein interactions lead to the selective packaging of miRNAs into sEVs in healthy pregnancy and GDM. Funding: This research was funded by National Health and Medical Research Council (NHMRC 1195451) 

Amélie Légaré 1 ; Arianne Morissette 1 ; Christine Dallaire 1 ; Joanie Dupont-Morissette 1 ; Geneviève Guèvremont 1 ; Thibault Varin 2 ; Geneviève Pilon 1 ; Julien Boucher 3 ; Valérie Chénard 3 ; André Marette 1  Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Québec, Canada, Québec, Canada;  Institut sur la Nutrition et les Aliments Fonctionnels, Université Laval, Canada, Québec, Canada;  Centre de recherche du CHU de Québec, Canada, Québec, Canada Introduction: Numerous studies, notably carried out by our team, have shown that cranberries counteract obesity-related metabolic disorders. It has been reported that fruit vesicles can alleviate gut-associated diseases, such as obesity. Yet, the impact of cranberry extracellular vesicles (CranEVs) on metabolic and liver health has never been studied. The objective of this project is therefore to evaluate the effect of CranEVs intake on metabolic health in a mouse model of diet-induced obesity. Methods: CranEVs were isolated from freshly pressed cranberry juice by differential ultracentrifugation followed by a sucrose density gradient. The vesicles were then characterized by Nanoparticle Tracking Analysis, Dynamic Light Scattering, and transmission electron microscopy. Male C57bl/6J mice were subjected to an obesogenic diet for 10 weeks. The first group received daily gavage containing CranEVs diluted in PBS, while the control group received a daily gavage of PBS (n = 12 per group). The food intake and body weight were monitored daily. Insulin and glucose tolerance tests were performed at week 6 and week 8 respectively. Liver transcriptome profiles will be obtained by Illumina NovaSeq sequencing. Results: Despite a similar calorie intake, mice given CranEVs gained significantly less weight than the control group, in addition to experiencing a reduction of adipose tissue weight. In the liver, a significant decrease of the triglyceride and cholesterol content was observed in the mice given the extract. Analysis of the liver transcriptomic data will perhaps shine a light on the mechanisms underlying the observed benefits of CranEVs on mice health. Summary/Conclusion: This project is the first to assess the metabolic impact of CranEVs. Based on our results, CranEVs reduce adiposity and hepatic lipid accumulation. Thus, these cranberry-derived vesicles could represent a promising therapeutic avenue to counteract obesity-related metabolic disorders, such as nonalcoholic fatty liver diseases. Funding: This work was funded by the Cardiometabolic Health, Diabetes and Obesity Research Network. A. Légaré was financially supported by the Canadian Institutes of Health Research, Quebec Research Funds, and Natural Sciences and Engineering Research Council of Canada.

Shalil Khanal; Vijay Shah; Enis Kostallari Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, USA Introduction: Hepatic stellate cells (HSCs) are the main drivers of liver fibrosis by secreting high amounts of collagen. Our group has previously demonstrated that activated HSCs increase the release of fibrogenic small extracellular vesicles (sEVs). However, the mechanism leading to their release is not fully understood. Activated HSCs also display a highly glycolytic profile, where hexokinases (HK) catalyze the first committed step in glucose metabolism by phosphorylating glucose. The aim of this study is to investigate the role of glycolysis, and more specifically of HK2, in driving fibrogenic sEV release and liver fibrosis. Methods: In vitro, primary human HSCs were treated overnight with glucose to induce glycolysis or 2-deoxy-glucose (2DG) to inhibit HK2. sEVs were purified by differential ultracentrifugation and characterized by western blot and mass spectrometry. In vivo, mice lacking one allele of Hk2 gene selectively in HSCs (PDGFRβCreERT2/HK2fl/+) and littermate controls (HK2fl/+) were utilized. Liver fibrosis was induced by carbon tetrachloride (CCl4) administration. Results: Our publicly available single cell RNA sequencing dataset demonstrated that HK2 mRNA expression was 1.7-fold higher in fibrogenic HSCs isolated from CCl4-treated mice than in non-fibrogenic HSCs (adjusted p = 1.82E-09, GSE175939). In vitro, 1 g/L of glucose enhanced HK2 protein expression by 2-fold compared to no glucose condition (n=4, p< 0.05). Glucose treatment also increased sEV release by 7-fold as demonstrated by CD81 protein levels, which was abrogated by treating cells with the HK2 inhibitor, 2DG (1 g/L n=3, p< 0.05). Mass spectrometry analysis demonstrated that sEVs derived from HSCs cultured in the Introduction: Intercellular signaling between cancer cells and stromal cells within the tumor microenvironment (TME) and distant metastatic sites is known to be critical for regulating the progression of cancer. A number of recent studies have indicated a role for ex vivo isolated cancer cell extracellular vesicles (EVs) in communication between distinct cell types of the TME and in promoting metastasis; however, the precise role of endogenous EV transfer in tumorigenesis and autochthonous cancer progression has been elusive. Here, we developed novel mouse models to fluorescently track the endogenous exchange of CD9+ EVs and evaluate the functional consequences of CD9+ EV transfer in the development and progression of pancreatic cancer. Methods: In order to generate a conditional mCherry-CD9 allele, a cassette encoding loxP-STOP-loxP followed by mCherry-CD9 (LSL-mCherry-CD9) was knocked into the ROSA26 locus. Mice containing this allele were crossed to Pdx1-Cre; LSL-KrasG12D (KC), and Pdx1-Cre; LSL-KrasG12D; LSL-Trp53R172H (KPC) mice with LSL-YFP to express a cytoplasmic YFP, which was used to identify pancreatic epithelial or cancer cells. Transfer of mCherry CD9+ EVs was evaluated by confocal microscopy analysis of cells with mCherry signal and lacking cytoplasmic YFP signal. Single cell RNA sequencing of YFP-mCherry-and YFP-mCherry+ cells from the pancreata of KPC mice was used to evaluate transcriptional changes in stromal cells containing mCherry CD9+ EVs. Results: Exchange of epithelial cell derived CD9+ EVs (EC EVs) with local cells in the microenvironment of KPC pancreata occurs prior to formation of precancerous lesions and increases throughout cancer progression. Such EC EVs exchange with surrounding stromal cells in the TME, including fibroblasts and immune cells, was associated with transcriptional reprogramming of stromal cells. Analysis of the metastatic organs lung and liver revealed an accumulation of EC EVs with cancer progression and metastasis in KPC mice. EC EVs were also detected in the lung and liver of KC mice in which metastasis does not occur, but at a lower frequency than KPC mice with metastasis. Transfer of EC EVs was detected in organs lacking metastasis, including the kidney and brain; however, such transfer was less common in non-metastatic organs compared to metastatic organs. Summary/Conclusion: Together, these data suggest that EC EVs are endogenously transferred prior to tumor formation, suggesting that EV transfer may play a role in intercellular communication in the context of normal physiology. EC EV accumulation in both non-metastatic and metastatic organs indicates that EVs may act to promote metastasis and exert other yet to be uncovered functions. 

The clinical application of EVs as natural nanoparticles still requires a deeper understanding of their in vivo behavior after exogenous administration. Nuclear imaging emerges as useful tool due to its non-invasive feature and high selectivity and sensitivity. We present a simple method for the radiolabeling of small EVs (sEVs) and its validation as non-invasive tool for the study of EVs pharmacokinetic properties by nuclear imaging. Methods: sEVs were isolated from goat milk by combined ultracentrifugation and size exclusion chromatography and radioactively labeled with commercial pertechnetate ([99mTc] NaTcO4) in presence of SnCl2 at 37 • C/30min. Purity and in vitro stability (37 • C/PBS) were assessed by HPLC and TLC. After physicochemical characterization, 99mTc-sEVs were administrated to healthy mice by intravenous (310-350μCi) and intraperitoneal injection and intranasal instillation (140-170μCi). In vivo circulation time was assessed by blood extraction and pharmacology profile was evaluated by in vivo SPECT/CT, ex vivo biodistribution and autoradiography. Results: Radioactive 99mTc-sEVs presented high purity (>95%) and stability (>95% at 48h), maintaining similar properties to non-labeled sEVs (cup-shape morphology, 114.0±8.0nm). In vivo tracking revealed major changes in biodistribution; intravenous injection showed main sEVs liver uptake (36.6±7.5%ID/g) and short blood half-life (4min). sEVs intraperitoneally injected showed higher circulation time (16min) but typical free 99mTc signal at thyroid (22.0±7.2%ID/g at 24h), suggesting sEVs degradation. Nuclear signal in brain was observed after intranasal instillation, with main uptake in stomach. Summary/Conclusion: We present an easy method for EVs radiolabeling and its validation as non-invasive tool to study the EVs biological behavior after exogenous administration, demonstrating drastic changes in their pharmacokinetics among administration routes Funding: This study was supported by the Ministerio de Ciencia, Innovación y Universidades, Instituto de Salud Carlos III, project "PI16/02037", co-funded by the European Regional Development Fund (ERDF), "A way of making Europe", and by Comunidad de Madrid, project "Y2018/NMT-4949 (NanoLiver-CM)" and "S2017/BMD-3867 (RENIM-CM)", co-funded by European Structural and Investment Fund. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505) . dynamics between prostate cancer-and mesenchymal stem cell recipients. However, heterogeneous sEV uptake was observed for both systems and associated with cell area. Summary/Conclusion: Overexpression of the tetraspanin CD81 fused to a fluorescent protein caused significant alterations to the phenotype and proteome of secreted sEVs, which are under-appreciated in similar studies. Furthermore, the understanding of uptake and distribution of these sEVs in 2D dynamic cell systems provided a quantifiable benchmark and starting point for further investigations in 3D microenvironments. Funding: Cancer Research UK Keywords: CD81, tetraspanins, tdTomato, fluorescent proteins, sEVs, MVB, SWATH-MS, proteomics, uptake, retention, fluorescence microscopy

Weija Luo 1 ; Xiao Du 1 ; Zhishi Chen 1 ; Yuan Dai 1 ; Wei Yu 1 ; Jiang Chang 2  Texas A&M University, Houston, USA;  Texas A&M University College of Medicine, Houston, USA Introduction: Exosomes emerge as new messengers for in vivo communication amongst cells and organs. The beneficial effects of exosomes on the injured heart and potential roles in the prognosis of varied cardiovascular diseases have been suggested with promising results. Understanding the biodistribution and kinetics of endogenous cardiac exosomes during post-myocardial infarction (MI) will significantly impact exosome-mediated basic and translational studies. This study uncovered the tissue distribution and kinetics of the exosomes released from the MI hearts using an inducible exosome reporter mouse model. Methods: We generated a transgenic mouse expressing the bioluminescent reporter NanoLuc-fusion protein. We fused the NanoLuc reporter with exosome surface marker CD63 for specific labeling of exosomes. The cardiomyocyte-specific αMHC promoter was introduced, followed by a loxP-STOP-loxP cassette for precise spatial labeling of exosomes originating from cardiomyocytes. The cardiomyocyte-specific exosome reporter mice were bred with tamoxifen-inducible Cre mice. The exosome labeling and distribution were assessed by luciferase assay and non-invasive bioluminescent live imaging. We performed a permanent ligation of the left anterior descending artery. All of the assessments were conducted on day three and day fourteen of post-MI, representing the acute and chronic stages of the post-MI injury healing process. Results: The uptake levels of exosomes among thirteen different organs, including plasma, were detected and quantified. Profound differences of cardiac exosomes in various tissues during the MI healing process were exhibited. Summary/Conclusion: Exosome-mediated communications between the heart and other organs are differential and dynamic during post-MI recovery. Meanwhile, the exosome tracking mouse model enables elucidating the endogenous exosome trafficking pattern and allows the study of exosome behavior in different biological and pathological conditions. Funding: American Heart Association Innovative Project Award: 18IPA34180012

Chair: Lucia R. Languino -Thomas Jefferson University

Karen Crasta 1 ; Rekha Jakhar 2 ; Yen Nguyen 3 ; Matius Robert 3  National University of Singapore, Singapore, Singapore;  Research Fellow, Singapore, Singapore;  Research Assistant, Singapore, Singapore Introduction: Breast cancer is a leading cause of cancer death in women. Among the chemotherapeutic drugs used as front-line therapy are microtubule-targeting agents (MTAs) which include paclitaxel. The use of such drugs can lead to therapy-induced senescence (stable cell cycle arrest). The aim of this study was to investigate the effect of extracellular vesicles released from therapy-induced senescence breast cancer cells in breast cancer pathogenesis. Methods: MDA-MB-231 human breast cancer cell line and mouse mammary 4T1 cells treated with MTA commonly used in lab settings, Noc (100 ng/μl) for 72 h to induced therapy-induced senescence (TIS). For sEV/exosome isolation, senescent conditioned media (CM) was prepared after Noc and DMSO treatment by incubating cells in growth media containing exosomedepleted FBS for 72 h. CM was then collected and centrifuged at 500×g 10min, 2,000×g 30min, and 15,000×g 30min at 4 • C to remove cells and large debris. Supernatant was filtered, exosomes (sEVS) pelleted at 120,000×g, 2h, 4 • C, washed with PBS, centrifugation at 100,000×g,1h, 4 • C. Izon fractionation collector was used to collect sEVs/exosomes. Presence and size evaluation Introduction: Membrane-bound metalloproteinase membrane type 1-matrix metalloproteinase (MT1-MMP) plays a central role in extracellular matrix (ECM) degradation during breast cancer progression. MT1-MMP is exposed on the plasma membrane of cancer cells, from where it can degrade the ECM. In addition, MT1-MMP has been found to be enriched on EVs, although the contribution of such EVs to cancer progression has been overlooked. We previously showed that sEVs/exosomes can be tethered at the surface of producing cells by the protein tetherin. Interestingly, tetherin is highly expressed in metastatic breast cancer where it modulates ECM degradation, tumour cell migration and metastasis formation. The molecular mechanism behind tetherin overexpression and cancer progression remains elusive. Methods: Tetherin KO and tetherin overexpressing MDA-MB-231 cell lines were generated. Biochemistry, fluorescence microscopy, and electron microscopy were used to characterise MDA-MB-231 cell lines, sEV release, and to study the localisation and function of MT1-MMP and tetherin. In some cases, Bafilomycin-A1 was used to induce release of exosomes. sEVs were isolated by size exclusion chromatography and characterised using biochemistry, electron microscopy and nano-flow cytometry (nanoFCM). Extracellular matrix degradation assays were also performed.

Our data show that MDA-MB-231 cells release sEVs containing MT1-MMP, that are likely derived from multivesicular endosomes (exosomes). MDA-MB-231 cells express tetherin. Tetherin is released through sEVs and can tether sEVs at the surface of producing cells, modulating the release of (MT1-MMP) sEVs in the extracellular space. Tetherin expression modulates ECM degradation by MDA-MB-231 cells. Summary/Conclusion: Our data show that breast cancer cells release MT1-MMP through exosomes that can be tethered at the cell surface by tetherin. This study will contribute to elucidate the function of exosomal metalloproteases and tetherin during breast cancer progression. Funding: JRE and RP are supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (216370/Z/19/Z). HKJ is funded by Exosis.

Paula Carpintero-Fernandez; Marta Barturen-Gomez; alejandro Garcia-Yuste; Joaquin Mosquera; Benigno Acea; Maria D. Mayan

Introduction: Connexins are channel-forming proteins involved in cell-to-cell communication. These channel proteins modulate the tumour microenvironment via channel-dependent and channel-independent functions. Connexin43 (Cx43) has been reported to be dysregulated in breast cancer, however there is still controversy of its role in the pathogenesis and development of the disease depending on the subtype. The therapeutic response of tumour cells are also affected by these transmembrane proteins. On the other hand, small extracellular vesicles (sEVs) are already being used in different trials as new advanced therapies as drug vehicles to treat different types of cancer. Methods: The human ER+/HER2-breast cancer cell line MCF7 was used in this study. Lentiviral vectors and sEVs-enriched in Cx43 isolated from different sources , were used to restore Cx43 in breast cancer cells. sEVs were isolated by ultracentrifugation and characterized by NTA, electron microscopy and WB. Standard methods were used to study the effect of sEVs-enriched in Cx43 in combination with CDK4/6 inhibitors in cellular senescence and apoptosis. The CDK4/6 inhibitors (Palbociclib, Abemaciclib, Ribocilcib) were obtained from Selleckchem. Results: Here, we have demonstrated that the restoration of Cx43 in ER+/HER2-breast cancer cells using lentiviral vectors enhances senescence and sensitizes breast cancer cells to cell death by apoptosis increasing the efficacy of the CDK4/6 inhibitors (Pabociclib, Abemaciclib, Ribociclib) . We obtained similar results using sEVs-enriched in Cx43, in combination with CDK4/6 inhibitors, which significantly increases CDK4/6 inhibitors efficacy in 2D and 3D models. Further, the combination of Cx43, CDK4/6 inhibitors and senolytic drugs, such as Navitoclax, has been assayed and this combination resulted in the best strategy to enhance senescence, reduce the proliferation of breast cancer cells and increase cell death increasing efficacy and preventing drug resistance. Summary/Conclusion: The results presented here has been protected in an EU patent application. We propose a new and effective drug combination strategy based on the use of sEVs enriched in Cx43 to reduce CDK4/6 inhibitors resistance by improving their efficacy in breast cancer models. The results obtained could impact in the manage and treatment of tumours that respond to CDK4/6 inhibitors treatment and with a potential clinical benefit in patients. Introduction: Transforming growth factor-β (TGF-β) signaling forms a complex pathway known to suppress early-stage hyperplasia, but yet induces epithelial-mesenchymal transition, assisting tumors to achieve metastasis. Extracellular vesicles (EVs) secreted by diverse human tumor cells have been associated with the process of metastasis. Although some molecules associated with the TGF-β pathway have been implicated as cargo of EVs, a role of TGF-β signaling on EV biology has been poorly studied. Here, we described the impact of TGF-β signaling on the protein content of breast cancer cell-derived EVs and the biological role of EVs induced by TGF-β (TGF-β+EV). Methods: To this end, EV-fractions enriched in CD81 or Cholerae toxin B chain (CTB) were isolated from the vesicular secretome fraction (VSF) of human breast cancer MDA-MB-231 cells stimulated or not with TGF-β. The EVs were characterized based on morphology, size distribution and presence of EV-specific antigens. Results: Of the 971 proteins detected in the CD81+EVs by LC-ESI-MS/MS, 38 were specific to TGF-β+EVs while 137 were present in the Control (Ctrl EV). From the 2,406 proteins identified in the CTB+EVs, 86 were present only in the TGF-β+EVs, while 124 were specific to the Ctrl. PANTHER and STRING database analysis indicated that the content of TGF-β+EVs was related to integrin signaling and inflammation mediated by chemokines. Moreover, such EVs induced TGF-β signaling. Blocking EV uptake did not suppress such effect, whereas EV incubation with a monoclonal anti-TGFβ antibody significantly reduced the activation of TGF-β signaling induced by these EVs. Furthermore, TGF-β pathway activation by EVs led to higher levels of TGF-β responsive genes in the recipient cells, also increasing their motility and resistance to cytotoxic drugs. Summary/Conclusion: Hence, we uncovered new functions of the TGF-β pathway regulating the cargo of tumor-derived EVs, which drives tumor aggressiveness and may assist the prediction and assessment of cancer patients. Introduction: Cancer cells crosstalk with the tumor microenvironment (TME) by releasing small extracellular vesicles (sEVs) which are enriched in the epithelial-specific αVβ6 integrin. sEVs mediate protein transfer of αVβ6 to microvascular endothelial cells and monocytes in the TME; this transfer results in de novo αVβ6 expression and affects these cells' functions. As evaluated by our proteomic and immunoblotting analyses, αVβ6 expression in donor cancer cells and their sEVs inhibits the levels of molecules that have a tumor suppressive role, such as STAT1; in recipient cells, sEV-mediated αVβ6 expression affects STAT1 signaling. Methods: sEVs were isolated from DU145 prostate cancer cell culture media and characterized by electron microscopy, density gradients, NTA, and immunoblotting analysis, as per MISEV2018 guidelines. sEVs were mixed with non-silencing siRNAs, ITGB6 targeting D1 or D13.1 siRNA, or Cy3 control DsiRNAs, and electroporated; then, they were incubated with RNase A, washed, centrifuged, and tested in functional assays using PC3 cells.

We have now selected αVβ6, which is expressed in many cancer cells but absent in normal cells, as candidate for a sEV-mediated therapeutic approach. We show an efficient strategy to target αVβ6 in cancer cells by using siRNA loaded into sEVs. We first demonstrate that fluorescently labeled siRNAs can be efficiently loaded into sEVs by electroporation; by confocal microscopy, we show internalization of siRNA-loaded sEVs into recipient cells. We then provide evidence that sEV-mediated delivery of β6 targeting siRNAs to cancer cells specifically downregulates αVβ6 expression, without changing the expression of the β5 integrin subunit. As a consequence, sEV-mediated delivery of β6 targeting siRNAs, but not of non-silencing siRNAs, significantly reduces cell adhesion and migration of cancer cells on αVβ6-specific substrates. Summary/Conclusion: Overall, this study shows that sEVs from cancer cells may contribute to a horizontal propagation of integrin-associated phenotypes from cancer cells to the TME and demonstrates an efficient approach for specific targeting of the αVβ6 integrin in cancer cells using sEVs carrying ITGB6-specific siRNAs. Funding: NIH-R01CA224769; NIH-P01CA140043

Emily LaPlante; David Chen; Robert Fullem; Matthew Roth; Eric Van Nostrand; Aleksandar Milosavljevic Baylor College of Medicine, Houston, USA Introduction: RNA binding proteins (RBPs) play a critical role in the release and transport of extracellular RNA (exRNA) by sorting and loading exRNA into vesicles/lipoproteins and even directly carrying exRNA. However, the current understanding of RBPs as carriers of exRNA fragments in human biofluids is still lagging behind work on other exRNA carriers. Methods: We leverage ENCODE eCLIP data, which determines the bindings sites for 150 RBPs and intersect it with 6,930 human small RNAseq samples from the exRNA Atlas to determine the landscape of exRNA fragments bound by these RBPs. We identify exRNA fragments with RBP binding sites from eCLIP experiments that are significantly correlated in the human exRNA Atlas to discover RBPs with detectable exRNA "footprints". We then perform computational deconvolution of RBP exRNA cargo to place RBPs into different carrier types -vesicles, lipoproteins, or RNPs. All files are made available for the community. Results: At least several hundred thousand short RNA fragments appear to be carried into the extracellular space by RBPs. RBPs with enriched exRNA patterns are identified across different biofluids, including plasma, serum, saliva, CSF, and urine. We find 33 RBPs with detectable footprints in both cell supernatant and healthy human plasma. These RBPs are enriched for 5 different functions -RNA stability and decay, translation regulation, RNA localization, miRNA processing, and splicing. We also place 44 RBPs into vesicular and non-vesicular carrier classes. Summary/Conclusion: We present the first map of exRNAs carried by specific RBPs within extracellular vesicles, lipoprotein particles, and free RNPs across human biofluids. To empower the community, we extend the exRNA Atlas resource with intersections between all 150 RBPs eCLIP "footprints" and all 6,930 human small RNAseq samples in the exRNA Atlas. The map and the new data resource will improve understanding of the role of RBPs as carriers of extracellular RNA in both normal physiological and disease contexts. Introduction: Malaria is the most serious mosquito-borne parasitic disease, caused mainly by the intracellular parasite Plasmodium falciparum (Pf). This parasite invades human red blood cells (RBCs) and releases extracellular vesicles (EVs) carrying DNA, RNA, proteins and lipids to alter its host immune responses. Recent findings from various mammalian systems indicate the heterogeneity of the EV pool, based on distinct cargo components. Our aim was therefore to identify the various EV subpopulations produced by malaria infection. Methods: EV samples were subjected to size-separation analysis, using Asymmetric Flow Field-Flow Fractionation (AF4). The EV subpopulations were then collected and characterized both by atomic force microscopy (AFM) and cryo-transmission electron microscopy (cryo-TEM). Proteins were also isolated and subjected to proteomic analysis. Furthermore, using Förster resonance energy transfer (FRET)-based fusion assay we examined the fusion capabilities of the EV subpopulations. Finally, Laurdan staining and AFM puncture analysis combined with machine learning methods were applied in order to characterize the membrane biophysical properties of the subpopulations. Results: Interestingly, we identified two distinct EV subpopulations secreted by parasite-infected RBCs that differed in size (26 nm and 69 nm, in average). A mass spectrometry (MS) analysis of each respective EV subpopulation revealed a mixture of human and parasitic proteins. Surprisingly, the enriched cellular pathways differed between the two subpopulations, with the small EVs containing complement-system proteins and the large EVs a pronounced amount of proteasome subunits. We then measured the membrane fusion abilities of each subpopulation with three types of host cellular membranes: plasma, late and early endosome.

Remarkably, the small EVs fused to early endosome liposomes at significantly greater levels than did the large EVs. Atomic force microscope puncture tests evaluated with machine-learning methods further emphasize the difference between the two subpopulations through their mechanical properties. Summary/Conclusion: Our data demonstrate that Pf-iRBCs release two distinct subpopulations of EVs which differ in their protein content, biophysical properties, and fusion abilities. Therefore, this study may shed light on how malaria parasites utilize the powerful EV pathway to secrete particles responsible for discriminated functions in the parasite-host interaction. Introduction: Coagulation is the first step of wound healing, and reduces blood loss and the risk of infection. Tissue factor (TF) is the transmembrane receptor of (activated) coagulation factor (F)VII(a), and together they form the "extrinsic tenase" complex, which triggers coagulation by activating FX to FXa. Surprisingly, TF occurs not only in the vessel wall, but is also commonly present on extracellular vesicles (EVs) in body fluids such as amniotic fluid (AF), milk, saliva, and urine. Here we investigated whether such EVs expose extrinsic tenase complexes. Methods: Milk, saliva, and urine were collected from healthy breastfeeding women (n=6), and AF was collected from healthy women undergoing routine amniocentesis (n=7). EVs were isolated by size exclusion chromatography (SEC) and coagulation (clotting) experiments were performed in the presence and absence of antibodies against TF and FVIIa in normal plasma and in FVII-deficient plasma. The extrinsic tenase activity, i.e. the activation of FX to FXa, was determined by a chromogenic assay. Results: All body fluids triggered clotting of normal plasma. Milk was the most coagulant body fluid, followed by AF, saliva, and urine. The body fluids also triggered clotting of FVII-deficient plasma, which was inhibited by anti-FVII and anti-TF (milk: p=0.031, p=0.044; AF: p< 0.000, p=0.001; saliva, p< 0.000, p=0.055; urine: p=0.024, p=0.024). SEC showed that only fractions containing EVs triggered clotting and generated FXa, which was inhibited by anti-FVII and by anti-TF. Summary/Conclusion: Normal human body fluids contain EVs exposing functional extrinsic tenase complexes. These EVs activate FX directly, thereby bypassing a major part of the coagulation cascade and providing direct hemostatic protection. Funding: Y.H. was supported by a scholarship from the China Scholarship Council (CSC). J. T. was supported by an unrestricted travel grant from the International Society on Thrombosis and Haemostasis.

We have shown that bovine milk and colostrum provide abundant small extracellular vesicles, also referred to as exosomes, for drug delivery. We now report a novel exosome-polyethyleneimine (PEI) matrix (EPM) for delivery of siRNA targeting KRAS (siKRAS) and nuclear factor erythroid 2-related factor, NRF2 (siNRF2) to inhibit lung cancer by downregulating target protein expression. These two oncogenic targets are known to be highly expressed in lung, as well as other types of cancer. Methods: Exosomes were isolated from bovine colostrum powder by rehydration and differential centrifugation and characterized for size, pdi and charge (Zetasizer) and hallmark proteins (Western blot). Test siRNAs were entrapped in the EPM by brief incubation of siRNA with the vector, followed by PEG precipitation. The siRNA entrapment efficiency was determined using tracer 5'-32P-labeled siRNA. The transfection efficiency was determined in vitro by measuring gene knockdown via Western blot analysis, while anti-cancer effects were assessed in vivo using lung tumor xenografts in immunocompromised mice. Results: Colostrum exosomes exhibited similar size and charge as mature milk exosomes and carried similar surface proteins markers. Based on the presence of radioactive siRNA, we found that the EPM entrapped >90% of added siRNA (up to 20 μg). siRNA entrapped in EPM was protected from enzymatic degradation upon exposure to RNases, as detected by gel electrophoresis using radioactive tracer siRNA. EPM-siKRAS decreased mutant KRAS expression in A549 lung cancer cells dose dependently. When composed of folic acid-functionalized exosomes, EPM-siKRAS resulted in significant inhibition of orthotopic lung xenografts (>70%; p< 0.01) and downregulation of KRAS expression in the tumor (>50%; p< 0.05). Likewise, EPM-siNRF2 delivery resulted in dose-dependent decrease of NRF2 expression in the same cell line and correlated with the inhibition of cell survival. EPM-siNRF2 formulation also inhibited the growth of subcutaneous lung tumors (>57%; p< 0.01) in immunocompromised mice. Summary/Conclusion: The colostrum exosome-based EPM technology provided significant gene knockdown and lung tumor inhibition by targeted delivery of siRNA and represent a simple and effective exosome-based gene therapy approach. Funding: Supported from funds from 3P Biotechnologies and, in part, from the USPHS grant CA221487-01. Introduction: Bone biomineralization is a process mediated by osteoblasts through the release of matrix vesicles (MVs). The most accepted theory describes the MVs´biogenesis by budding from cell membranes, secreted at specific sites in the bone extracellular matrix. The internal MVs reservoir is composed by enzymes such as PHOSPHO1, a phosphatase that hydrolyzes phosphocholine and phosphoethanolamine, and generates Pi through phospholipids degradation, that may be also involved in MVs biogenesis. In this study, we aimed to investigate the interactions of PHOSPHO1 with lipids enriched in the membrane of MVs and to compare the results with monolayers composed by native lipids extracted from MVs. Methods: For this, we isolated MVs from the femurs of chicken embryos (16-17 days) and purified the lipids using a sequence of dispersion in organic solvents/centrifugation steps. We used DPPC, DPPS and cholesterol (Chol), and Langmuir monolayers as a mimetic membrane model. 1M of lipid chloroformic solutions were dripped at the air-liquid interface on a Tris/HCl (containing 100 mM NaCl and 2 mM MgCl2) buffer, pH 7.4. PHOSPHO1 (0.76 μg/mL) was added to the subphase, then the monolayer was compressed with the aid of a Langmuir trough. Results: The compressional moduli of the DPPS monolayers was increased in the presence of the enzyme when compared to pure DPPS. Moreover, the area occupied per lipid molecule was increased from 56.9 Å2 to 63 Å2 in the presence of PHOSPHO1, which indicated interaction between DPPS and the enzyme. Although PHOSPHO1 also increased the compressional moduli of DPPC monolayers, its presence reduced the area occupied per lipid, revealing different interaction guided by the composition of the lipids polar head In situ fluorescence microscopy images of the monolayers revealed the formation of larger domains rich Introduction: There is a need for technologies that profile extracellular vesicles (EVs) on the single vesicle level to uncover functionally different subpopulations which are masked in bulk analyses. Here we report a single EV multiplexed proteomics platform named Digital Omics of Single EVs (DOSE) based on DNA exchange imaging (DEI). Methods: We use EVs derived from HT29 and A431 cells that are purified using 220 nm filters followed by a size exclusion column step (IZON). The EVs are characterized according to MISEV2018 with TRPS, TEM and their protein content is validated by Western blot. Results: DOSE uses a flow cell on top of a glass coverslip with antibody functionalized surface which is mounted in an inverted fluorescence microscope (Nikon Ti2). The EV capture on the surface is monitored by label-free live imaging using interferometric scattering imaging (iSCAT). We proceed to block the surface and incubate a panel of antibodies, each of which is conjugated to a target specific DNA oligo. Detection is done using complementary oligos with fluorescent labels which can be hybridized in less than five minutes and be removed after imaging by toehold mediated strand displacement. Liquid handling and imaging are fully automated so that thousands of individual vesicles can be characterized with an initial panel of twenty protein markers. The image analysis is done with a custom Matlab pipeline using the label-free images to detect vesicles, suppressing noise and non-specifically bound labels in the fluorescence channels. We validate the label free EV detection using silica beads as size standards as well as a CD63-GFP expressing A431 cell line. Furthermore, we show proof-of-principle DEI experiments on HT29 EVs where specific capture of vesicles positive for low abundance targets results in enrichment of subpopulations compared to general capture of EVs. Summary/Conclusion: The method overcomes several challenges in single EV techniques including spectral limitations in multiplexed fluorescence imaging and a background from non-specific interactions or imperfect EV isolation techniques. It allows for fast cycling times and high throughput at relatively low added cost making it a promising technique to be incorporated into the single EV toolbox.  University of Nebraska-Omaha, Omaha, USA;  Department of Clinical Chemistry, Vesicle Observation Center, Department of Biomedical Engineering and Physics, Amsterdam UMC, University of Amsterdam, Amsterdam, the Netherlands, Amsterdam, Netherlands;  University of Nebraska in Omaha, Omaha, USA Introduction: Nanoscale flow cytometry (nFC) can enumerate thousands of extracellular vesicles (EVs) within minutes using fluorescence and light-scattering detection, thus allowing us to evaluate the clinical utility of EVs as liquid biomarkers in human diseases. However, gating strategy for large sample cohorts can be time-consuming and subjective with low abundant EV populations and background noise. Here, we developed a computer-assisted gating strategy for rapid and objective EV quantification. Methods: Blood samples of 4 healthy donors and 92 localized prostate cancer (locPCa) patients were prospectively collected using institutional review board approved protocols. Optimized multiplex labeling of EVs with antibodies was performed to detect different EV subsets (prostate, platelet, leukocyte). The Apogee A60-MicroPlus nanoscale flow cytometer was used to detect EVs. An automated gating model, written in R programming language, transformed flow cytometry data to hyperbolic arcsine and detected dense background noise by 'Kmeans' clustering. Based on the slopes of the density plots around the cluster locations, polygon boundaries were generated to select target EVs for each file. To distinguish EVs of interest from non-specific events, multiple machine learning models were evaluated. Results: Comparing to manual data processing, computer assisted-data processing generated similar concentrations of prostate (p>0.08) and platelet (p>0.06) EV subsets in an unbiased and faster manner (less than 10 minutes). Both processes resulted in no significant differences in prostate EV levels between healthy donor and locPCa patients. However, levels of leukocyte EVs and platelet EVs from locPCa patients was significantly higher (1.5-fold, p< 0.05 and 10-fold, p< 0.01 respectively) than healthy donors. Summary/Conclusion: This study provides reproducible clinical EV measurement using nFC and automated data analysis, and ultimately will serve as framework for the future development of EV-based liquid biopsies in human diseases. Funding: This research was supported by departmental grant (FL) and generous benefactors (FL). FL was recipient of a postdoctoral fellowship from the Fonds de Recherche du Quebec-Sante (FRQS). 

The expanding EV field needs reproducible and sensitive methods to characterize single EVs and EV-mediated effects on single cells. Although single-EV techniques are now commonly applied in the field, high-dimensional single-cell analyses to elucidate the functional impact of EV uptake on recipient cells are lacking. Here, we developed the novel EV labeling method TeLEV based on L-2-tellurienylalanine (TePhe), a phenylalanine mimic containing the heavy metal tellurium (Te). TePhe is applied as an amino acid substitute to EV-producing cells, resulting in the release of Te-labeled EVs (TeLEVs). TeLEV recipient cells can be easily identified by mass cytometry (MC). As MC and imaging MC (IMC) can discriminate over 52 heavy metal isotopes conjugated to antibodies, both emerged as technologies for the phenotypic dissection of heterogeneous cell populations and tissues amongst others for PBMCs. Combining TeLEV with MC, we became thus able to explore EV uptake at a single-cell level in over 30 different cell types in parallel and could also study the impact of EVs on various cellular functions such as apoptosis, DNA-damage response, migration, proliferation and tyrosine-kinase signaling. Methods: In our studies, we separated TeLEVs from serum-free cultured cells of eight different cell lines (incl. HEK293T, HeLa, JVM-3) and from primary cells (incl. chronic lymphocytic leukemia cells) by combining 0.2 μm filtration, 10 kDa ultrafiltration and size-exclusion chromatography (qEV/35nm). Results: Upon characterizing TeLEVs according to the MISEV2018 criteria and comparing them with non-labeled EVs of the same cells, we did not observe any differences in composition or morphology. Next, we analyzed the uptake of TeLEVs by PBMCs cultured in the presence of TeLEVs for 16 h. Our analyses revealed that independent of the EV source, classical, transitional and non-classical monocytes, as well as myeloid dendritic cells, were the main recipients of TeLEVs. Since we found differences in the individual label intensities of these EV incorporating cells depending on the EV source, we conclude that the differences emerged from different cellular uptake behaviors rather than being unspecific. Notably, although we analyzed T cells from 14 subsets (incl. Tregs and γδ T), we did not identify EV uptake in any T cell. Summary/Conclusion: In summary, TeLEV is a broadly applicable and easy-to-operate labeling method for EVs secreted by various cell types. Due to the advantages of MC and IMC, TeLEV will provide new avenues for studying the biodistribution of EVs and their functional impact on recipient cells. Introduction: Endogenous EV labeling via membrane-localizing peptides and EV-associated tetraspanins serve as an attractive solution for long-term in vivo EV analysis. In this work, we systematically evaluated the labeling efficiency of various EV labeling tags at the single EV level. Methods: We generated a library of lentiviruses encoding a panel of fluorescent proteins (GFP, ZsGreen, Venus, tdTomato, or RFP) that are tagged with: 1) Src myristoylation sequence (myr-) ; 2) Lyn kinase myristoylation and palmitoylation sequence (myrpalm-) ; 3) full-length human CD63; or 4) full-length human CD9; as well as the GAP43 palmitoylation sequence (palm-) tagged with GFP or tdTomato (a kind gift from Professor Charles P. Lai). A panel of cell lines (293T, A431, T47D, BT459, and MDA-MB-231) were stably transfected. Their EVs were purified using IZON columns and analyzed using a 3 laser CytoFLEX flow cytometer and an in-house interferometric scattering microscope capable of label-free single EV imaging. Results: All EV tags successfully labelled the plasma membrane of all cell lines screened. We also observed fluorescence signal in the cytosol of cells transfected with the myr-, myrpalm-and palm-tags, suggesting an accumulation of non-membrane bound fluorescent proteins. When benchmarked using GFP as a model fluorescent protein, palm-and the tetraspanin-based tags achieved EV labeling efficiencies of over 75%, as measured by both instruments. Meanwhile, myr-and myrpalm-GFP only labeled around 20∼30% of the total EV population. While some cell-type-dependent fluctuations in labeling efficiency was observed, the excitation profile of the fluorescent protein also influenced the measurable labeling efficiency, most notable on the CytoFLEX platform that lacks a laser optimal for orange/red fluorescent proteins. It recorded a labeling efficiency of 89.4% for palm-GFP but only 10.3% for palm-tdTomato. Similarly, myr-ZsGreen (21%) recorded a labeling efficiency much greater than myr-Venus and myr-RFP (1.4∼5%).

Our work demonstrated that single EV labeling efficiency can vary greatly depending on the tag and fluorescent protein of choice. Ongoing work entails proteomic profiling of the labelled EVs vs. their non-transfected counterparts to examine any potential alterations in the EV proteome caused by each EV labeling tags. Introduction: This presentation summarises our efforts towards ultra-correlative microscopy that can allow single-EV analysis with utmost clarity. We developed new approaches based on direct stochastic optical reconstruction microscopy (dSTORM) and correlative light-electron microscopy (CLEM). dSTORM uses multi-iterative localisation of fluorophores between light and dark states to provide a super resolution image that can visualise EVs ≥30nm. CLEM allows alignment of fluorescently labelled molecules under light microscopy with their ultrastructural details observed under electron microscopy, providing extensive information at the nanoscale. Ultra-correlation involves advanced CLEM that includes high-content compositional correlation. Methods: For dSTORM (complemented by NTA, qNano, DotBlot-CD63,TSG101,GM130, TEM and cryoTEM analysis), cell uptake and sonication experiments, DiD-labelled-EVs were isolated using Qiagen exoEasy Maxi Kit (from primary mouse stem cells). For CLEM, Du145 prostate cancer derived EVs were isolated (ultracentrifugation) and labelled with AF488 C5-maleimide. For compositional correlation at 3D nanoscale, two approaches were developed: 1) 3D ORBISIMS using the HYBRIDSIMS platform and 2) Cryo-Biopsy approach using the 3D FIBSEM platform, for omics analysis of native-state biology. Results: d-STORM approach can simultaneously provide size, shape and count analysis with high range and speed, and complements cryo-TEM data for detection of EV damage/changes due to low-power sonication. CLEM allowed structural correlation of single fluorescent EVs with their lipid bilayers. 3D cryo-ORBISIMS approach allowed identification of subcellular mass spec data (e.g. phosphatidylcholine, phosphatidylinositol) of DAPI correlated cells. Cryo-biopsy approach allowed detection of 18s RNA and transcriptomic analysis from fluorescence correlated subcellular microsurgeries (< 200μm2) as confirmed by PCR and RNA sequencing, respectively. Summary/Conclusion: dSTORM and CLEM show high potential for ultra-correlative single-EV analysis allowing native-state structure-function-composition correlation, providing valuable information about EVs in situ and after isolation. Future work will focus on standardisation of protocols. Funding: BBSRC, EPSRC, MIUR OF. Imaging flow cytometry, a sensitive tool to measure urinary extracellular vesicles with a simple and isolation-free protocol Introduction: The measurement of urinary extracellular vesicles (uEV) is still hindered by low sensitivity of detecting techniques, or bias induced by isolation. Imaging flow cytometry (IFCM) is a highly sensitive technique to detect small single uEV. Centrifugation, dithiothreitol (DTT) treatment, and dilution are commonly used to remove contaminants, increase uEV yield, or adjust urine pH. Here, we present a reliable, simple and isolation-free protocol to measure uEV by IFCM. Methods: Urine from healthy volunteers (n=5) and kidney transplantation recipients (KTR) (n=5) were collected. Supernatant of a colorectal cancer cell line (COLO 205) were used as a positive control. Samples were labeled with CD63-Alxa488 and CD63-APC and isotype labeling was set as negative control. Detergent treatment identified biological structures. Coincidence events were excluded from analysis, double positive events represented EV. Results: 14-times repeated IFCM detecting positive control showed a coefficient of variance of 4.6%. Centrifugation (10,000 g 10 min) removed autofluorescent particles in unstained urine, therefore optimizing the gating strategy enabling to distinguish uEV from autofluorescent background. In stained urine samples, this centrifugation removed 6.13 ± 4.15 × 10ˆ6/ml uEV (P < 0.05), representing 20.8 ± 15.8% uEV (P < 0.05). Addition of DTT (25 mg/ml) did not affect uEV numbers in healthy urine, but showed a 17.5 ± 25.8% higher concentration of uEV in KTR urine (P = 0.06). This might be associated with a higher total protein/creatinine ratio in patient urine (89.6 ± 44.4 g/mol) than healthy urine (4.0 ± 1.6 g/mol) (P < 0.01). Dilution resulted in a logarithmic decrease of uEV concentration with no significant effects on total uEV numbers. Summary/Conclusion: In summary, IFCM is a sensitive and reliable tool to characterize single uEV in both urine from healthy as well as diseased individuals, with 25 mg/ml DTT incubation, and no need of centrifugation or uEV isolation. Introduction: Extracellular vesicles (EVs) are fundamental for proper physiological functioning of multicellular organisms. By shuttling nucleic acids and proteins between cells, EVs regulate a plethora of cellular processes. However, our mechanistic understanding concerning the biophysical principles underlying EV-based communication is still incomplete. Mechanisms explaining why and when cells apply EV-based communication and how EV might agument the signalling potency of proteins presented on their surface, are sought. In our study, we focuse on immune-modulatory protein ligands presented on EVs, aiming to eludciate any amplfying biophysical effects of the EV membrane on ligand-receptor interactions. Methods: We apply an in vitro reconstitution approach to engineer fully-synthetic EVs based on vesicles released into T-cell immune synapses and between osteoblast and osteoclasts. This approach allows to prescicly control the biophysical properties of the EV mimetics. Based on this, we verify the proposed mechanism using natural EVs isolated from human primary T-cells and combine our analysis with in silico diffusion simulations. Results: We identify vesicle-induced receptor sequestration (VIRS) as a potentially universal mechanism augmenting the signalling potency of proteins presented on EV-membranes. By bottom-up reconstitution of synthetic EVs, we show that immobilization of the receptor ligands FasL and RANK on EV-like vesicles, increases their signalling potential by more than 1000-fold compared to their soluble forms. Moreover, we perform diffusion simulations within immunological synapses to compare receptor activation between soluble and EV-presented proteins. By this we propose vesicle-triggered local clustering of membrane receptors as the principle structural mechanism underlying EV-based protein presentation. Summary/Conclusion: We conclude that EVs act as extracellular templates promoting the local aggregation of membrane receptors at the EV contact site, thereby fostering inter-protein interactions. Our results uncover a potentially universal mechanism explaining the unique structural profit of EV-based intercellular signalling. Introduction: Extracellular vesicles (EVs) hold the potential as a native and multifunctional nanovesicle-mediated drug carrier. Both the drug loading strategy of EVs, and the single-EV level of characterization and tracking in live cells, are crucial for EV application in drug delivery. In this study, we optimized two different EV loading strategies: electroporation and liposome fusion, and characterized the difference of interactions between cross-species EVs and cells on a single EV level. Methods: We isolate EVs from four different origins and three different species -mouse plasma, C2C12 cell culture, human plasma, human MSC cell culture, and bovine milk. The EVs were characterized following the MISEV 2018 guidelines. The siRNA loading efficiency was analyzed at the single-particle level using state-of-the-art TIRF microscopy. Then single-particle tracking of fluorescent-labeled EV in live cells (HEK293 cells, bEnd-3 cells, and ARPE-19 cells) was performed using HILO microscopy. The data was analyzed by an in-house developed python script. The EVs were loaded with siRNAs against GADPH and the knockdown efficiency of GADPH in recipient cells was evaluated by QRT-PCR. Results: In our results, milk EVs showed the highest loading efficiency of above 90% EVs loaded with siRNAs, while other EVs showed 70-80%. The interactions between individual EVs and cells are species/origin-dependent with different efficiency of EV co-localization with the cell membrane, uptake in the recipient cells, and co-localization with an endosome. Our data also supported that EVs can deliver siRNAs into recipient cells and knock down the gene expression with origin-dependency. Summary/Conclusion: Here we demonstrate the origin/species dependency of EV interaction with recipient cells by our single EV tracking technique. The technique allows us to determine the mechanistic interspecies differences. We envision that this is an important step for specific EV-mediated therapeutic and native drug delivery. Introduction: Tumour-derived small extracellular vesicles (sEVs) are known as potent promotors of tumour progression and metastasis. However, the attribution of such effects to specific sEV subpopulations remains challenging. Syntenin-1 (ST1) is a key regulator for the biogenesis of an endosomal-derived sEV subpopulation and overexpressed in many tumour entities. Moreover, ST1 contributes to the assembly of protein complexes at the membrane. Indeed, the PDZ domains of ST1 directly bind various transmembrane interactors like syndecans and tetraspanins supporting the formation of higher order signalling nexi. Therefore, this study aimed to characterize the ST1-dependent sEV protein signature and its functional relevance in breast cancer. Methods: sEVs from murine wild type and ST1 knockout breast cancer cells (4T1) were isolated by serial ultracentrifugation and classified by proteomics and nanoparticle tracking analysis. Several differentially expressed proteins (DEPs) were selected for immunoblot validation, database screening and further functional analysis. Direct protein interaction was quantified via surface plasmon resonance (SPR) spectroscopy. Results: Proteomic analysis revealed a subset of 52 ST1-dependent sEV marker proteins including direct ST1-binding partners like ALIX, CD63 and SDC4 as well as tumour-associated proteins with clinical impact on patient survival. Intriguingly, many of the downregulated DEPs on ST1-deficient sEVs were attributed to cell adhesion, extracellular matrix assembly and cell-cell contacts via pathway enrichment analysis. A ST1-dependent expression on sEVs was validated in 4T1 and human MCF 7 breast cancer cells for selected DEPs with such adhesive potential including the epithelial cell adhesion molecule (EpCAM). By SPR technology we show that EpCAM directly binds to ST1 via its PDZ-binding motif. Functionally, EpCAM seemed to act merely as a cargo protein without evident contribution to sEV biogenesis. Summary/Conclusion: ST1 regulates the formation of a sEV subpopulation associated with cell adhesion. Thus, ST1-dependent sEVs might be potential drivers for enhanced tumour-derived sEV distribution and pre-metastatic niche formation in breast cancer by promoting the disappearance of adhesion complexes from cancer cells and their secretion in the matrix on sEVs. Introduction: Extracellular RNAs have emerged as a novel mechanism for cell-to-cell communication and drive many physiological and pathological processes including cancer. The major vehicle for transmission of these extracellular RNAs is extracellular vesicles (EVs). However, the underlying mechanisms by which these RNA-containing EVs are generated are poorly understood. Here, we identify VAP-A and CERT linkages at endoplasmic reticulum-endosome membrane contact sites (ER MCS) as key subcellular locations for the biogenesis of RNA-containing EVs. Methods: We used bioinformatics, RNA-sequencing, lipidomic, confocal and transmission electron microscopy, tumor xenograft and various biochemical techniques to analyze EV biogenesis and cargo content in colon cancer cell lines.

Results: High-resolution imaging data indicated small RNA localization at ER-endosome membrane contact sites. RNA-Seq analysis revealed several small RNAs that are changed in VAP-A KD small and large EVs compared to control EVs. Density gradient fractionation revealed that VAP-A regulates a select subpopulation of small EVs that are enriched with RNA. Confocal microscopy data revealed that key RNAs are altered in multivesicular endosomes in VAP-A KD cells. Lipidomics analysis of small and large EVs revealed that VAP-A controls levels of ceramide, a key lipid involved in EV biogenesis. In addition, KD of the VAP-A binding ceramide transporter CERT led to a similar defect in EV biogenesis as that of VAP-A KD. Furthermore, imaging data revealed that the ceramide producing enzyme neutral sphingomyelinase 2 co-localizes with VAP-A positive ER membranes. Summary/Conclusion: Altogether, we discovered that VAP-A-CERT linkages at ER membrane contact sites are critical for biogenesis of RNA-containing EVs. These data suggest a model in which ceramide transfer at ER MCS drives biogenesis of a select subpopulation of EVs containing RNA-RBP complexes. Beyond improving our understanding of EV biogenesis, we expect that these findings may be useful for exploring the functions of RNA-containing EVs. Funding: Funding was provided by NIH grants U19CA179514 and PO1CA229123.

Biao Lu 1 ; Annie Brown 2  Department of Bioengineering, Santa Clara University, Santa Clara, USA;  Department of Bioengineering, Santa Clara, USA Introduction: Exosomes are cell-derived nano-sized extracellular vesicles that hold a great promise for targeted delivery of therapeutic proteins and enzymes. However, an exosome-based strategy to deliver preloaded enzymes into targeted intracellular action sites is still lacking. Here we describe a novel genetic method to produce enzyme-loaded exosomes for the targeted intracellular delivery and treatment of Gaucher disease, one of the most common lysosomal storage disorders. Methods: We designed and synthesized a cohort of fusion genes composed of human β-glucocerebrosidase (GBA), green fluorescence protein, and tetraspanin CD63 in different configurations. We then cloned these fusion genes into mammalian vectors for the production of enzyme-loaded exosomes. Results: Using transfection and confocal microscope, we show the successful incorporation of therapeutic GBA into presecreted exosomes in 293T cells. We also show that GBA-loaded exosomes are eventually released into culture medium. Isolation and characterizations of exosomes confirm the successful enzyme loading without significantly affecting vesicle size as compared to non-modified controls. Image study further demonstrates that addition of enzyme-loaded exosomes to the cultured human cells results in intracellular delivery of fusion enzymes to the endosomal compartments, the targeted therapeutic sites of GBA. Summary/Conclusion: Genetic fusion of lysosomal enzyme with CD63 enables exosomal loading. This strategy is useful to produce exosome-based nano-medicine for the treatment of lysosomal storage disorders. Funding: This work is supported by the National Institute of General Medical Sciences of the National Institute of Health under Award Number R15GM137449. Introduction: Extracellular vesicles (EVs) are essential in mediating cell -cell communication across physiological processes and pathologies, yet the mechanism by which EVs release their content into recipient cells remains a key open question. Enveloped viruses such as Influenza, HIV and VSV, utilize fusion between the viral-and cellular-membrane to deliver their genomes into the cytosol. This membrane fusion is mediated by glycoproteins on the viral enveloped known as fusogens, which are typically triggered by the low pH and environment of the endosome to mediate virus-to-host membrane fusion. Here, we tested the hypothesis that EVs release their cargo into the cytosol via pH-dependent membrane fusion and sought to identify the fusogens driving this process. Methods: EVs samples were isolated from cell culture supernatant via density gradient ultracentrifugation. Membrane fusion of EVs and enveloped viruses was evaluated using an in vitro FRET-based membrane mixing assay. Transmission Electron Microscopy (Cryo-TEM) and Cryo Electron Tomography (Cryo -ET) were used to visualize the fusion process and resolve the 3D ultrastructure of the EV-liposome interaction. CRISPR-Cas9 genome editing was used to delete CD9p-1, an EV protein from the immunoglobulin superfamily, to investigate its role in EV fusion. Results: Our results show that EVs fuse with membranes mimicking the lipid composition of the late endosome in a pH dependent manner, reminiscent of enveloped viruses. Unlike most envelope viruses, exposure of EVs to low pH in the absence of target membranes did not result in their irreversible inactivation. Analysis of EVs mixed with liposomes by cryo-TEM and Cryo-ET revealed clear fusion intermediates only upon acidification. Finally, CRISPR-Cas9 mediated knockout (KO) of the immunoglobulin superfamily protein CD9-p1 in ovarian cancer cells resulted in up to 40% reduction in the EV fusion capability. CD9-p1 KO also results in lower EV biogenesis. Summary/Conclusion: Our work suggests that EVs utilize a virus-like mechanism to transfer their cargo into the cytoplasm. Our results are consistent with a model wherein EV are produced in the low pH environment of the multivesicular bodies which would render them inactive. Upon secretion, exposure to neutral pH would prime the fusogen on the EV membrane. While the identity of the fusogen/s remain unknown, we identified a novel protein, CD9p-1, involved not only in the EVs cargo delivery process but also in EVs biogenesis, as it has been observed for virogenesis upon deletion of key viral proteins. These findings provide novel insight and shed a light on still obscure aspects of EVs biology and their interaction with recipient cells. Introduction: COVID-19 infection can present with a heightened inflammatory state and an increased thrombotic risk. Extracellular vesicles (EVs) are pro-coagulant and pro-inflammatory mediators of intercellular communication and frequently attributed to inflammation, however their role, if any, in COVID-19 remains poorly characterised. We hypothesised that circulating EV signatures differ between COVID-19 patients and healthy controls (HC) as well as COVID-negative (COVID-) general medicine (GM) patients. Methods: Platelet poor plasma samples from COVID-19 positive (COVID+) patients requiring intensive care (severe) or hospital care (non-severe), as well COVID-general medical inpatients and healthy controls were enriched for EVs by ultrafiltration. Immunoblotting confirmed successful EV enrichment. Proteomic signatures were established by mass spectrometry. Results: Quantitative proteomic profiling of circulating EV enrichments robustly quantified over 300 proteins. Strikingly, hierarchical clustering of EV protein levels distinctly separated all patient cohorts. Moreover, compared to HC, COVID+ patients displayed fundamentally increased expression of several highly proinflammatory proteins and complement factors. Intriguingly, EV protein levels clearly separated severe and non-severe COVID patients. Although GM patients were also hospitalised for pro-inflammatory conditions of the respiratory system, no differences in protein expression were observed between non-severe COVID+ and GM patients. Our data highlight that severe COVID-19 presents with a distinct inflammatory state and indicate that plasma EV protein signatures may be a useful tool in assessing the severity of COVID-19. Summary/Conclusion: In conclusion, we have established that EV protein signatures mirror the pro-inflammatory state of COVID-19 patients and may be surrogate markers of COVID-19 severity. These findings may be of translational relevance towards characterising the underlying differences in severe and non-severe COVID-19 disease progression. Funding: This study was funded by Science Foundation Ireland.

Introduction: COPD is one of the most common lung diseases, characterized by progressive and irreversible airflow limitation as a result of increased tissue destruction and defective tissue repair. Since current therapeutics do not alter disease progression, new therapies that pharmacologically reactivate lung repair are needed. Recently, the secretome of lung fibroblasts, consisting of Extracellular Vesicles (EVs) and other soluble factors (SF), has been associated with alveolar regeneration. We aimed to elucidate the supportive function of lung fibroblast (MRC5)-derived EVs and SF on the regenerative potential of alveolar epithelial progenitor cells in an organoid assay. Methods: EVs and SF derived from MRC5 cells (∼ 3.0 E+08 cells) were purified using ultrafiltration and size exclusion chromatography. EVs were characterized by a combination of nanoparticle tracking analysis, total protein analysis, dot blot analysis for different EV-markers (CD9, CD63, and CD81), and cryogenic transmission electron microscopy. Murine organoids were established by co-culturing 10,000 alveolar epithelial progenitor cells (EpCAM+/CD45-/CD31-) with 2,500 CCL206 lung fibroblasts in Matrigel. MRC5-derived EVs (1E+09 particles/ml) or SF (30 μg/ml) were added once on day 0 or consecutively for 14 days. On day 14 total number and size of the organoids formed was determined. Immunostainings were used to assess the number of differentiated alveolar organoids. Results: Treatment with EVs or SF on day 0 significantly increased the number of organoids formed, i.e. a 29.50% ± 8.11% increase for EVs and 33.00% ± 20.34% for SF. Neither single treatment with EVs nor SF affected the size of organoids. Immunostaining for prosurfactant protein C revealed that the alveolar organoid count was significantly enhanced upon single treatment with EVs or SF (i.e. a 13.33% ± 5.01% increase for EVs and 13.50% ± 5.75% for SF). In addition, treatment with EVs or SF over the course of 14 days resulted in enhanced organoid count (i.e. a 58.17% ± 39.60% and 91.67% ± 33.28% increase respectively) and organoid size (i.e. a 36.50% ± 10.46% and 37.50% ± 27.02% increase respectively). Summary/Conclusion: Both lung fibroblast-derived EVs and SF support the formation of alveolar epithelial organoids, making them an interesting potential regenerative treatment to further pursue for COPD. Introduction: With the worldwide spread of emerging SARS-CoV-2 variants, it is critical to develop vaccines that can induce broadly protective responses with increased potency and durability. Small extracellular vesicles (sEVs), key intercellular communicators characterised by low toxicity and immunogenicity, offer a novel platform for effective SARS-CoV-2 vaccine development. sEVs containing multiple SARS-CoV-2 antigenic regions were tested for their ability to stimulate SARS-CoV-2 specific T cells. Methods: FLAG tagged fusion constructs containing regions from SARS-CoV-2 proteins were cloned to scaffold proteins that are enriched in sEVs. Constructs were designed with multiple distinct sites on scaffolds accessible for inserts -allowing viral regions to be placed inside or outside sEVs. Constructs were confirmed by sequencing, and protein localisation verified by flow cytometry and immunofluorescence. sEVs were isolated by ultracentrifugation and characterised using western blotting, electron microscopy and nanoFCM. Immunogold electron microscopy confirmed antigen topology on sEVs. In vitro confirmation of sEV antigen processing was assessed by stimulation of T cells using sEV-exposed B cell lines. Results: The expression of fusion proteins containing SARS-CoV-2 antigenic regions on or within sEVs, that are likely endosomeorigin exosomes, did not alter steady-state trafficking of sEV proteins or yields. B cells exposed to bioengineered sEVs potently stimulated virus-specific T cell clones at significantly lower doses compared to control recombinant viral proteins. Summary/Conclusion: We have developed a novel prototype exosomal vaccine against multiple SARS-CoV-2 antigens that is able to elicit potent T cell responses. The SARS-CoV-2 vaccine demonstrates the potential development of exosome-based vaccines to target multiple coronaviruses and novel vaccines to emerging pathogens. Funding: Exosis, Inc University of Duisburg-Essen, Essen, Germany, Essen, Germany;  School of Pharmacy and Pharmaceutical Sciences, Trinity Biomedical Sciences Institute, and Trinity St. James's Cancer Institute, Trinity College Dublin, Dublin , Ireland., Dublin, Ireland Introduction: Rheumatoid arthritis (RA) is an autoimmune disease characterised by inflammation, progressive damage, and pain in joints. Currently available therapy is inadequate to alleviate the inflammation and reduce the joint damage. While the immune-regulatory effect of MSC-EVs has been tested in many diseases, little is known with respect to their effect on RA. Thus, we aimed to assess the effect of human MSCs and MSC-EVs on T cells and synovial fibroblasts (RASFs) of RA patients. The effect of EVs derived from MSCs primed with interferon beta (IFNb) was also assessed. Methods: MSC-EVs were collected using a PEG precipitation, followed by ultracentrifugation-based protocol and were characterised via NTA, TEM, imaging flow cytometry (IFCM) and immunoblotting analysis. Immune-regulatory properties of MSCs and MSC-EVs were assessed on CD4+ T cells stimulated with CD3/CD28. Also, the effect of MSC-EVs on RASFs stimulated with TNFa was assessed. Results: EVs from naïve and IFNb primed MSCs were prepared and all fulfilled MISEV2018 criteria as evaluated by NTA, TEM and immunoblotting. Additionally, IFCM confirmed the recovery of CD9+ and CD63+ small-sized EVs. Applied onto the stimulated CD4+ T cells, EV preparations from IFNb primed MSCs suppressed the expression of more inflammatory cytokines (GM-CSF, IL-2, IL-4 and TNFa; p < 0.05, in all cases) associated with the pathogenesis of RA. However, while MSCs suppressed T cell proliferation, all MSC-EVs had a tendency to increase numbers of T regulatory cells. Furthermore, MSC-EVs inhibited (p < 0.05) the migration of RASFs and reduced (p < 0.05) the expression of the RA surface markers HLA-DR and CD34. Introduction: Mesenchymal stromal cells (MSC)-derived extracellular vesicles (EVs) are being investigated as a therapy for acute respiratory distress syndrome (ARDS). Previously, we demonstrated that MSC EVs restore functional activity of the injured cells through mitochondrial transfer. However, the impact of this mechanism on the balance of oxidative phosphorylation and glycolysis is not known. Here we investigated how EV mitochondrial transfer modulates metabolic alterations in the primary human pulmonary cells exposed to LPS or ARDS plasma. Methods: EVs were isolated from bone-marrow MSCs with normal or dysfunctional mitochondria by ultracentrifugation. Mitochondrial dysfunction in MSCs was induced by Rhodamine6G. EVs were characterized for number, size distribution, tetraspannin expression, and mitochondrial content. Primary human distal lung epithelial and endothelial cells, and monocyte derived macrophages were stimulated with LPS or plasma from ARDS patients and treated with EVs. Mitochondrial respiration and glycolytic flux were assessed by Seahorse metabolic analyser, barrier properties were assessed by xCELLigence, phagocytosis was assessed by flow cytometry. Also, single cell transcriptomic analysis was performed on mouse lungs in the in vivo LPS-induced lung injury model. Results: Inflammatory stimulation resulted in pronounced reduction of mitochondrial respiration, increase in glycolysis and functional impairment in all cell types. MSC EVs isolated from normal MSCs inhibited glycolytic flux, restored mitochondrial respiration and cell function while mitochondria-depleted EVs were not effective. Analysis of the single cell seq data showed that MSC EVs administration regulates expression of essential genes involved in mitochondrial metabolism in vivo. Introduction: Mesenchymal stromal cells-derived extracellular vesicles (MSC-EVs) can prevent the development of Bronchopulmonary Dysplasia (BPD) in animal models and in preliminary clinical trials. We investigated the effects of MSC EVs on the development of fibrosis and on some functions of lung epithelial cells in an animal model of hyperoxia-induced BPD. Methods: Good Manufacturing Practice-grade (GMP-grade) EVs were produced by human Wharton-Jelly derived MSCs (Exo Biologics, Belgium) isolated by tangential flow filtration (TFF) and characterized according to MISEV2018. Rat pups were divided in 3 groups: normoxia + PBS vehicle, hyperoxia with PBS, hyperoxia with MSC EVs in PBS. Both PBS and EVs were injected intratracheally (IT) on days 3, 7 and 10 and pups were sacrified on day 14. It was evaluated the expression of the genes involved in fibrosis pathways such as TGFβ1 and alpha-SMA. To evaluate epithelial secretory function, the expression of glycosaminoglycans (Alcian blue staining) and of surfactant protein C (SFTPC) was analyzed by immunohistochemistry and immunofluorescence. Collagen deposition was assessed by Sirius Red staining. Macrophages from bone marrow were cultured and analyzed for alpha-SMA and CD90 expression by flow cytometry after TGFβ1 treatment. Results: Pups under hyperoxia exhibited an increase both in collagen deposition and in the expression of pro-fibrotic genes in the lungs. Both parameters were reduced by treatment with MSC EVs. Both Glycosaminoglycan and SFTPC lung content were significantly increased in MSC EV-treated rat pups compared to untreated animals. In vitro, MSC EVs suppressed the induction of alpha-SMA in macrophages. Summary/Conclusion: Intratracheal administration of clinical-grade MSC-EVs counteracts the development of fibrosis and improve pulmonary epithelial function in a neonatal model of hyperoxia-induced lung injury. These results can contribute to unraveling the mechanism of action of these nanoparticles in preventing the development of BPD.

Chair: Antonio Marcilla -Universitat de Valencia

Introduction: The intestinal microbiota influences mammalian host physiology in health and disease locally in the gut but also in organs devoid of direct contact with bacteria such as the liver and brain. Extracellular vesicles (EVs) or outer-membrane vesicles (OMVs) released by microbes are increasingly recognized for their potential role as biological shuttle-systems for inter-kingdom communication. However, physiologically relevant evidence for the transfer of functional biomolecules from the intestinal microbiota to individual host cells by OMVs in vivo is scarce. Methods: By introducing Escherichia coli engineered to express Cre-recombinase (E. coliCre) into mice with a Rosa26.tdTomatoreporter background, we leveraged the Cre-LoxP system to report the transfer of bacterial OMVs to recipient cells in vivo. Results: Colonizing the intestine of these mice with E. coliCre, resulted in Cre-recombinase induced reporter gene-expression in cells along the intestinal epithelium, including intestinal stem cells as well as mucosal immune cells such as macrophages. Furthermore, even far beyond the gut, bacterial-delivered Cre induced extended marker gene expression in a wide range of host tissues, including the heart, liver, kidney, spleen, and brain. Summary/Conclusion: Together, our findings provide a method and proof of principle that OMVs can serve as a biological shuttle system for the horizontal transfer of functional biomolecules between bacteria and mammalian host cells. Introduction: Salmonella Typhimurium is a causative agent of nontyphoidal salmonellosis, for which there is a lack of a clinically approved vaccine in humans. As an intracellular pathogen, Salmonella impacts many cellular pathways, which may play a role in the immunity towards this pathogen. However, the intercellular communication mechanism facilitated by host-derived small extracellular vesicles (sEVs) is an overlooked aspect of the host responses to this infection. Methods: We used a proteome-based network analysis of proteins associated with sEVs derived from Salmonella-infected macrophages. Next, we performed in vitro and in vivo studies using BALB/c mice to identify the role of sEVs in adaptive immunity against the challenge with Salmonella. Results: Our bioinformatics-based analysis of vesicular proteins generated by infected macrophages predicted that the hostderived sEVs generated during macrophage infection stimulate macrophages and promote activation of T helper 1 (Th1) cell activation. We also identified that sEVs generated during infection contain Salmonella proteins, including specific antigens that were previously shown to stimulate protective immune responses against Salmonella in vivo. Furthermore, we showed that sEVs generated by infected macrophages stimulate a mucosal immune response against Salmonella infection when delivered intranasally to BALB/c mice, a route of antigen administration known to initiate mucosal immunity. Specifically, sEVs stimulated the production of anti-Salmonella IgG and IgA antibodies. These sEVs also lead to an antigen-specific cell-mediated immunity. In particular, splenic mononuclear cells isolated from mice administered with sEVs derived from Salmonella-infected macrophages increased CD4+ T cells secreting Th1-type cytokines if restimulated with Salmonella antigens. Moreover, sEVs led to a reduced bacterial burden in organs in the challenge study and increased survival of the animals. Summary/Conclusion: Our results demonstrate that sEVs formed by macrophages during infection contribute to Th1 cell bias in the anti-Salmonella responses. Collectively, we unraveled the novel role of sEVs as vehicles transmitting antigens to induce Th1-type immunity against Gram-negative bacteria. Understanding the sEVs-mediated defense mechanisms will allow the development of future approaches to combat intracellular bacterial infections. Funding: R03 AI135610-02

Tiziana Corsello-Gorgun; Yue Qu; Teodora Ivanciuc; Antonella Casola; Roberto Garofalo Department of Pediatrics, Division of Clinical and Experimental Immunology and Infectious Diseases, Galveston, USA Introduction: Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infections in children and elderly. Extracellular vesicles (EVs) are microvesicles known to carry biologically active molecules, including RNA, DNA and proteins. EVs have the potential to modulate cellular responses of recipient cells following a variety of stimuli. Viral infections can induce profound changes in EV composition, but EV contribution to the replication and pathogenesis of respiratory viruses is largely unknown. In this study, we investigated the composition/function of EVs released from airway epithelial cells infected with RSV and tested whether RSV-EVs affect the antiviral response of recipient cells. Methods: EVs were isolated from uninfected (control-EVs) and RSV infected (RSV-EVs) A549, a human alveolar type II-like epithelial cell line, or from primary small airway epithelial (SAE) cells. EVs were purified using a two-step enrichment procedure and characterized using particle sizing (size and concentration) and Western blot for the EV markers. Recipient A549 and SAE cells were cultured for 24 hours in the presence or absence of control-EVs or RSV-EVs, then infected with RSV for 24 hours. Viral titers by plaque assay were measured in recipient infected cells. IFNs and cytokine assays were used to detect their levels in EVs and in recipient cells. Results: Recipient cells, both A549 and SAE cells, treated with RSV-EVs showed significantly lower viral replication than control EV-treated cells in response to RSV infection, indicating that EVs released from A549 and SAE cells infected with RSV (RSV-EVs) can transfer antiviral activity to infected neighboring cells. In addition, significant amount of immuno-reactive interferon beta (IFN-β) and lambda (IFN-λ) were detected by ELISA in preparations of isolated RSV-EVs. To test the bioactivity of IFNs associated with RSV-EVs, recipient cells were treated with Cerdulatinib, an IFN receptor signaling inhibitor. Following RSV infection, increased viral replication was observed in recipient cells treated with Cerdulatinib and RSV-EVs compared to recipient cells treated with RSV-EVs alone, suggesting that IFNs carried by epithelial EVs are indeed biologically active.

The microbiota-gut-brain axis is beginning to take centre stage in Parkinson's disease (PD) research, as the spotlight becomes fixed on microbiotic dysbiosis as a facilitator and driver of PD pathogenesis. It is understood that the gut microbiota promotes local, systemic and neural inflammation, which can promote neurodegeneration in PD, however how bacteria influence these processes remains unclear. We propose that Outer membrane vesicles (OMVs) released from Gram-negative bacteria are a mediators of PD, due to their immune stimulatory composition, including containing potent lipopolysaccharides (LPS) and capability to impact gastrointestinal permeability and influence global inflammation. Methods: OMVs isolated from bacterial culture or murine faeces were characterized by density, size, morphology, LPS content and ability to stimulate immune cells in vitro. OMVs from bacterial culture or an equivalent dose of LPS were orally administered to a PD model mouse to determine if OMVs contribute to the acceleration and exacerbation of PD inflammation, and gastrointestinal and neurological dysfunction. Results: Escherichia coli OMVs exacerbated dysfunction in the gut of PD model mice evidenced by reduced faecal pellet output and increase of some proinflammatory cytokines. OMVs also promoted motor dysfunction in PD model mice and accelerated neurodegeneration. These results indicate OMVs can influence gastrointestinal and neuronal pathology and function. Summary/Conclusion: This is the first study to demonstrate that orally administered OMVs promote inflammation, behavioural dysfunction and neurodegeneration in a PD mouse model, and thus could contribute to the pathogenesis of PD. The greater implications of this work include drawing functional connections between microbiotic dysbiosis, inflammation and the gutbrain axis, which is relevant to PD, but also many other neurodegenerative disorders.

Regis Stentz; Emily Jones; Rokas Juodeikis; Andrew Goldson Goldson; Arlaine Brion; Catherine Booth; Simon Carding Quadram Institute, Norwich, United Kingdom Introduction: It is increasingly apparent that bacterial extracellular vesicles (BEVs) produced by members of the intestinal microbiota contribute to microbe-host cell interactions. Unresolved questions are, what is the nature of the cargo packaged into BEVs and how do they impact on host cell function? Here we analyzed and compared the proteome of BEVs produced by the major human gut symbiont Bacteroides thetaiotaomicron (Bt) produced in vitro and in the mouse intestine and identified proteins that are exclusively enriched in BEVs produced in vivo suggesting that their increased abundance is induced by host-related factors. Methods: In vitro conditions consisted of cultures of Bt grown in a complex medium and in vivo studies consisted of orally administering Bt to germfree mice and collecting cecal contents 3 days later. Vesicles from cultures and cecal contents were concentrated by crossflow filtration, separated by size exclusion chromatography (SEC) and recovered by ultracentrifugation followed by protein extraction. Differential expression of BEV proteins obtained under different conditions were explored using tandem mass tagging (TMT) combined with liquid chromatography mass spectrometry (LC-MS/MS). Data sets were analyzed using the Proteome Discoverer v2.1 software. Enzyme assays were performed using intact BEVs fractionated by SEC and obtained from the same conditions.

We identified 102 proteins highly enriched in BEVs in vivo of which the majority (66/102) were enriched independently of their expression in parental cells. These abundantly BEV-secreted proteins included a dipeptidyl-peptidase IV (CD26) and an asparaginase and the highly efficient degradation of substrates by intact BEVs derived from the mouse caecum was demonstrated. The potential significance and impact of these activities on host physiology will be discussed. Summary/Conclusion: These findings provide new insights into the role BEVs play in microbiota-host interactions with their contents capable of playing key roles in the maintenance of intestinal homeostasis and host metabolism. Funding: This work was supported in part by the UK Biotechnology and Biological Sciences Research Council under grant numbers BB/J004529/1, BB/R012490/1, and BBS/E/F000PR10355 (SC).

Andrea L. Hahn 1 ; Aszia Burrell 2 ; Kylie I. Krohmaly 3 ; Claire Hoptay 2 ; Robert J. Freishtat 1  Children's National Hospital, Washington, USA;  Children's National Research Institute, Washington, USA;  George Washington University, Washington, USA Introduction: Cystic fibrosis (CF) is an autosomal recessive disease caused by dysfunction of the CF transmembrane conductance regulator (CFTR) gene. Persons with CF suffer from recurrent and chronic lung infections, with Pseudomonas aeruginosa (Pa) being one of the most impactful on lung inflammation and disease progression. One particular virulence factor carried by Pa extracellular vesicles (EVs), Cif, induces lung inflammation through modification of CFTR expression and induction of the NFkB signaling pathway. We hypothesized that Pa EVs would induce increased NFkB signaling in wild-type (WT) compared to CF human bronchial epithelial (HBE) cells due to increased presence of CFTR protein.

Methods: EVs were isolated from the sputum supernatants of persons with CF using precipitation and size exclusion chromatography (1 mL starting volume). Pa antibodies were attached to releasable magnetic beads to isolate Pa-specific EVs. Life extended WT and CF HBE cells (F508del/F508del) were grown to 80% confluency, and equivalent volumes of Pa EVs (250 uL) were added and incubated for 22 hours before cell harvesting. Cells were immediately lysed with TRIzol and RNA extracted with a DirectZol MiniPrep kit. RNAseq was performed using NextSeq and HiSeq. A Galaxy workflow incorporating HISAT2, Stringtie, Gffcompare, featureCounts, and DESeq2 was used to determine differential gene expression. Ingenuity pathway analysis (IPA) was used to identify differences in canonical pathways. Results: RNA quality assessment showed RIN values near 10, with an average of 35 million reads per sample (range 14-55 million). A total of 246 transcripts were differentially abundant between CF and WT HBE cells after Pa EV exposure with an unadjusted p-value < 0.05, which were imported into IPA. Importantly, NFkB signaling was significantly upregulated (Z-score 2.12, genes BMP4, CSNK2B, FGFR3, IL1A, LTBR, MAP2k7, PRKACA, RELA), while interleukin (IL)-6 and IL-8 signaling pathways also trended toward upregulation in WT HBE cells compared to CF HBE cells. Summary/Conclusion: NFkB, IL-6, and IL-8 inflammatory signaling pathways were increased in WT compared to CF HBE cells, suggesting CFTR expression is an important variable in Pa EV induced inflammation. Introduction: Extracellular vesicles (EVs) are the emerging alternative as the therapeutic delivery and vaccine platform. However, low efficiency on EV cargo loading and cellular secretion remains the biggest roadblock. Therefore, we developed a "smart" polymeric micelles as the first nanoparticle-based cellular transfection tool for enhancing the site-specific packaging of EVs with desired cargoes. Methods: The PCL-PEG functionalized polymeric micelles have been designed and synthesized for carrying therapeutic peptides/drugs via photo-cleavable chemical groups and self-assembled as nanoparticles. The human dendritic cells were used for fast uptaking which allows the internalization of carried cargos. Owing to the conjugated photo-cleavable group, the photocontrolled release of cargos are enabled on demand for releasing carried cargos into endocytic pathway specifically, which is subsequently the precursor for packing EVs and secreting into extracellular environment. The characterization of synthesized nanoparticles has been confirmed by NMR and Dynamic Light Scattering (DLS) analysis. The secreted EVs have been characterized by nanoparticle tracking analysis in terms of size, concentration, and zeta potential, NanoView surface marker profiling, as well as the SEM and TEM for morphological study. Results: Compared to conventional transfection of cargo, the small hydrodynamic size and uniform spherical morphology has been observed from synthesized nanoparticles allowing for rapid cellular uptake and internationalization. Here we used the tumor antigenic peptide gp-100 as the model cargo. The time elapse confocal imaging of internalization and cargo packing demonstrated the cargo release into the endocytic pathway for forming the secreted EVs. Such highly efficient cellular transfection is specific to endocytic pathway, which provides a new solution for EV therapeutic cargo loading. The entire cellular transfection and EV loading protocol is simple by adding polymeric micelle nanoparticles into culture medium and treat with 365nm light at the incubation time of 30-60 minutes, which is compatible with conventional cell culture system for scaling up and GMP manufacturing without special facility setup. The further investigation on therapeutic function in vivo is underway. Summary/Conclusion: We introduce a viable nanotransfection method for promoting cellular production of cargo loaded EVs via the endocytic pathway. The photo-controlled release strategy during the cell culture is simple and effective, which is desirable for large-scale EV cellular manufacturing. Funding: NIH NIGMS MIRA award 1R35GM133794

Dhanu Gupta 1 ; Samir El Andaloussi 2  Department of laboratory medicine, Karolinska Institutet, Stockholm, Sweden;  Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden, Huddinge, Sweden Introduction: The unique properties of extracellular vesicles (EVs), such as biocompatibility and the ability to cross biological barriers, render EVs a next-generation drug delivery tool. Therapeutic EV research has seen tremendous development in the past decade, from in vitro studies towards pre-clinical models to various clinical trials. Even so, the road towards successful clinical translation has various hurdles such as manufacturing and fast plasma clearance of exogenously delivered EVs. Here we have utilised gene therapy modalities to engineer cells in vivo to utilise endogenously secreted EVs to deliver biotherapeutics. Methods: For the generation of in situ engineered EVs in vivo, the EV engineering transgenes were delivered either as an mRNA or DNA using LNPs or in adeno-associated viruses. Importantly, these delivery vectors have tropism to the liver and in part to the spleen. Therefore the in situ produced engineered EVs primarily have a hepatic origin. For tracking and assessing pharmacokinetics in situ engineered EVs in vivo, endogenous bioluminescent (BL) EV labelling strategies were used. The therapeutic application of this platform was evaluated in an in vivo intestinal inflammation model which mimics Crohn's disease. Results: BL tagging of in situ EVs showed a body-wide distribution of EVs to all major organs, including hard-to-reach tissues such as CNS and muscle. This system could achieve a sustained release of engineered EVs in circulation and showed a dramatic increase in pharmacokinetic profile over exogenously administered EVs. For therapeutic applications, using the liver as a bio factory, in situ engineered EVs potently delivered an anti-inflammatory protein to alleviate intestinal inflammation in an in vivo disease model. Summary/Conclusion: By harnessing the latest innovations in nucleic acid therapy, this approach utilises the power of both synthetic and natural drug delivery vectors for efficient biotherapeutics delivery to hard-to-reach organs. Introduction: The physical stimulus of shockwave therapy (SWT) has a pro-angiogenic impact on ischemic tissue, representing a promising regenerative approach. The Hippo signaling pathway YAP/TAZ plays a key role in angiogenesis and can be regulated by mechanical signals. Both SWT stimulation and YAP/TAZ activation cause a release of extracellular vesicles (EVs). We aim to substantiate the mechanotransduction of SWT via YAP/TAZ facilitated EV release and subsequent angiogenic response. Methods: Human umbilical vein endothelial cells (HUVECs) were stimulated with 300 impulses at a frequency of 3 Hz and an energy flux density of 0,1mJ/mm2. 4h thereafter, mRNA expression of YAP/TAZ target genes (ANKRD1, CYR61) was measured and the nuclear localization of YAP/TAZ was examined by immunofluorescence. The culture supernatant was collected. EV release was characterized by flow cytometry (Cytoflex LX calibrated with fluorescent silica beads) using the membrane dye Cell Mask Orange (CMO) and tetraspanin markers CD63 and CD81. Furthermore, EVs were analyzed by a bead-based flow cytometry assay with CD63-coupled magnetic beads. Results: SWT of HUVECs resulted in a higher concentration of CMO+ EVs (9,953±2,039 vs.6,092±1,503 EVs/μl) in the culture supernatant as compared to the untreated control. This was confirmed by a higher percentage of EV-decorated beads after SWT, accompanied by higher mRNA expression of YAP/TAZ target genes ANKRD1 (p=0.0005, respectively) and CYR61 (p=0.0006, respectively). Immunofluorescence staining showed nuclear translocation of YAP/TAZ upon SWT compared to untreated controls. These effects were abolished and the EV concentration was decreased upon pharmacological inhibition of YAP/TAZ nuclear translocation. Summary/Conclusion: The mechanical stimulus of SWT activates the Hippo-Pathway YAP/TAZ and causes EV release. Our study provides substantial insight into cellular mechanotransduction and serves as the basis for translation into clinical practice.

Qi Hui Poh; Alin Rai; David W. Greening Baker Heart and Diabetes Institute, Melbourne, Australia Introduction: A major cause of infertility is failure of the embryo implanting to the maternal endometrium. This process of implantation requires precise coordination between the endometrium and embryo and is mediated, in part, by extracellular vesicles (EVs) released by the outer layer of the embryo (trophectoderm). Hence, engineering nanovesicles that recapitulate trophectoderm EV function is an attractive therapeutic strategy to improve embryo implantation and reproductive success. Methods: Human stem cell-derived trophectoderm cells (Tsc) were serially extruded (10, 5, 1 μm) to generate nanovesicles (NVs), purified using density-based separation (6.5 x 105 cells, 1.13g/cm2), characterized using cryo-electron microscopy, single particle tracking analysis, and quantitative proteomics, and functionally assessed for attachment of human trophectoderm spheroids and mouse embryo outgrowth post-hatching from the zona pellucida. Results: Tsc-NVs were spherical in morphology, ∼150 nm in diameter, transferred to endometrial cells (24 h), and significantly improved trophectoderm spheroid attachment to low receptive human endometrium (HEC1A) cells (p< 0.05, n=5) and mouse embryo outgrowth on fibronectin matrix, similar to native trophectoderm EVs. Using quantitative proteomics, nanovesicle transfer reprogrammed low receptive endometrial cell proteome landscape, upregulating expression of factors (ALPP, CD44, ITGA2, ITGB1, PHB, TNC) and molecular pathways (non-integrin membrane-ECM interactions, ECM proteoglycans) involved in embryo implantation and endometrial remodeling and receptivity. To enhance therapeutic efficacy, engineered nanovesicles can also be modified to encapsulate pro-implantation factors, including epidermal growth factor (EGF). Summary/Conclusion: Therefore, this strategy of nanovesicle generation is potentially a highly scalable and modifiable therapeutic approach for personalized reproductive medicine. 

Wenyi Zheng 1 ; Xiuming Liang 1 ; Rui He 2 ; Jeremy Bost 3 ; Samir El Andaloussi 1  Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden, Huddinge, Sweden;  Karolinska Institutet, Huddinge, Sweden;  Department of laboratory medicine, Karolinska Institutet, Stockholm, Sweden Introduction: Small extracellular vesicles (EVs) from immunomodulatory cells, such as mesenchymal stem cells, are potential therapeutics for inflammatory disorders, while the efficacy thereof is limited by fast elimination from circulation as well as low abundance at target tissues. Endothelial cells at inflammatory sites typically upregulate E-selectin and other adhesion molecules to facilitate leukocytes infiltration via binding to the surface glycan sialy Lewis-X (sLeX).In this study, we aim to boost the antiinflammatory efficacy of small EVs by displaying the glycan ligand sLeX. Methods: EV source cells, primarily HEK-293T cells, were genetically engineered in a modular manner to display the glycan sLeX on EV surface. Small sLeX-EVs were harvested and characterized in terms of ligand expression by single-vesicle flow cytometry, size distribution, affinity to recombinant E-selectin protein, cellular uptake by endothelial cells, and circulation time in mouse. Results: Through simultaneous expression of fucosyltransferase 7 and a modified glycoprotein based on the EV scaffold protein CD63, sLeX was successfully displayed on source cells as well as on small EVs. The small sLeX-EVs had typic spheric shape and size distribution as exosome, like their counterpart without glycoengineering. In difference, only small sLeX-EVs displayed increased uptake in activated murine as well as human endothelial cells. Moreover, displaying the glycan sLeX extended the circulation time of small EVs in multiple mouse models. Summary/Conclusion: Displaying the glycan sLeX on small EVs through genetic engineering achieved higher uptake by endothelial cells in inflammatory state and retention in mouse plasma. The next steps involve using immunomodulatory mesenchymal stem cell as the source cell and examining the anti-inflammatory functions of sLeX-EV. Funding: S.E.-A. is supported by H2020 EXPERT, SSF-IRC, ERC CoG, and SSMF. S.E.-A. is a founder, consultant, and stock holder of EVOX Therapeutics.

Ludovic Giloteaux; Maureen Hanson Cornell University, Ithaca, USA Introduction: ME/CFS is a debilitating disease of unknown etiology lasting for 6 months or more, featuring fatigue, cognitive impairment, myalgias, and immune system dysfunction. These symptoms are exacerbated upon physical or mental activity, a phenomenon known as post-exertional malaise. EVs are known to be released following exercise and their cargo could be involved in cell-to-cell signaling that results in abnormalities in patients' immune function and metabolism at baseline, but particularly after exertion. We aimed to characterize the content of EVs in ME/CFS and controls before and after exercise. Methods: Blood samples were collected from 18 female with ME/CFS and 18 female controls before, 15 minutes and 24h after a cardiopulmonary exercise test. EVs were isolated from plasma by SEC and characterized by NTA,TEM and immunoblotting. An LC-MS/MS based shotgun proteomics analysis strategy was used to perform longitudinal proteomic profiling of EVs. Linear mixed models assessed differences between groups and changes in response to exercise at each time point along with gene ontology (GO), pathway enrichment and protein-protein interaction network analysis. Linear regressions were applied to find analytes associated with physiological parameters. Results: Morphological analysis showed a homogeneous population of vesicles in both groups with no significant differences of size at the 3 time points. ME/CFS individuals had significantly higher levels of EVs at baseline and post exercise. There was a significant increase of EVs in the control group post exercise not observed in patients. EV proteomics analysis identified of 886 proteins, including 164 and 95 unique to the controls and ME/CFS subjects respectively. Several proteins were found to be significantly and consistently dysregulated at the 3 time points, while other were only dysregulated during recovery (15min. and 24h). GO and pathway enrichment analysis of differentially expressed proteins evidenced redox imbalance in ME/CFS, disruption of the ephrin signaling pathway and dysregulation of carbon metabolism. Furthermore, we found EV proteins that were solely associated with peak oxygen consumption in the ME/CFS group post-exercise. Summary/Conclusion: The abundance of EVs changes following exercise and the protein cargo of EVs differs at each time point and between cases and controls. This work demonstrates that patients and controls differ in their response to exercise with regard to EV proteins involved in immune response, energy metabolism, muscle contraction, and axon guidance. The findings of this study may open new windows to reveal ME/CFS pathogenic mechanisms and may aid in the development of better biomarkers. Funding: This project was funded by NIH NNINDS/OD/NIDA/NHLBI/NHGRI through NINDS U54NS105541 to the Cornell Center for Enervating Neuroimmune Disease.

Carlos J. Nogueras-Ortiz 1 ; Pamela Yao 1 ; Liz Calzada 1 ; Erden Eren 1 ; Christopher Dunn 2 ; Olga Volpert 3 ; Francheska Delgado-Peraza 1 ; Maja Mustapic 1 ; Christopher Ramsden 1 ; Erez Eitan 3 ; Dimitrios Kapogiannis 4  Laboratory of Clinical Investigation, National Institute on Aging, Baltimore, USA;  Flow Cytometry Unit, National Institute on Aging, Baltimore, USA;  NeuroDex Inc., Natick, USA;  National Institutes of Health, Baltimore, USA Introduction: The neuronal cell adhesion molecule L1CAM has been considered until recently as an extracellular membrane marker of blood-borne neuron-derived extracellular vesicles (NDEVs) and hence targeted via immunoprecipitation (IP) to derive EV biomarkers of brain pathology. Although cumulative evidence showing enrichment of neuronal cargo after L1CAM IP supports this rationale, recent publications claimed that L1CAM is not EV-associated. To address this challenge, we performed single-vesicle studies showing L1CAM in plasma-derived EVs in association with bona fide neuronal markers. Methods: The existence of L1CAM+ NDEVs was evaluated by measuring both EV-associated and soluble L1CAM in human plasma fractionated via size-exclusion chromatography (SEC; Izon® qEV10/70nm columns) using a previously validated Simoa® assay and an intact EV Luminex assay designed to detect L1CAM in pan-tetraspanin positive EVs. The detection of EV-associated L1CAM in SEC EV fractions as well as in the L1CAM IP eluate was confirmed using high-resolution confocal microscopy, immune-electron microscopy and high sensitivity nanoscale multiplex flow cytometry analysis (FCA) under non-swarming conditions. Results: L1CAM Simoa® detected L1CAM in early SEC EV fractions devoid of soluble plasma proteins. Findings were confirmed by: 1) intact EV Luminex assay showing EV-associated L1CAM in early SEC fractions; 2) immune electron and confocal microscopy allowing visualization of single L1CAM/Alix and L1CAM/VAMP2 double-positive nanoparticles; and 3) FCA demonstrating a sub-population of L1CAM+ EVs among total plasma EVs. These methods also showed that L1CAM IP results in the capture of L1CAM-bearing EVs double-positive for bona fide EV markers, as well as neuronal markers VAMP2 and GAP43, and a 25-fold enrichment of L1CAM-positive EVs over SEC EV fractions. Summary/Conclusion: Multiple EV analyses at the single-vesicle level confirmed the presence of plasma NDEVs bearing surface L1CAM, thus validating L1CAM as a target for NDEV immunocapture.

Dmitry Ter-Ovanesyan 1 ; Emma J. K. Kowal 2 ; Wendy Trieu 3 ; Bogdan Budnik 3 ; Siddharth Iyer 3 ; Aviv Regev 4 ; George Church 3 ; David Walt 3  Harvard University, Cambridge, USA;  MIT, Cambridge, USA;  Wyss Institute for Biologically Inspired Engineering, Boston, USA;  Genentech, San Francisco, USA Introduction: Extracellular vesicles (EV) are an exciting class of biomarkers since they contain RNAs and proteins from the cell of origin. Isolating neuron-specific EVs from human biofluids such as cerebrospinal fluid (CSF) or plasma represents a promising approach for understanding the state of neurons and diagnosing neurological disease. Previous attempts to isolate neuron-derived EVs have relied on immuno-isolation of L1CAM, which we have previously found to be a free protein in CSF and plasma. Thus, new candidate markers are needed. Methods: We developed an unbiased pipeline for the identification of cell-type specific EV markers based on gene expression and EV proteomics data and applied it to prioritizing candidates for the isolation of neuron EVs. We generated high quality data for the EV proteome of human CSF and plasma based on a novel EV purification technique involving multimodal chromatography resin. We then analyzed transmembrane proteins to assess their cell type of origin based on organ-level and cell-level RNA-Seq datasets. Results: We have characterized the EV proteome of human CSF and plasma to higher depth than previous studies after purifying EVs to exceptionally high purity. Using this data, we suggest several new candidate markers that found in EV biofluids as well as displaying neuron specific expression within the brain and brain-specific expression within the body. Summary/Conclusion: Our work represents a framework for the identification of cell type-specific EV markers and an important step towards the isolation of neuron-specific EVs for diagnostics of neurological disease. Funding: Chan Zuckerberg Initiative; Open Philanthropy Project; Wyss Institute OS. Tau, antioxidant proteins, and cellular markers are altered in brain tissue extracellular vesicles in Alzheimer's disease Introduction: Alzheimer's disease (AD) is a slowly progressive neurodegenerative disease characterized by intracellular or extracellular protein accumulation and associated neuroinflammation. Extracellular vesicles (EVs) have emerged as important participants in AD pathophysiology. Brain tissue derived EVs (bdEVs) act locally at the source and also leave the brain to betray the state of the central nervous system (CNS). Recent advances allow us to separate bdEVs and reveal bdEV proteome changes in AD brains. To find additional candidates for AD detection and peripheral bdEV capture, selected AD pathogenesis-related proteins and CNS cellular origin markers were tested on bdEVs from AD and controls. Methods: Post-mortem human AD (n=23) and control (n=7) brains were obtained from the Johns Hopkins Alzheimer's Disease Research Center. Brain tissue-derived EVs were separated as previously described (Huang, et al., JEV, 2020; Vella, et al., JEV, 2017) and characterized in accordance with the recommendations of the Minimal Information for Studies of EVs (MISEV). Lysed and intact bdEVs were then subjected to electrochemiluminescence-linked (ECL) immunoassay for detection of AD pathogenesis related proteins and selected cellular of origin markers. Results: Levels of total tau, phosphorylated tau, and antioxidant proteins peroxiredoxin 1 (PRDX) 1 and 6 were significantly elevated in AD bdEVs compared with controls. CNS cell marker detection indicated bdEVs from different cell types, including astrocytes, endothelia, microglia, and neurons. Moreover, several EV cellular origin surface markers were elevated in AD patients. Summary/Conclusion: Elevation of several EV markers was observed, indicating cell activation and/or increased release of these EVs in AD. The dysregulated molecules may be involved in AD mechanisms, constitute new biomarkers for disease monitoring, and, in the case of surface molecules, serve as targets for bdEV capture. Funding: This work was supported in part by grants AI144997, MH118164, and UG3CA241694 to Kenneth W. Witwer.

Simran Rastogi 1 ; Fredrik Nikolajeff 2 ; Saroj Kumar 1  All India Institute of Medical Sciences, New Delhi, New Delhi, India;  Lulea University of Technology, Lulea, Sweden Introduction: The earlier studies of our group proved the link between saliva-derived small-sized extracellular vesicles (sEVs) and Parkinson's disease (PD). PD, a manageable condition although remains asymptomatic in the early stages. The current study used Fluor NTA (nanoparticle tracking) and explores how it could be a potent biomarker for PD. Methods: The salivary sEVs of the subjects (PD=70, healthy controls=26) were isolated by PEG-based chemical precipitation followed by antibody-based validation through CD63 (universal surface marker) and confirmed neuronal origin by CD171 as well morphologically characterized through cryo-EM. The sEVs quantification via fluorescence-tagged sEVs NTA and antibodybased NTA using CD63. The α-syntotal in sEVs cargo was determined by ELISA. The confirmation of the disease severity staging was done by 99mTc-TRODAT-SPECT. Results: We observed a significant increase of fluorescence-tagged sEVs in PD (p< 0.0001) than the HC via NTA (sensitivity of 94.34%) as well it was in line with the antibody-tagged sEV p=0.006 (sensitivity of 94.12%). A significant increase of αsyntotal concentration in the sEVs of PD when compared to HC (sensitivity of 88.24%). The fluorescence-tagged sEV depicted a positive correlation with the hallmark protein of PD α-syntotal r=0.4709, p=0.0486. The striatal binding ratios in the 99mTc-TRODAT-SPECT shown to have a positive correlation with the fluorescent sEVs concentration r=0.3000, α-syntotal concentration r=0.8000. The findings suggested that the salivary sEV concentration and the expression level of α-syn are not only higher in PD patients than HC but also followed the results of the TRODAT scan. Summary/Conclusion: This study is the first to address that the fluorescence tagged sEVs can screen the progression of the disease with clinically acceptable sensitivity and can be a potent early detection method for PD. Funding: The funding for this study was provided by Lulea University of Technology, Sweden, Department of Health Research (DHR) and Indian Council of Medical Research (ICMR), India for extramural and AIIMS intramural research grants. Introduction: Extracellular vesicles (EVs) are emerging vehicles in studying neurodegenerative diseases including Alzheimer's disease (AD). Almost every cell type in the central nervous system including neurons and glia are known to shed EVs, which can be detected in the cerebrospinal fluid (CSF). Monitoring cell type-specific EVs from patient-derived CSF and profiling their contents by transcriptomic or proteomic analyses provide a useful method to study the pathophysiology of AD. We have recently identified EV unique molecules to human neurons, astrocytes, microglia-like cells, and oligodendrocytes. We applied these datasets for the characterization of CSF EV proteins and their changes in mild cognitive impairment (MCI) and AD cases. Methods: We performed combined label-free and tandem mass tag-labeling based quantitative mass-spectrometry of EVs isolated from human induced pluripotent stem cells (hiPSCs) by using size exclusion column method and CSF by MagCapture method to conduct a comprehensive EV proteomics study on controls, AD and MCI risk cases. The enrichment of EV was determined by quantitative mass-spectrometry and immunoblotting of EV and non-EV markers. The changes in the cellular composition of the EV profiles were assessed for their correlation with disease status, pathway analysis and correlation with cognitive function. Results: Novel cell type-specific EV protein markers were identified from induced pluripotent stem cell-derived excitatory neurons (e.g., NCAM1, ATP1A3), astrocytes (e.g., LRP1, ITGA6), microglia-like cells (e.g., LCP1, ITGAM) and oligodendrocytes (e.g., LAMP2, FTH1). Furthermore, cell type-specific EV molecule analysis revealed significant enrichment of astrocyte and microglia-derived EVs in AD CSF compared to control or MCI EV samples. Moreover, Quantitative proteomics analysis showed that EVs expressed 1284 unique proteins in AD, MCI and control groups. Statistical analysis identified three proteins (HSPA1A, NPEPPS, and PTGFRN) involved in AD progression. Finally, the PTGFRN showed a correlation with amyloid plaque (rho = 0.404, p = 0.027) and tangle scores (rho = 0.500, p = 0.005) in AD, MCI cases. Summary/Conclusion: Our study presents novel human neural cell type-specific EV markers, highlights the key role of gliaderived EVs in AD CSF. The study also indicate that three proteins, HSPA1A, NPEPPS and PTGFRN, may be used to monitor the progression of MCI to AD. Introduction: Extracellular vesicles (EV) are small membrane structures released by cells that act as potent mediators of intercellular communication. In the field of biomedicine they have been studied as a novel source of biomarkers and drug delivery vehicles. Detection and potential characterization of freshly isolated EV is of great interest for subsequent EV analysis and studies. Thus, lateral flow immunoassay is a potential, low-cost, and user-friendly tool for rapid EV characterization.

Methods: Fluorescent nanoparticles (EuNPs) were functionalized with an antibody against tetraspanin CD63 (detection antibody). For LFIA assays, antibodies anti-CD9 and anti-IgG were dispensed on a nitrocellulose membrane as test line and control line, respectively. Plasma-derived EV were isolated by using a precipitation reagent or by size exlusion cromatography (SEC). Size characterization and concentration were determined by nanoparticle tracking analysis (NTA) at Nanovex (Spain). Protein content in the obtained fractions was measured by BCA assay. Fluorescent LFIA was then performed to enable rapid on-site detection of EV. Results: The high sensitive fluorescent LFIA system can detect CD63+CD9+ EVs is freshly isolated plasma-derived EV, by SEC or by using a precipitation reagent. EV could be clearly detected in both cases, even in high diluted SEC fractions, and employing a only minimum volume of the sample. The limit of detection (LOD) of our system was 5.27×104 EVs /μl. Summary/Conclusion: This study shows a fluorescent LFIA may be used for rapid on-site detection of plasma-derived EVs isolated by SEC or by using a precipitation reagent. This system achieved low LOD. In addition, our system may be adapted to other surface markers to characterize further freshly isolated EV fractions according to the field of interest. Funding: This research was funded by the Ministerio de Ciencia e Innovación (Spain), grant number: PID2020-119087RB-I00. This study was also financed by European Union through the European Regional Development Fund (Principado de Asturias, FICYT, PCTI 2018 -2022 , under the grant number: IDI/2021/000112.

Alice Gualerzi; Silvia Picciolini; Francesca Rodà; Valentina Mangolini; Marzia Bedoni

Introduction: Extracellular vesicles (EVs) from mesenchymal/stromal cells (MSC) are one of the emerging strategies for cellfree regeneration therapies. Problems of purification, yield, and reproducibility of the isolation procedure are some of the hurdles in their translation to clinics. Differently from their cellular counterpart, MSC-derived EVs cannot rely for their quality control only on the superficial markers because EV-markers are more difficult to be detected, require dedicated batches and sophisticated technologies. Raman spectroscopy (RS) can be used as quality control as it provides the label free biochemical characterization of the sample; it is fast, cost-effective and sensitive, it does not focus on a specific component, but rather looks at the true complexity of their composition. Methods: The RS bulk characterization of EVs was performed on preparations from different research laboratories and small industries . The reproducibility of different isolation methods, the purity of the preparations and the quality of the cell sources were assessed by calculating the spectroscopic protein-to-lipid ratio and performing the multivariate statistical analysis of the collected spectra.

The results obtained by the Laboratory of Nanomedicine and Clinical Biophotonics demonstrate that the Raman approach is effective in the quality control of both EVs and conditioned media obtained from MSC cultures, demonstrating that RS can be a valuable tool in the reproducibility assessment of cell-free products to be used in the pipeline of stem cell-derived preparations for regenerative medicine. Summary/Conclusion: The RS approach to the massive production of EVs can help in the assessment of the proper isolation procedure for a downstream application, in the evaluation of reproducibility among batches and operators and in the comparison of data obtained between laboratories by the generation of online databases that can merge Raman data of EVs from various sources to foster transparency of data. Funding: The project was supported by the Italian Ministry of Health. Introduction: DNA-assisted proximity assays are powerful and versatile tools for sensitive, specific and high throughput detection of macromolecules such as DNA, RNA and proteins as well as posttranslational modifications in in situ and in liquid biopsies. Methods: Commonly, in these assays the target molecules are recognized by several proximity probes, each equipped with a DNA oligonucleotide. Upon binding of the target molecules, the DNA oligonucleotides are brought in proximity, subjected to enzymatic ligation or polymerization, which results in formation of an amplifiable reporter molecule. The use of multiple recognition events in combination with signal amplification allows highly specific and sensitive detection of the target molecules. Results: We have developed a large number of affinity-based proximity assays for single-and multiplex detection of large complexes. Several of these technologies, such as proximity ligation assay (PLA) combined with flow cytometry readout, multiplex proximity extension assays (PEA) and proximity barcoding assays (PBA) are used for sensitive detection and characterization of extracellular vesicles in bulk or at single molecule level. In addition, we have applied these technologies in combination with mass spectrometry for identification of exosomal surface proteins, allowing establishment of assays for detection of organ-specific extracellular vesicles in liquid biopsies. Summary/Conclusion: Here, we discuss the application of proximity assays for screening, characterization and sensitive detection of extracellular vesicles in body fluids. Introduction: Surface Enhanced Raman Spectroscopy (SERS) has been extensively applied for both labeled and label-free detection and classification of extracellular vesicles (EVs). However, the heterogeneity of EVs and their diverse biochemical compositions have restricted the clinical translation of EV SERS. Novel machine learning techniques are capable of dealing with this heterogeneity and mitigating the disadvantages of labeled methods. In this research, we developed bottleneck classifiers (BC) that can extract the unique differences between highly heterogeneous placental EV SERS data, enabling the label-free classification and visualization of preeclampsia. Methods: Placental extracellular vesicles (EVs) were harvested from donated placentae by culturing 400 mg explants in netwell inserts. EVs were isolated from conditioned media using differential ultracentrifugation and size exclusion chromatography. Following validation using transmission electron microscopy and nanoparticle tracking analysis, EV SERS spectra were obtained using EVs from both healthy and preeclamptic placentae. We then constructed an autoencoder-like network by changing the decoder to a classifier to achieve the desired BC. Encoders with both linear and nonlinear activations were considered, as linear activation achieves highly interpretable results nonlinear achieves maximum classification accuracy. Results: We show that the linearly activated encoder leads to a classification accuracy of 92%, which is greater than any other conventional machine learning, while also producing interpretable results. In fact, Raman bands of 1745 cm-1 and 1330 cm-1 play the most vital roles in classification between Normotensive (NT) and Preeclamptic (PE) EVs. Both of these bands are associated with phospholipids and this finding is consistent with previous biological research in this area. Summary/Conclusion: We have developed novel bottleneck classifiers for both the visualization and classification of EV SERS data. The superiority of the presented technique over classical techniques is shown via interpretable classification and visualization of placentae NT and PE EVs. Funding: HRC New Zealand, AMRF, and Goodfellow

Andrea Galisova; Jiri Zahradnik; Hyla Allouche-Arnon; Mattia Morandi; Ori Avinoam; Neta Regev-Rudzki; Gideon Schreiber; Amnon Bar-Shir Weizmann Institute of Science, Rehovot, Israel Introduction: Understanding viral-induced pathologies is of great interest, particularly nowadays with the prolonged COVID19 pandemic. In this regard, extracellular vesicles (EVs) offer a unique platform for studying viral infection paths and mechanisms due to similar properties as viruses and possibility of visualization by imaging methods (Han Z. et al, JEVs 2021, e12054) . Here, we show the development of genetically engineered EVs displaying the receptor-binding domain (RBD) of SARS-CoV-2 on their surface as corona-virus-like mimetics that can be labeled both magnetically or fluorescently. Methods: HEK293 cells were genetically modified to express the RBD of SARS-CoV-2 on their surface. Then, these cells were incubated with SPIO nanoparticles (2.5-40 μg/mL) and the secreted labeled EVs (EVsRBD as corona-mimetics or EVsnoRBD as controls) were isolated by differential centrifugation (400 g, 2000 g, 10 000 g, 100 000 g) and characterized by several methods (MRI, NanoSight, Western blot, flow cytometry and transmission electron microscopy -TEM). For in vivo examination, two types of subcutaneous tumors were induced in nude mice (HEK293T as control or HEK293T overexpressing ACE2) to study in vivo targeting of EVsRBD to the ACE2 receptor. EVs (3x10E11) were intravenously injected in mice followed by in vivo MRI and ex vivo fluorescence imaging. Results: Cryo-TEM analysis showed that the secreted genetically engineered EVs have typical EVs size (∼100 nm) and shape. Binding to the ACE2 receptors was confirmed by a higher uptake of EVsRBD into ACE2-expressing cells compared to control EVsnoRBD. MRI of isolated EVs showed concentration-dependent accumulation of SPIONs as depicted from the lower MRI signal on T2-weighted images. In vivo targetability of EVsRBD was demonstrated by MRI and fluorescence imaging of mice following EVs injection. The higher accumulation of the EVsRBD in ACE2-expressing cells as compared to control cells was confirmed by the lower T2/T2* MRI signal. Similarly, ex vivo fluorescence imaging showed higher accumulation of EVsRBD in the ACE2 expressing cells as compared to EVsnoRBD. Summary/Conclusion: Here, we show a multimodal imaging platform for mapping the binding of coronavirus mimetics to the ACE2 receptors both in vivo and in vitro by genetically engineered EVs expressing the RBD of SARS-CoV-2. The proposed platform can be implemented to study other viruses by engineering a tailored peptide on the EVs surface and thus to explore the role of receptors in a wide spectrum of viral pathologies and to evaluate antiviral treatments. Funding: This study was supported by the Ben B. and Joyce E. Eisenberg Foundation. Introduction: Proper isolation of Extracellular vesicles (EVs) and lipoproteins (LPs) from plasma and serum samples remains challenging. Due to their similar size and density distributions, EVs and LPs often co-isolate when using physical-based enrichment methods; detecting reciprocal contamination is in itself nontrivial. We apply a recently developed single-particle nanomechanical screening method to EVs and LPs, showing that it can be used to detect co-isolated subpopulations, obtain their size distributions and estimate their relative abundance. Methods: EVs were obtained through tangential flow filtration from a culture of human cardiac progenitor cells (CPCs) and characterized via cryo-Transmission Electron Microscopy, Atomic Force Microscopy (AFM), Nanoparticle Tracking Analysis, Western Blot. LPs (VLDL, IDL, LDL, HDL, chylomicrons) were purchased from commercial sources. All samples were characterized via a nanomechanical screening method we recently developed (Ridolfi et al 2020, doi:10.1021/acs.analchem.9b05716; Frigerio et al 2021, doi:10.3390/cells10030544) Results: Each LP subtype is found to display a specific mechanical fingerprint, which is different from that of EVs and of other LPs. Only Very-Low Density Lipoproteins (VLDL) and chylomicrons exhibit substantial reciprocal overlap in term of size and mechanical stiffness. While VLDL and chylomicrons exhibit a nanomechanical behavior associated with pressurized elastic vessels, other LPs do not -suggesting major (but still unclear) structural differences. Summary/Conclusion: To the best of our knowledge, these results represent the first single-particle mechanical investigation of lipoproteins. Our AFM-based method can resolve the nanomechanical properties of co-isolated EVs and LPs, hence providing a useful tool for assessing the purity of plasma-and serum-derived preparations. Funding: We acknowledge the grants H2020 FETOPEN-801367 "evFOUNDRY", H2020 FETPROACT 952183 "BOW", H2020-FETPROACT-951768 "MARVEL".

Introduction: EVs are of interest as a potential source of biomarkers and mediator of intercellular communication. Trace metals are essential elements involved in various physiological and pathological processes, however there is limited information on the metal content of EVs or their potential role in metal homeostasis. This study aims to comprehensively examine the metal content of EVs, isolated from a variety of in vitro and in vivo sources, and the capacity of EVs for intercellular metal transfer. Methods: 15 biologically important metals were measured in sEVs from 3 cell lines and human nasal secretions, frontal cortex or plasma using inductively coupled plasma mass spectrometry. sEVs were isolated by differential centrifugation followed by ultracentrifugation (UC), size exclusion chromatography (SEC), density gradient-UC (DGUC) or combination of DGUC and SEC. Immunoblotting and transmission electron micrography were used for validation. SH-SY5Y cells treated with Fe57 isotope or Pb were used to determine metal packaging into EVs and transfer to recipient cells. Results: Relative to cells or neat clinical samples, sEVs from in vitro and in vivo sources, were enriched in essential trace metals, particularly Fe and Cu. While Al and Pb were undetectable in neat plasma, these non-physiological metals were detected in sEVs, implying a role for sEVs in removal of toxic metals. In vitro, Fe 57 and Pb were enriched in sEVs and in recipient cells after loading donor cells with these rare metals, confirming the intracellular packaging of metals into sEVs and the capacity of sEVs for intercellular metal transfer. Summary/Conclusion: This is the first study to comprehensively characterize the metal content of EVs and demonstrate transfer of essential trace and heavy metals via EVs. This study highlights the potential roles of EVs in trace metal homeostasis and cellular defense against toxic metals and has implications for biomarker and therapeutic target discovery of diseases with metal dysregulation. (12):1240-54). Methods: Supermeres, EVs and exomeres are purified using a combination of methods including differential ultracentrifugation, density-gradient purification and size exclusion chromatography. Because producing EVs and nanoparticles can be labor intensive using a traditional 2D cell culture method, a hollow fiber bioreactor is used to produce these in high concentration. Proteomic and RNA analysis of the hollow fiber secretome was compared with the same cells grown on tissue culture dishes. Results: Supermeres have consistent protein cargos within DiFi cells grown in 2D and in the bioreactor; small RNA cargo differs somewhat between the two growth conditions. Supermeres contain various RNA binding proteins as well as proteolytically cleaved membrane proteins, including proteins associated with neurodegeneration. A major RNA cargo in supermeres is miR-1246, derived from the RNU2 snRNA. Functionally, supermeres have the ability to traverse diverse tissues including crossing the blood brain barrier, transfer drug resistance and can metabolically reprogram hepatocytes in vivo. Summary/Conclusion: Supermeres are a new class of secreted nanoparticle replete with disease biomarkers and therapeutic targets. Funding: NCI grant nos R35 CA197570, UG3 241685, P01 CA229123 and P50 236733 to R.J.C. We acknowledge the generous support of the Nicholas Tierney GI Cancer Memorial Fund Introduction: Extracellular vesicles (EVs) are membrane-based structures produced by cells, with capacity to carry miRNAs, non-coding RNAs with around 22 nucleotides. MiRNA enrichment into EVs is promoted by specific motifs, designated as Exo-Motifs. Therefore, the aim of this work was to investigate whether ExoMotifs would promote packaging of engineered miRNA-based silencing sequences into EVs to use them as therapeutic vehicles to treat Machado-Joseph Disease (MJD). MJD is a neurodegenerative disorder caused by abnormal over-repetition of a CAG tract within the ataxin-3 (ATXN3) gene, conferring toxic properties to the ATXN3 protein.

Methods: An ExoMotif signal was associated with silencing sequences targeting mutant ATXN3 (mutATXN3) to promote its packaging into EVs, which was evaluated by qRT-PCR. Additionally, neuronal targeting proteins were expressed at EVs surface, and their neuronal targeting efficiency was evaluated in vitro and in vivo by immunocytochemistry and flow cytometry. To evaluate target engagement, engineered EVs were administered by daily intranasal administration to mice expressing a mutATXN3 dual luciferase reporter system. Results: We found that silencing sequences with the ExoMotif retained the capacity to silence mutant ataxin-3 and were effectively incorporated into EVs. Furthermore, the bioengineered EVs significantly decreased mutATXN3 mRNA levels in neuronal cells. Importantly, continuous intranasal administration of therapeutic EVs significantly decreased the brain luminescence associated with mutATXN3 dual luciferase reporter system. Summary/Conclusion: MutATXN3 silencing sequences enriched in EVs reach the brain via intranasal route and are therapeutically active reducing in vivo the disease-causing ataxin-3. Introduction: Non-Typhoidal Salmonella (NTS) causes over 95 million infections each year, and despite the severity and disease burden there is no approved vaccine to combat infection. A potential vaccine strategy that has not been deeply explored, are extracellular vesicles (EVs) produced during NTS infection. Methods: To study the role of EVs isolated after NTS infection, we infected macrophages with Salmonella and isolated EVs using ultracentrifugation. Next, we treated 35 mice with either PBS control, live Salmonella vaccine (∆aroA) control, EVs isolated from NTS infection of macrophages, or disrupted EVs; and collected blood and stool samples to assess the effect of EVs on mucosal immunity. We performed ELISAs to measure IgG in serum and IgA in stool. Finally, we challenged mice orally with NTS.

Results: Our analysis of serum IgG and fecal IgA showed a significant increase in Salmonella-specific antibody responses in the EV treated mouse group. IgG and IgA increased over time for 12 weeks in both live vaccine and EV treatment groups. Challenge results showed that mice with protective IgA and IgG responses had a lower bacterial burden and increased survival compared to PBS control mice and mice given disrupted vesicles. Summary/Conclusion: Our results display the ability of EVs isolated during Salmonella infection to generate Salmonella-specific protective antibody responses. Our findings demonstrate a previously unknown role of EVs in bacterial infection and their potential as a vaccine strategy. Understanding the full immune potential of EVs will aid in EV vaccine approaches to fight the bacterial infection. Funding: R03 AI135610-02

Patricia Introduction: Osteoarthritis (OA) is a degenerative disease in which there is a loss of articular cartilage and chronic inflammation. Currently, only palliative treatments are available. In this context, mesenchymal stem cells (MSC) and their derived small extracellular vesicles (sEVs) appear as the candidate of choice for the development of new therapies for OA due to their chondroprotective and immunomodulatory properties. Methods: sEVs were isolated from metabolically reprogrammed MSC through ultracentrifugation. sEVs were then quantified by nanoparticle tracking analysis and characterized by flow cytometry. Then, sEVs were added to the culture media of either chondrocytes for regeneration or macrophages for inflammation evaluation, respectively. After 24 hours, chondrocytes were collected to measure classical OA markers through RT-qPCR and macrophage polarization was assessed through flow cytometry. Results: sEVs derived from MSC with an induced metabolic reprogramming promote the polarization of macrophages towards an anti-inflammatory phenotype by increasing expression of M2 marker CD206+. On the other hand, we observed that the sEVs were able to protect the key components of cartilage tissue by increasing the ratio of COL2/COL1 expression and decreasing the expression of aggrecan degrading enzyme ADAMTS4. Summary/Conclusion: Our results show that the metabolic reprogramming of MSC improves the therapeutic properties of their sEVs. Indeed, we demonstrate that these sEVs have an anti-inflammatory effect, evidenced by the polarization of macrophages towards an M2 phenotype. Furthermore, sEVs from metabolically modulated MSC showed a chondroprotective effect, evidenced by the increase of the COL2/COL1 expression ratio and a decrease of ADAMTS4, indicating a recovery of the hyaline cartilage phenotype. Funding: This research was supported by the Agencia Nacional de Investigación y Desarrollo (ANID) from Chile through the grants FONDECYT regular n • 1211353; FONDECYT iniciación n • 11220549; FONDEF ID: 21I10194. Introduction: Full restoration of tendon structure and function after injury has been a clinical challenge. This is mainly due to profound inflammation and insufficient regeneration after injury. We found EVs from inflammation-primed adipose-derived stem cells (iEVs) reduce inflammation after injury. However, if iEVs may improve tendon structural and functional recovery after injury and the active subgroup and mechanism of iEVs in these actions remain elusive. Methods: Bulk iEVs were prepared from conditioned medium of IFNγ-primed mouse ASC culture containing 2% EV-free FBS via differential centrifugation, separated by tetraspanin markers, and analyzed via TEM, western blot, DLS, NTA, and flow cytometry. With IACUC approval, the dose-effect of iEVs (0, 1e+09, or 5e+09 iEVs/repair) on tendon healing was assessed in a preclinical mouse Achilles tendon injury and repair model. The active subgroup and the mechanism of action of iEVs were determined in vitro. Results: iEVs dose-dependently reduced injury-site NF-κB activity in the first week after injury and led to over 2-fold increases in Arg1, Il13, and Il1rn expression and dramatic improvements in collogen deposition and organization at 4 weeks after injury. iEVs also improved mouse ankle movement by up to 45% and 64% at 3 and 4 weeks after injury and reduced the incidence of postoperative tendon rupture/gap formation by 35% at 4 weeks after injury. In vitro studies revealed that iEVs blocked TLR4/NF-κB signaling in IFNγ+LPS activated macrophages and promoted tendon cell proliferation, anabolic genes Postn, Scx, Tnmd, and Igf1 expression, and type I collagen release. The effects of CD9+/CD63+/CD81+ small iEVs were more dramatic than those of bulk iEVs. Summary/Conclusion: iEVs reduced inflammation and improved tendon structural and functional recovery after injury via modulating macrophage and tendon cell functions. It is to be determined if the identified active subgroup is more effective than bulk iEVs in enhancing tendon repair. Funding: NIH R21AR075274, WU ICTS/CTRFP4805. Introduction: Rheumatoid arthritis (RA) is an inflammatory disease of which there is no cure. Cell therapies using umbilical cord mesenchymal stromal cells (UC-MSCs) are becoming a popular possibility for treatment due to their ability to promote immunosuppression. Extracellular vesicles (EVs) mimic this but offer the advantage of safety (no malignant transformation or rejection), being easily stored and an extended shelf life. Hence, we sought to determine the ability of UC-MSCs, and their derived EVs, to treat RA using an antigen induced model of arthritis (AIA). Methods: Mice were injected with 5x105 UC-MSCs, or equivalent EVs from the same cell number, 24 hours post-arthritis induction. Cells were characterised by flow cytometry and EVs by Tunable Resistive Pulse Sensing (IZON) and cryo-electron microscopy following isolation using ultracentrifugation with a 30% sucrose cushion. Joint swelling was assessed over 72 hours, after which histological analysis was performed. Results: Cells displayed the expected phenotype markers for MSCs. EVs from 5x105 cells yielded 2.15x109 particles/ml with a mean size of 166±70nm, of which most particles displayed an EV-like morphology of a double lipid membrane bilayer. Joint swelling decreased for both treatments, in comparison to control, but only MSC-EV treated mice achieved significance (P< 0.01) at the 72-hour timepoint. EV superiority over UC-MSCs was reflected in the histological scoring, with EVs achieving a consistently lower mean score in comparison to controls (improved clinical outcome), whilst cells showed higher mean scores (worse clinical outcome). Summary/Conclusion: In this study, EVs appear to be superior to their cell counterparts when treating an arthritis model. This supports the argument that MSC-EVs may be a suitable therapy to treat RA. gene. HGSNAT is a non-secretive, exclusively trans-lysosomal-membrane enzyme. AAV gene therapy offers a great tool for treating neurogenetic diseases. All cells are known to continuously release extracellular vesicles (EVs) and communicate by exchanging large molecules via EV trafficking. Targeting the unmet need, we developed a vector with an EV-mRNA packaging signal to engender by-stander effects for the normally non-secreted HGSNAT protein.

We constructed a rAAV vector with an EV-mRNA packaging zip code (ZC) signal linked to human HGSNAT cDNA. We tested it in human MPS IIIC skin fibroblasts to determine rHGSNAT expression, EV packaging of hHGSNAT-mRNA and the correction of lysosomal GAG storage. The conditioned media from transduced MPS IIIC cells were processed for EV purification and characterization. Purified EVs were incubated with non-infected MPS IIIC cells to assess the EV-facilitated hHGSNAT expression and the cross-correction of lysosomal GAG storage. Results: rAAV-hHGSNATzc vector mediated not only efficient expression of functional HGSNAT protein, but also the release of abundant EVs containing hHGSNAT mRNA to the media. Importantly, incubation with purified EVs from transduced cells resulted in the expression of functional HGSNAT protein and the clearance of GAG contents in non-treated recipient cells. Summary/Conclusion: This study demonstrates that the incorporation of the ZC signal sequence in rAAV-hHGSNAT vector mediates EV packaging of hHGSNAT-mRNA, which can be transported to recipient cells and translated into functional rHGSNAT protein. Our data strongly support the therapeutic potential of rAAV gene therapy with EV-mRNA-cargo facilitated by-stander effects for treating MPS IIIC. It may therefore reduce the vector dose and potential risks. Further, this technology may also be applicable to broad disease targets involving transcription factors and proteins that are not secreted. Funding: R21AI146653 NC Biotech Center #2020-GTF-6903

The other abstracts in this session were late breaking and will be included in a late breaking journal addendum after the conference. Introduction: Inspired by extracellular vesicle (EV) released by blood flow, we propose to boost EV release by exerting a controlled turbulence shear stress on adipose stromal cells (ADSCs) during their culture in bioreactors in an unprecedented approach. We evidenced that turbulent flow tuning elicited massive EV release (10 times more and 10 times faster than the classical starvation method). We identified turbulence micro-vortex size as a physical parameter that ensures consistent and massive EV shedding and process scalability. The aim of this study was to investigate the regenerative effect of turbulence EVs. Methods: The therapeutic potential of turbulence EVs for regenerative medicine was investigated in 5 animal models: (i) a myocardial infarction in mice; (ii) an inflammatory perianal fistula in rats; (iii) a post-surgical colo-cutaneous fistula in rats; (iv) a post-surgical gastro-cutaneous fistula in pigs and (v) an esophageal stricture in pigs. Results: Our results in the myocardial infarction model in mice evidenced that, when compared to state-of-the-art EVs at the same dose, our turbulence EVs produced the same therapeutic effect. A healing therapeutic effect was also observed in an inflammatory perianal fistula model in rats. Our investigation in a post-surgical colo-cutaneous fistula model in rats demonstrated that turbulence EV biodistribution was improved when EV administration was performed locally combined to a thermo-responsive hydrogel. The therapeutic healing effect of this combined therapy was demonstrated in this colo-cutaneous fistula rat model as well as in the gastro-cutaneous fistula model and the esophageal stricture model, both in pigs. Summary/Conclusion: The proposed turbulence method is expected to be advantageous in terms of high yield, costeffectiveness, and time-saving. The therapeutic effect of turbulence EVs featuring the markers and potency of bona fide EVs was demonstrated in 5 animal models indicating the robustness and versatility of the turbulence production method. Funding: ERC Exocyther (number 852791), ANR Fisther Introduction: Idiopathic Nephrotic Syndrome (NS) is the most common glomerular disease in children with immune-related pathogenesis. Corticosteroids are the first-line treatment, but patients not responsive may progress to end-stage renal disease, requiring dialysis or kidney transplant. In this context, biofluid-derived extracellular vesicles (EVs) can function as carriers of biomarkers for monitoring immune activation and kidney damage in NS. Methods: PBMCs, sera and urines were obtained from thirty NS patients (steroid-sensitive and resistant) and seven healthy subjects (HS) (approval by local ethical committee). Immune cell and EV (serum and urine) profile was conducted by multicolour flow cytometry. EVs concentration, size and structure were analyzed by NTA and transmission electron microscopy. Filtration/Ultracentrifugation followed by size exclusion chromatography were used to separate NS-EVs for molecular analysis. Results: Total CD19+, naïve, and switched memory B cells in NS patients were increased compared to HS. Moreover, decreased transitional B cells and increased plasmablasts and plasma cells were found in NS with active proteinuria compared to therapyresponsive patients (remission). We also observed a reduction of Th1 and Th17, an increase of Th2 cells in NS in respect to HS, and reduced levels of Foxp3+ Treg during remission. NTA analysis revealed no differences in EV concentrations among groups, with a slight increase of NS-EV diameter (mode distribution). Biofluid NS-EVs expressed Tetraspanins with a heterogeneous distribution among the patients' subgroups; the highest expression was observed in serum EVs from proteinuria patients. Similarly to the cell compartment, serum NS-EVs showed abnormal lymphocyte markers (CD4, CD25, CD19 and HLA-DR). Interestingly, B/T cell markers were already present in serum EVs at the disease onset. Urine NS-EVs were enriched in adhesion, epithelial and leucocyte molecules, and their number positively correlated with proteinuria levels. Summary/Conclusion: Change in the biofluid NS-EV profile might represent the bloodstream NS immune dysfunctions, and EVs can act as facilitators of kidney damage in NS children. Funding: This research was supported by Grant P-0038 from IMPACTsim S.p.A. and by Fondazione La Nuova Speranza ONLUS -lotta alla sclerosi focale. Introduction: Glomerular endothelial dysfunction plays a key role in the development of chronic kidney disease (CKD). We have previously shown that glomerular endothelial cells (GEC) are damaged in Alport syndrome mice (AS, characterized by mutations in collagen IVα3α4α5), manifested by enlarged fenestrations and damaged glycocalix in the early stage of the disease. In the present study we report on the role of altered fatty acid utilization pathways leading to GEC dysfunction in AS, and the role of extracellular vesicles derived from amniotic fluid stem cells (AFSC-EVs) in re-establish lipid homeostasis. Methods: GEC were isolated from tdTomato-reporter AS and WT mice at 4 months of age by FACS and transcriptome was analyzed and compared by bulk RNA-seq. Tissue samples from patients with AS were used to confirm our findings by immunohistochemistry. In vitro, silencing experiments using human primary GEC were performed to study the role of decreased fatty acid synthase (FASN) in GEC dysfunction, and AFSC-EVs (which contain FASN in their cargo) were applied as a rescue strategy to normalize FASN level and restore lipid homeostasis. Data were confirmed using AFSC-EV FASN-/-. Results: AS GEC were highly enriched for differentially expressed genes (DEG) associated with cellular metabolism, and lipid metabolism in particular. Genes associated with fatty acid transport (CD36, FATP-1, FATP-2, Fabp3) and synthesis (FASN) among others were downregulated, which was further associated with glomerular accumulation of lipid droplets in mice. We observed similar findings in human biopsy samples from AS patients by histology. In vitro, AFSC-EVs were able to rescue FASN deficiency and improve GEC function, unlike AFSC-EV FASN-/-. Summary/Conclusion: We report for the first time a lipid metabolic dysfunction in Alport GEC, and the ability of AFSC-EVs to rescue this phenotype. Therefore, better understanding of the functional role of GEC in AS could lead to the development of targeted new therapies for the treatment of this and other forms of CKD. Introduction: Diagnosis of chronic kidney disease (CKD) progression through urinary markers could be of great potential to complement renal biopsies and imaging approaches. We aimed at identifying proteome changes of urinary extracellular vesicles (uEV) in progressive versus stable diabetic nephropathy (DN). Methods: Large and small uEV were isolated by differential (ultra-)centrifugation (1) . DN patients were stratified based on stable (n=17) or progressive pathophysiology (n=19) (mean GFR change of 1.9±1.8 and -9.4±1.2 mL/min per year, respectively), and cell-free urines sourced from two CKD-biorepositories upon ethical approval. Tryptic peptides were subjected to RP-HPLC-ESI-MS/MS using a TripleTOF6600+ mass spectrometer, in data dependent (DDA) mode and SWATH®2.0-data independent acquisition (DIA) (SCIEX-OneOmics) mode. Results: Analysis of uEV via atomic force microscopy, scanning EM and nanoparticle tracking confirmed size (50-250 nm), positive EV-markers (alix, flotillin, TSG101) and absence of negative markers (TOM20, GS28). Urinary EV proteomics versus full urine proteomics was firstly investigated by shot gun proteomics (DDA-MS/MS): the uEV displayed 4-5 more protein identities (at 1% false discovery rate) compared to the full urine depleted of abundant serum proteins, showing the advantage of uEV. Next, cohorts of stable and progressive patients (12 each) were selected for large and small uEV isolation. Qualitative proteomics of small uEVs led to 1439 protein identities, of which 30% were uniquely detected in the progressive cohort. However, only a fraction (12%) were displayed consistently in at least 4-10/12 progressive patients. Similarly, large uEV revealed 1323 protein identities, of which 37% were solely present in progressive patients. Comparative quantitative SWATH-MS showed that a lysosomal membrane component and a nephrotic syndrome-linked lipoprotein are significantly associated with large uEV of progressive patients (N=11 progressive; N=10 stable; >60% confidence), whereas proteins regulating filaments dynamics and plasma membrane protrusions are associated with small progressive uEV (N=12 progressive; N=10 stable; >60% confidence). Validation of three of these progression markers by immune-blotting confirmed their differential expression in the progressive cohort, both in large and small uEV. Summary/Conclusion: Urinary EV (large and small) have a great potential as a source of progression biomarkers in alternative to full urine, with possible application in clinical trials or clinical routine in the future, if their reproducibility can be ascertained. Funding: Nottingham Trent University external engagement award and Vice-Chancellor studentship.

(1) Furini et al (2018) Introduction: Extracellular Vesicles (EVs) represent stable, tissue specific nano-sized particles that reflect the conditional state of their tissue of origin. Normothermic Machine Perfusion (NMP), aimed at restoration of cellular metabolism and function to organs, offers the possibility to assess graft status prior to transplantation through analysis of biomarkers in the perfusion fluids.

Here, the dynamic release and phenotype of kidney EVs released during NMP were analyzed to examine whether EVs could function as a potential biomarker for assessing kidney quality before transplantation. Methods: Eight discarded kidneys (∼13 ± 5 hours of cold ischemia, age 68 ± 7 (mean ± standard deviation), all male) were perfused in a closed system at 37C for 6 hours. Perfusates were taken before and at 1, 3 and 6 hours and examined with Nanoparticle Tracking Analysis (NTA) and Imaging Flow Cytometry (IFCM). For IFCM, perfusates were stained with the tetraspanin EV markers CD9, CD63 or CD81, or a mix of the three markers in combination with CFSE to identify, quantify and characterize EVs. Results: Analysis of perfusates with NTA revealed that the majority of nanoparticles present in the perfusates are < 300 nm. Using IFCM, we selectively studied these small nanoparticles. For CFSE and the mix of tetraspanin double-positive EVs, we observed a ∼700 / 740 / 560 fold increase compared to EV levels before perfusion at 1, 3 and 6 hours of NMP, respectively. Especially after 1 hour of NMP, double-positive EV levels were found to be positively correlated with donor age whilst negative correlations were found for cold ischemia time. Furthermore, tetraspanin CD81 was found to represent the majority (∼70%) of the excreted double-positive EV (CD9: ∼15% / CD63 < 10%). Summary/Conclusion: EVs are excreted during NMP with highest excretion levels during the first hour of perfusion. Tetraspanin CD81 is predominantly present on these EVs. The characterization of the excreted EVs as well as their correlation with clinical parameters provides a starting point to study their role as potential biomarkers of kidney quality. Funding: The authors declare no funding-related or other conflicts of interest. Introduction: Extracellular Vesicles (EV) -regarded as "snapshots" of their cell of origin -represent promising liquid biomarkers to monitor allograft function post transplantation. Recently, we developed an imaging flow cytometry (IFCM) based protocol to identify and characterize EV ≤ 400 nm in molecularly complex samples such as human plasma without prior isolation of EV. Using this protocol, we measure allograft derived EV based on HLA phenotype as a first step to detect allograft specific EV in the circulation of kidney transplant (KTx) recipients. Methods: EDTA blood samples from kidney transplant donors (HLA-A2+, n=21) and recipients (HLA-A2-, n=33) were collected before transplantation as well as 3 days, 7 days, 6 months and during 'for-cause' biopsies (recipients only) after transplantation. Platelet-poor plasma (PPP) was stained with a donor-specific HLA antibody (HLA-A2) in combination with a common EV marker (tetraspanin CD9) and measured using standardized IFCM. Results: Quantification and comparison of CD9+/HLA-A2+ double-positive EV showed 1.1E7 ± 8.9E6 vs 3.5E5 ± 2.5E5 objects/mL for donor and recipient (pre-KTx) EV respectively, with recipients A2-EV concentrations representing background level of the machine. CV values for inter-and intra-assay variability were 16% and 11%, respectively. Serial dilution of A2+ PPP in A2-PPP (n=5) showed a linear reduction in the numbers of CD9+/HLA-A2+ EV according to the dilution rate whilst total CD9+ EV levels remained unchanged. The lower limit of detection of our protocol was defined as the dilution at which point CD9+/HLA-A2+ EV dropped below baseline (A2-PPP), and was determined to be ∼1% of the concentrations measured in undiluted A2+ PPP. Measurement of longitudinally collected recipient samples revealed the detection of allograft derived EV as soon as 3 days -but up to at least 6 months -after KTx. Summary/Conclusion: Here we demonstrate for the first time the detection of allograft derived EV in the circulation of KTx recipients in unprocessed human plasma samples. Identification, quantification and characterization of these EV opens up the possibility to monitor these EV over time after transplantation, and may prove to be a minimally-invasive biomarker. Funding: The authors declare no funding-related or other conflicts of interest. Introduction: Extracellular vesicles (EVs) are being explored for regenerative medicine, and one of the target diseases is osteoarthritis (OA), whose current treatments present secondary effects and limited efficacy. Since platelet rich plasma (PRP) has shown promising effects in OA and platelet lysate (PL) is being studied as its alternative since it presents some advantages compared to PRP, specifically EVs present in PL seem to be responsible for its regenerative effect. However, EVs from other sources such as mesenchymal stromal cells (MSCs) are also being studied as a possible treatment for OA. Thus, we aimed at evaluating the effectiveness of PL-derived EVs (pEVs) as a new treatment for OA in an in vitro OA-induced model using human cartilage explants compared to EVs derived from conditioned media of human umbilical cord MSC (hUC-MSCs). Methods: EVs isolated by size exclusion chromatography from PL or conditioned media of hUC-MSCs were characterized in terms of size and particle concentration by transmission electron microscopy, nanoparticle tracking analysis and in terms of Introduction: One of the major concerns is to deplete contaminating EVs present in FBS. EVs present in FBS are co-isolated with cell-derived EVs and therefore act as contaminants that, affect any quantitative and/or qualitative assessments of EVs derived from the cultured cells. Methods: For preparation of Freeze drier EV-depleted FBS (FD-dFBS), the directions below was followed to deplete EVs from FBS: 1-4 mL FBS was loaded onto 50 mL falcon tube and stored at −80 • C overnight. 2-The falcon tube containing frozen FBS was put in a freeze-drier machine and processed for 6 hours. In this step, the water was evaporated and precipitation of FBS was sedimented in falcon tube. • Critical step: Do not close the door of falcon tube, just seal the door with sterile aluminum foil and make some pore by piercing the aluminum foil to exchange the temperature and vacuum. 3-The precipitation collected and dropped in a clean porcelain mortar. Then, it was rubbed at least for 2 minutes to completely become powder. 500 μL of PBS or media was added to soft powder of FBS and rubbed again at least 2 minutes. Finally, additional 3.5 mL of PBS or media added. Pipette it or use vortex until it dissolves completely. 4-The dissolved FBS was centrifuged for 30 minutes at 10,000 g in 4 • C to remove debris and aggregate proteins.

• Hint: A portion of the FBS proteins may not dissolve very fast, therefore before centrifugation, it is better to incubate it 4-6 hours in 4 • C. Ultrafiltration EV-depleted FBS (UF-dFBS) was done. Briefly, FBS was loaded onto Amicon Ultra-15 Centrifugal Filter Units, and spun for 55 min at 3,000 g. Ultracentrifugation EV-depleted FBS (UC-dFBS) was subjected to centrifugation for 19 hours at 100,000× g at 4 •C. Results: Analysis of the different EV-depleted FBS Based on DLS analysis, while untreated FBS contained a heterogeneous population of EVs, with sizes ranging from < 50 nm to >500 nm, no vesicles could be detected in FD-dFBS. After an 18-h UC of the FBS, a small population of vesicles structures and protein aggregates persists in UC-dFBS, which may indicate low efficiency of ultracentrifugation. The UF-dFBS also displayed scarce vesicle-like structures. Characterization of the EV samples by TEM mostly supported the DLS analysis. While FD-dFBS had no EVs, UF-dFBS still contained some EVs or other EV-like particles that were mainly small. The diameter of most membrane vesicles was between 30 and 120 nm, with the spherical shape typical of EVs. The morphological analysis of UC-dFBS also displayed EVs population and protein aggregates. As expected, regular FBS contained both large and small EVs. Summary/Conclusion: In this study, we have developed a novel, cost-and time-effective, standardized and simple protocol based on freeze drying to deplete EVs from FBS, and addressed the purity of this freeze drying EV-depleted FBS in comparison with ultrafiltration, ultracentrifugation, and regular FBS.

Hala Saneh 1 ; Christine Finck 2 ; Joanne Walker 3 ; Heather Wanczyk 3 Introduction: Extracellular vesicles (EVs) play a key role in many physiological and pathophysiological processes and hold great potential for therapeutic and diagnostic use. Despite significant advances within the last decade, the key issue of EV storage stability remains unresolved and under investigated. Here, we aimed to identify storage conditions stabilizing EVs and comprehensively compared the impact of various storage buffer formulations at different temperatures on EVs derived from different cellular sources for up to two years. Methods: EV features including concentration, diameter, surface protein profile and nucleic acid contents were assessed by complementary methods, and engineered EVs containing fluorophores or functionalized surface proteins were utilized to compare cellular uptake and ligand binding. Results: We show that storing EVs in PBS over time leads to drastically reduced recovery particularly for pure EV samples at all temperatures tested, starting already within days. We further report that using PBS as diluent was found to result in severely reduced EV recovery rates already within minutes. Several of the tested new buffer conditions largely prevented the observed effects, the lead candidate being PBS supplemented with human albumin and trehalose (PBS-HAT). Summary/Conclusion: We report that PBS-HAT buffer facilitates clearly improved short-term and long-term EV preservation for samples stored at -80 • C, stability throughout several freeze-thaw cycles, and drastically improved EV recovery when using a diluent for EV samples for downstream applications.

Maria F. Fernandez Rhodes; Soraya Williams; Bahman Adlou; Mark P. P. Lewis; Owen G. Davies Loughborough University, Loughborough, United Kingdom Introduction: Optimising effective skeletal muscle extracellular vesicle (SM-EV) isolation methods offering high levels of purity will be important to appreciate their composition and functionality in physiological and pathophysiological systems. Sizeexclusion chromatography (SEC) is a ubiquitously applied method for the isolation of EV-enriched fractions that can be combined with ultrafiltration (UF) to increase sample throughput and allow for scalable and selective EV isolation. However, SEC has not previously been optimised from SM-EV isolation and the impact of UF column choice on recovery remains undefined. Methods: Murine myoblast cultures were collected after 48 hours differentiation, pre-concentrated using Amicon® Ultra 15, 100KDa or Vivaspin®20, 100KDa UF columns and processed by SEC (IZON). Thirty individual fractions were collected and individually analysed using nanoparticle tracking analysis (NTA) and bicinchoninic acid (BCA) assay to calculate particle number and particle to protein (PTP) ratio. Transmission Electron Microscopy (TEM), western blotting (WB) and NanoFCM were applied to validate the presence of EVs. Results: Differential patterns of EV markers were identified following pre-concentration with Amicon and Vivaspin UF columns. Alix and TSG101 could be detected up to fraction 13, while CD9 and Annexin A2 until fraction 6. The presence of high-density lipoproteins (ApoA+) was detected from fraction 6 onwards for both protocols by WB. Pooling identical fractions (2-10) to maximise EV recovery led to qualitative and quantitative variations in EV marker profiles and PTP ratios of 129 (Amicon) and 60 (Vivaspin). CD63+ and CD81+ particles were quantitatively (NanoFCM) increased following Amicon pre-concentration (CD63: 7.21x108 vs 4.35x108 and CD81: 6.5x108 vs 4.14x108 for Amicon and Vivaspin respectively; p< 0.05). Eliminating lipoprotein coisolation by reducing the fraction window (1-5) resulted in a net loss of particles (32%). However, an increase in PTP ratio (8%) and significantly increased CD81+ particle recovery was observed (5.64x108 vs 4.14x108 for Amicon and Vivaspin respectively; p< 0.05). Summary/Conclusion: Caution should be taken when pre-concentrating samples for SEC due to observed variations in EV outputs. The isolation of fractions 2-10 was tested to maximise EV recovery at the expense of sample purity but resulted in no significant increase in quantitative measures of EV tetraspanins. Reducing the fraction window (fractions 1-5) eliminated lipoprotein contamination while having no significant impact on EV recovery. 

Soraya Williams 1 ; Maria F. Fernandez Rhodes 1 ; Alice Law 2 ; Ben Peacock 3 ; Mark P. P. Lewis 1 ; Owen G. Davies 1  Loughborough University, Loughborough, United Kingdom;  NanoFCM, Nottingham, United Kingdom;  NanoFCM Co., Ltd., Nottingham, United Kingdom Introduction: Extracellular vesicles (EVs) continue to gain interest for therapeutic applications. However, progression to clinical translation is limited by the diversity of EV isolation methods and their compatibility with downstream analysis. Methods: We compared EV recovery from C2C12 mouse myoblast conditioned media, isolating by ultracentrifugation (UC), polyethylene glycol precipitation (PEG), Total Exosome Isolation Reagent (TEIR), an aqueous two-phase system with and without repeat washes (ATPS and ATPS/R) and size exclusion chromatography (SEC). Additionally, to understand parameters governing method implementation (e.g. cost and scalability), we distributed an international survey using Qualtrics. Results: Data collected by nanoparticle tracking analysis and a BCA protein assay indicated the highest particle and protein concentration was obtained with TEIR (1.15E+09) and SEC (4066μg/ml) respectively. Purity was measured by particle to protein ratio, with ATPS/R and UC displaying the highest purity and SEC the lowest. EV tetraspanin markers (CD63, CD9 and CD81) were analysed by western blot, ExoELISA and nano flow cytometry, indicating EV-enriched fractions isolated by all methods but with variable profiles across both isolation and analysis methods. However, evidence showed the potential interference of reagents with methods such as ATPS. To evaluate our collective findings, data was normalised to the highest output to plot radar charts. Outputs highlighted that although SEC displayed low purity, it provided the highest marker presence overall, followed by UC. Data also indicated a significant increase in CD81 when isolating by UC. When observing parameters governing implementation, SEC and UC were found to be favoured by respondents due to their overall efficiency. However, limitations in the scalability of SEC were reported. Summary/Conclusion: Overall, all methods isolated EV-enriched fractions but with variable outputs that could impact downstream biological functions and therapeutic utility. Funding: OGD acknowledges funding from the Academy of Medical Sciences, Wellcome Trust, Government Department of Business, Energy and Industrial Strategy, British Heart Foundation, Diabetes UK (SBF004∖1090) and the EPSRC/MRC Doctoral Training Centre in Regenerative Medicine Introduction: The molecular content of extracellular vesicles (EVs) is highly heterogeneous. Given that EV surface molecules determine their interactions with their environment, it is conceivable that EV functionality varies between EV subpopulations with different surface profiles. However, it is as of yet challenging to test this hypothesis as methods to isolate intact EV subpopulations based on surface markers are lacking. Here, we demonstrate recovery of intact and functional EV subpopulations from antibody-coated magnetic beads using an optimized release protocol. Methods: EVs from HEK293T, MDA-MB-231 and cardiac progenitor cells (CPCs) were isolated using tangential flow filtration and size exclusion chromatography (SEC). EV subpopulations were captured on antibody-coated magnetic beads targeting tetraspanins or phosphatidylserine (PS). Design-of-experiments (DoE)-based statistical modeling was applied to optimize an elution buffer to release EVs from the beads. EV release and integrity were monitored using flow cytometry and proteinase protection assays, respectively. Released EV subpopulations were further characterized by transmission electron microscopy (TEM), Nanoparticle Tracking Analysis (NTA) and western blotting. EV functionality was assessed in cellular uptake and scratch wound migration assays. Results: A DoE-based library of elution conditions was applied to magnetic beads which had captured MDA-MB-231 EVs via CD9 or CD81 antibodies. EV elution and integrity analyses revealed that pH of the elution buffer was the most critical factor affecting both EV elution efficiency for both antibodies and EV integrity. A second round of optimization was performed to identify optimal elution conditions to release EV subpopulations enriched for CD9, CD63, CD81 or PS from various cell sources. Released EVs appeared intact by TEM and NTA and lacked typical contaminants observed in the original SEC-derived EV isolates. Uptake experiments in various recipient cell types revealed differences in cell-type specificity and uptake efficiency between EV subpopulations. Furthermore, the regenerative capacity of CPC-derived EVs in scratch wound assays differed among EV subpopulations. Summary/Conclusion: Using DoE methodology, we readily optimized a protocol to recover intact EV subpopulations from magnetic beads based on their surface marker expression, and revealed functional differences among them. This universal Introduction: Size-exclusion chromatography (SEC) is emerging as a possibly preferential method for isolation of extracellular vesicles (EVs). This is because SEC separates EVs from soluble proteins, and preserves the EV structure. The method is based on sieving the sample through a porous stationary phase enabling faster migration and thus quicker elution of larger particles like EVs. Various materials can be used as stationary phase, whereby Sepharose-based SEC was already shown to be successful in EV separation. However, efficiency of different stationary phases in SEC separation of EVs has not been evaluated. This study compared Superose 6 PG, Sephacryl S-400, Sepharose CL-6B and commercial SEC column in separating EVs from human cerebrospinal fluid (CSF). Methods: Three severe traumatic brain injury patients were included into the study after obtaining informed consent from family. CSF samples were collected during external drainage for monitoring intracranial pressure at days 1-3 after injury and analysed by immunoblot for albumin, EV protein markers CD9 and CD81, and lipoprotein markers ApoE and ApoAI. Pool of 9 CSF samples in volume of 2.8 ml per experiment was separated by four SECs: columns packed with Superose 6 PG, Sephacryl S-400 or Sepharose CL-6B (S6B), and a commercial column for EV separation. 46 fractions were collected and analysed by slotblot followed by Ponceau staining and immunodetection for albumin, CD9, CD81, ApoE and ApoAI. Size and concentration of nanoparticles in fractions were determined by tunable resistive pulse sensing. Selected fractions were analysed by transmission electron microscope and western blot. Results: All CSFs contained EVs, lipoproteins and high level of albumin. All SECs provided separation of nanoparticles from soluble proteins and lipoproteins, but S6B with its mean 1.08E+09 of total isolated nanoparticles provided the highest enrichment of nanoparticles which also included CD9+ and CD81+ EVs. Summary/Conclusion: S6B is suitable for separation of EVs from CSF. Introduction: Currently standard EV isolation methods have issues with purity, efficiency, and EV functionality. More advanced strategies include alteration of streamlines within microfluidic channels; thus, utilizating differential lateral displacement of differently sized particles. Most available microfluidic systems use small volumes of sample and typically have low flow rates. Such low volumes and flow rates are not feasible if the goal is to extract EVs from bioreactor media and urine. Additionally, these devices predominantly are fabricated from PDMS (Polydimethylsiloxane), while it is well known to absorb and interact with lipophilic molecules. Therefore, new methods and devices for EV isolation from high volume samples are necessary. Methods: As the first step we evaluated two PDMS alternatives -OSTE (Off-stoichiometry thiol-ene polymer) and SEBS (styreneethylene-butylene-styrene) materials with respect to their optical, fat-soluble molecule, EV absorption properties and RT-qPCR inhibition. Next, we microfabricated EV separation devices from OSTE polymer based on asymmetric flow field flow fractionation (A4F) principle and optimised them with standard EV sample produced within bioreactor, isolated with SEC and characterised by NTA, WB and TEM by applying in-house developed pressure system. Finally, the devices currently are tested for EV isolation directly from bioreactor media and compared with EV isolated from media by SEC and UC. Results: Our results showed that OSTE have superior characteristics for EV isolation in comparison to PDMS and SEBS. Next, optimisation experiments showed that volume flow rate within the channel system should be below 100 μl/min since higher volume flow rates result in EV losses. Meanwhile, EV isolation directly from bioreactor media is ongoing and will be presented at the conference. Summary/Conclusion: A4F devices fabricated from OSTE for EV isolation are more suitable than PDMS devices. Funding: Nr. lzp-2019/1-0142 Introduction: Extracellular vesicles (EVs), particularly exosomes, exhibit great potential for the diagnosis, prognosis and treatment of brain disorders such as Alzheimer's disease, representing an advantageous tool for Precision medicine; the latter demands high-quality biomarkers, especially for complex brain disorders, where pathological heterogeneity and diverse clinical presentations complicate the development of precise patient-tailored treatments. Thus, the collection and characterization of physiologically relevant exosomes are of the utmost importance. However, it is still challenging to identify, isolate and quantify exosomes efficiently, accurately, and selectively. For instance, current approaches for exosome isolation from brain rely on tissue dissociation, which may contaminate the exosome fraction by cellular disruption/damage leading to purer/contaminated EVs yield and thus, conflicting, incongruent or inaccurate characterization of exosome profile. Methods: Overcoming this drawback, we developed a novel method to isolate exosome-enriched EVs from mouse and human brain, relying on their spontaneous release. Results: To confirm the efficacy of the release method and its advantages over the existing, digestion-based approaches, we have utilized different state-of-the-art and innovative analytical platforms and approaches (e.g., Cryogenic electron microscopy, Highsensitivity flow cytometry, proteomic analysis, ExoView analysis) that help us to structurally, biochemically and functionally characterize the captured EVs in in vitro and in vivo studies. Based on the above multiscale analytical platforms, we hereby present an efficient purification method that captures a more physiologically relevant, sEVs-enriched population spontaneously released by mouse and human brain tissue. Summary/Conclusion: This spontaneous release method may contribute to the characterization and biomarker profile of physiologically relevant brain-derived exosomes in brain function and pathology. Introduction: The development of methods to isolate intact and biologically relevant extracellular vesicles (EVs) from brain tissues is of great interest given the wide range of physiological and pathological roles they play in the central nervous system. Dissociating the brain tissue without damaging the cells and releasing intracellular content, minimizing contamination by non-EV and lipoprotein particles, avoiding protein degradation and EVs aggregation are some of the many technical challenges that need to be overcome. The aim of this study is to assess, both quantitatively and qualitatively, the advantages of isolating brainderived EVs by inertial microfluidics as compared to standard ultracentrifugation-based approaches. Methods: EVs were isolated from enzymatically dissociated adult mouse brains either by ultracentrifugation at 100,000 x g and iodixanol density gradients (Hurwitz et al., J Neurosci Methods 2018) or by using an inertial-based microfluidic device (Tay et al., Lab Chip 2021). EVs characterization by Western blot, nanoparticle tracking analysis, transmission electron microscopy (TEM) and cryo-electron microscopy is currently underway. Results: While brain-derived EVs were successfully isolated using both ultracentrifugation and microfluidic approaches, the latter allowed for an improved yield, simpler and less laborious operation, and a much shorter processing time. TEM analysis showed that the size distributions and morphologies of EVs obtained by both methods were similar. We will present the results from ongoing quantitative and qualitative assays. Summary/Conclusion: Microfluidic approaches are a useful alternative to isolate EVs from brain (or other) tissues while avoiding some issues such as the ultracentrifugation-induced aggregation of EVs. We will test this methodology to isolate EVs from human brain tissues and devise functional assays to assess their quality. Introduction: Bacterial extracellular vesicles (BEVs) are isolated from microorganisms that have been grown in culture media containing animal-derived products. Consequently, these media are likely to contain animal EVs that may have an effect on downstream analyses. Preliminary data showed that Brain Heart Infusion (BHI) broth, a bacteriological culture media, contained particles that were similar in size to bacterial and eukaryotic EVs, thereby suggesting that BHI broth may contain animal-derived EVs. Here, we developed an optimised protocol for BEV purification that would eliminate animal-derived EVs from the bacteriological culture medium. Furthermore, we compared the properties of BEVs isolated using this new protocol with those isolated from bacteria grown in standard medium.

To deplete BHI broth of animal-derived "extracellular vesicle-like particles" (EVLPs), we used tangential flow filtration with a Vivaflow membrane (50,000 MWCO). The filtrate was used to cultivate Helicobacter pylori. BEVs isolated from this new protocol with those isolated from bacteria grown in standard medium was then subjected to Nanoparticle tracking analysis (NTA), proteomics and biochemical analysis for comparison. Results: NTA results showed that BEVs prepared from the non-depleted BHI medium contained greater particle numbers. EVLPs within BHI did not react with any eukaryotic EV marker antibodies (Epcam, Alix, CD9 and CD69), suggesting that these are not true eukaryotic EVs. Despite BEVs isolated from the two methods inducing similar interleukin-8 responses in mammalian cells, proteomic data suggested differences in their protein composition. While 399 H. pylori proteins were common to BEVs purified by both methods, 57 were unique to BEVs isolated from H. pylori grown in depleted BHI. Five were uncharacterised H. pylori proteins. Furthermore, there were 10x less bovine proteins and 3.4x more H. pylori proteins in BEVs prepared from non-depleted BHI. The decrease in bovine protein improved the quality of the sample for Mass spectrometry analysis. Summary/Conclusion: Collectively, we showed that EVLPs present within BHI broth are not true EVs but may still interfere with downstream analyses, such as with proteomic studies where these hinder the full characterisation of BEV proteins. Furthermore, we have successfully described an optimised method for BEV isolation that increases the purity of isolated BEVs and reduces potential confounding effects of EVLPs.

Aliosha I. Introduction: small Extracellular Vesicles (sEV) are acquiring a major interest in the cell therapy field. However, their successful translation will rely ultimately on their large-scale production necessary to achieve the doses-required quantities. Methods: We aimed to compare the sEV production from various types of cells including mesenchymal stromal cells (MSCs) in several harvest time points (2, 4 and 6 days) using Oxium®EXO, standard DMEM and a commercially available medium developed specifically for sEV production. Phenotype and cell viability analyses of parental cells were performed after the sEV production cycle. sEV secretion rate to the supernatant was evaluated by nanoparticle tracking analysis (NTA). sEV isolation by ultracentrifugation allowed characterization by NTA, flow cytometry and in vitro cell uptake assays, a in mice in vivo biodistribution study and a comparison of sEV's miRNA cargo profile. Results: MSC's phenotype and differentiation capacity was maintained in the three mediums after sEV production cycle. Oxium®EXO allowed a better cell viability after 6 days of production, as well as a 3-fold increase in total particle secretion to the supernatant and a 4-fold increase in those particles sized between 50 to 200nm, compared to DMEM or the commercial medium. The isolated-sEV characterization showed the presence of CD63, CD9 and CD81 on the three isolated types of sEV. No differences were observed as well in the in vitro cell uptake assay nor the in mice in vivo biodistribution pattern. The miRNA cargo profile will be discussed. Summary/Conclusion: Oxium®EXO allowed an increased particle secretion rate while conserving the classic sEV functional properties of internalization into acceptor target cells and biodistribution in vivo, supplying the amount and quality of sEV for the development of potentially cell-free therapies. 

Martin Schlumpberger 1 ; Rachel Talis 2 ; Francisco Ramirez 2 ; Karolin Spitzer 1 ; Caroline Metzen 1 ; Nike Bahlmann 1 ; Chris Heger 2 ; Markus Sprenger-Haussels 1  QIAGEN GmbH, Hilden, Germany;  Bio-Techne, Analytical Solutions Division, San Jose, USA Introduction: Overlapping size between viruses and exosomes (extracellular vesicle; EV) is the major obstacle for the isolation of pure EVs by most popular commercial kits based on EV precipitation by ultracentrifugation, PEG salting-out, hydrophobic binding with silicon carbide, or size exclusion chromatography. Historically, these methods have been employed for viral isolation, so one can expect co-isolation of viruses with EVs, especially from virus-containing liquids. Earlier we demonstrated that SubX technology is appropriate for isolation of both cfDNA and EVs from the same bioliquid sample. The different affinity of the SubX molecules to EV membrane phospholipids and phosphate residues of nucleic acids allows for the efficient separation of EVs from cfDNA. Due to the high affinity to nucleic acids, SubX squeezes out proteins from the nucleoprotein complexes and forms SubX-DNA/RNA pellet insoluble in the exosome reconstitution buffer (ERB). Based on this observation we suggested that EVs can be purified of the viruses since viral proteins are substituted by SubX and viral DNA/RNA will remain in pellets. Methods: Cell line B95-8 shedding Epstein-Barr Virus (EBV) was used as to develop the protocol for exosomes and viral DNA isolation from culture medium utilizing SubX Exo-DNA isolation kit. RNA-carrying recombinant lentiviruses were used as spikes in different bioliquids. EBV DNA quantity was determined by qPCR with primers for EBNA 1 gene. Extracellular genomic DNA released by B95-8 cells was detected using primers for 36B4 gene. RNA quantity was determined by RT-qPCR using primers specific for lentivirus used. Detection of SubX-DNA and exosomes binding was done by measurement of particle sizes using Zetasizer Nano ZS and Nanosight NS300. Results: SubX molecules bind exosomes and form micron-size aggregates that are easily pelleted in a brief 14K x g centrifugation step. SubX-DNA/RNA complexes also precipitate at the same centrifugation force. Exosomes are easily solubilized in ERB from the pellet, while tightly bound DNA/RNA remains insoluble. Less than 0.5% of viral DNA was associated with exosome fraction, while post-exosome pellet contained >99% DNA. This is over 200-fold purification of exosomes from viruses. Thus, we separate two distinct types of extracellular material -intact exosomes and mixture of viral plus cfDNA in a single protocol from the same sample. Also, less than 0.25% of B95-8 genomic cfDNA is detected in exosome fraction. Current results suggest that the EV-associated DNA measured after application of the SubX technology does not contain cf-or viral DNA. Summary/Conclusion: SubX technology based on specific capture of phosphate-clusters permits isolation of exosomes free of non-phospholipid vesicles, protein aggregates, DNP/RNP, as well as viral contamination. It also appears efficient for isolation of viral DNA or RNA from bioliquids (plasma, urine, cell culture medium).

Introduction: Dendritic cells (DCs) are potent antigen-presenting cells that control adaptive immunity and balance effector and regulatory components of the immune response. Extracellular vesicles (EVs), released from human amniotic fluid stem cells (HAFSCs), are characterized by important immunoregulatory properties. In this study, we investigated the potential of HAFSC-EVs to promote tolerogenic effects on specific subsets of murine DCs. Methods: We first produced EVs from HAFSCs cultured in serum-free medium. Ultracentrifugation of HAFSC-conditioned cell medium allowed isolation of EVs that were characterized by scanning electron microscopy (SEM). Nanoparticle tracking analysis (NTA) distribution plots showed vesicle size.Western blot analysis confirmed that the isolated fraction contained EVs and exosomes markers. Lipidomic, proteomic and miRNA analysis were used to completely characterize EVs. Murine DCs and T cells were isolated from bone marrow and spleen respectively. Confocal microscopy and cytofluorimetric analysis were used to evaluate EV uptake to cells. Experimental autoimmune encephalomyelitis (EAE) model was induced in C57BL/6 female mice immunized with MOG35-55 peptide. Results: We demonstrated that HAFSC-EVs are preferentially internalized by conventional dendritic cell type 2 (cDC2), but not by other cDCs, both in vitro and in vivo. Protein and miRNA cargo analysis revealed the enrichment of several immunoregulatory pathways in HAFSC-EVs. Indeed, immunogenic cDC2 conditioned with HAFSC-EVs acquired strong tolerogenic functions. Transfer of cDC2 conditioned with HAFSC-EVs in vivo resulted in suppression of autoimmune responses and significant improvement in the clinical score of EAE. Summary/Conclusion: These results demonstrate that HAFSC-EVs, which are naturally loaded with immunoregulatory mediators, contribute to reprogram inflammatory cDC2 to tolerogenic functions, leading to the control of autoimmune responses.

Sunitha Kodidela 1 ; Asit Kumar 1 ; Namita Sinha 2 ; Santosh kumar 1  UTHSC, Memphis, USA;  UTHSC, Mrmphis, USA Introduction: Cigarette smoke condensate (CSC), a component of cigarette/tobacco smoking, increases pro-inflammatory molecules in myeloid lineage cells. However, the cellular mechanism by which CSC contributes to CNS inflammation is unclear. We have recently demonstrated that long-term exposure of CSC to HIV -uninfected(U937) and infected (U1) macrophages are known to induce packaging of pro-inflammatory molecules, particularly IL-1β, in extracellular vesicles(EVs). Therefore, we hypothesize that exposure of EVs derived from CSC-treated monocyte/macrophages to CNS cells can increase their IL-1β levels, contributing to neuroinflammation. Methods: The U937 and U1 differentiated macrophages were treated once-daily with CSC (10μg/ml) for 7-days. After 7 days of treatment, we collected the media, isolated EVs, and characterized those according to the ISEV guidelines. The EVs isolated from 1ml of media from above U937 and U1 cells were treated to 1ml of media containing human astrocytic and neuronal cells (0.1million cells/well; 12 well plate) once daily for three days and two days respectively in the presence of CSC. At the end of the treatment, the cells were harvested and examined for protein levels superoxide dismutase-1 (SOD1), catalase (CAT), IL-1β, and Glial fibrillary acidic protein (GFAP) using western blot. The differences in the relative expression of protein levels between groups were compared using ANOVA and p< 0.05 is considered significant.

Rong Ma 1 ; Naseer Kutchy 2 ; Guoku Hu 1  University of Nebraska Medical Center, Omaha, USA;  St. George's University, True Blue, Grenada Introduction: Tolerance leads to not only limited use of opioids, including morphine, as analgesics but also increased risk of overdose and death. Critical features of morphine tolerance involve both neurons and astrocyte functions. There is little understanding of how astrocyte-neuron communication that can normally provide the essential networks underlying brain function is modified by morphine and contribute to morphine tolerance. Recent studies by others and us have demonstrated that pharmacological inhibition of extracellular vesicle (EV) release prevents morphine tolerance. Methods: C57/B6 mice were administered morphine for five days to develop tolerance, determined using the tail-flick. Mouse primary astrocyte-derived EVs (ADEVs) were isolated using both size exclusion chromatography (SEC) and ultracentrifugation approaches followed by characterization of EVs using zetaview for EV size distribution and number, and Western blotting for EV markers as well as electron microscopy for EV morphology. Mouse primary neurons were treated with either control-ADEVs or morphine-ADEVs for 48h followed by assessment of primary cilia. Pharmacological and genetic approaches were used to determine the role of EVs in primary ciliogenesis. Primary cilia were assessed by fluorescent immunostaining for primary cilia markers. Results: We found that morphine-stimulated astrocyte-derived EVs (morphine-ADEVs) can be taken up by neurons and promote neuronal primary ciliogenesis. Moreover, we also found that SHH was increased on morphine-ADEVs that can activate SHH signaling in neurons. Our in vivo results demonstrate that inhibiting primary cilia in neurons prevents morphine tolerance in mice. Summary/Conclusion: ADEV-mediated neuronal primary ciliogenesis and SHH activation contribute to the development of morphine tolerance. Funding: NIH-R21DA046831, R21DA042704 and MH112848

Maria Elena Cicardi 1 ; Davide Trotti 2  thomas jefferson university, Philadelphia, USA;  thomas Jefferson Univesrity, Philadelphia, USA Introduction: Amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD) are two neurodegenerative disease which belong to the same disease spectrum. The most common genetic cause of ALS-FTD is a hexanucleotide repetition (G4C2) present in the intron 1 of the C9orf72 gene. This mutation results in three main toxic events: C9orf72 protein reduction, RNA foci formation and aberrant repeat associated non-AUG translation (RAN-T). The products of RAN-T are five dipeptide repeat proteins (DPRs: polyGA, polyGP, polyGR, polyPA and polyPR). Methods: Extracellular vesicles extraction from mouse blood/cell culture media by UC and size exclusion chromatography (IZON columns) Analysis of EVs by NTA, Cytoflex, proteomic, Bioactivity of extracellular vesicles through live imaging of neurons Bioactivity of extracellular vesicles through in vivo injections is mice Primary cortical nuerons, hiPSCS Results: Employing a mouse model which conditionally expresses polyGR we found that a fraction of the extracellular vesicles (EVs) present in the serum encloses polyGR. We thus analyzed whether neurons produce EVs loaded with RAN-T products. Indeed, DPRs were detected in neuronal EVs and glutamate stimulation was able to increase the number of polyDPR+ EVs. Interestingly, we found higher percentage of polyGR+ EVs compared to other DPRs. We then investigate if the internalization of polyGR+ EVs elicited toxicity in recipient neurons. We treated cortical neurons with polyGR+ EVs and by live imaging we found a significant reduction in cell viability compared to control EVs treated neurons paralleled by TDP-43 nuclear depletion which is hallmark of ALS-FTD pathology. When we performed in vivo experiments injecting polyGR+ EVs in mouse spinal cord we observed loss of NeuN positive cells, paralleled by astrocytes and microglial recruitment at the injection site. Treating human derived motor neurons (MNs) with polyGR+ EVs and we found that only C9-ALS derived MNs show increased neurodegeneration possibly owing to polyGR+ EVs induced RAN-T which we were able to measure thanks to a luciferase RAN-T reporter. Summary/Conclusion: In conclusion we demonstrated that polyGR reception into neurons was able to cause neuronal toxicity, TDP-43 nuclear depletion and increase in RAN-T substantiating the hypothesis that EVs mediated neuron-to-neuron spreading of polyDPRs is one of the possible pathways in which neurodegeneration spreads across the central nervous system during ALS or FTD. Introduction: After ischemic stroke, where blockage of a brain artery impedes the supply of glucose and oxygen, the directly affected neurons die by necrosis, whereas brain cells surrounding the ischemic area (penumbra) remain metabolically active and can potentially be rescued. Several extracellular signals, including those received via extracellular vesicles (EVs), will influence the fate of brain cells at the penumbra in the hours/days following the ischemic insult. To decipher the nature of these critical extracellular stimuli, is fundamental to understand the pathophysiology of stroke. Methods: The aim of the present study was to analyse the mRNA cargo of brain-derived EVs (BDEVs) at two time-points, 24h (acute phase) and 7 days (recovery phase), after transient Middle Cerebral Artery Occlusion (tMCAO, a widely used mouse model of stroke) to gain insight into their potential participation in brain recovery processes. In a previous study, we proved that a commercially available mRNA expression panel allowing for multiplexed assessment of 770 genes is suitable to analyze BDEV mRNAs without the need for previous RNA isolation, thus simplifying the protocol and limiting sample/information loss. We here applied the same targeted approach together with an improved BDEVs isolation protocol using a iodixanol gradient. Results: Nanoparticle Tracking Analysis (NTA) showed that the amount of BDEVs is significantly increased at 7 days after reperfusion, together with the presence of more mRNAs being significantly upregulated (160 mRNAs) compared to 24h after stroke (with 78 mRNAs significantly increased). The analysis of gene ontology (GO) pathways for biological processes indicated a decrease in the "inflammatory response" (GO:0006954) and an increase in "immune system process" (GO:0002376) and "immune response" (GO:0006955) at 7 days compared to 24h. Fittingly, many highly overexpressed mRNAs at 7 days can be ascribed to microglia, the resident immune cells of the brain. Lastly, we detected that the most upregulated mRNA in BDEVs at 7 days after stroke is Spp1, which encodes for osteopontin (OPN), a phospho-glycoprotein secreted by macrophages and microglia and acting protective after stroke. By immunofluorescence analysis, we observed that OPN is upregulated at the cortical penumbral area. Introduction: Cellular prion protein (PrPC) is a GPI-anchored receptor glycoprotein with highest expression in the nervous and the immune system. It is also abundantly expressed on surface of the extracellular vesicles (EVs). In Alzheimer's disease (AD), although a decline in brain PrPC levels is reported but EV-expressed PrPC, along with shed extracellular PrPC forms, is suggested to sequester Aβ oligomers (Aβo) and to accelerate Aβ fibrillation, yet formal evidence is still lacking. Effects of PrPC decline on the EV compositions also lack comprehensive studies. Here, we aim to study the role of PrPC expressing EVs in Aβ fibrillization and to highlight physiological alterations associated with EV levels of PrPC. Methods: PrPC-containing (WT) and PrPC-deficient (KO) EVs were obtained from WT and PrPC-KO Neuro-2a (N2a) cells, respectively. After achieving ∼70% confluency in DMEM (+ 10% FBS), cells were incubated (∼16h) with serum-free DMEM. EVs pellets were prepared from conditioned DMEM by differential centrifugation steps (1500xg, 15min; 10,000xg, 30min; and 100,000xg, 1h10min). EVs were characterized using NTA, immunoblotting, and cryo-EM. Small angle X-ray scattering (SAXS), super-resolution microscopy (SRM), Cryo-EM, proteomic and lipidomic profiling, and associative biochemical and biophysical methods were employed to further the study objectives. Results: SAXS studies helped us to identify potent Aβo-sequestering properties of WT-EVs, which were further confirmed by SRM and aggregation assays. Lipidomic and proteomic profiling of WT-and KO-EVs pointed towards marked differences in the lipid and protein make-up (i.e., higher abundance of certain kinases, RNA-and DNA-binding proteins) of the WT-and KO-EVs. Summary/Conclusion: Our findings provide new evidence for crucial roles carried out by PrPC expressing EVs in the pathophysiology of AD, i.e., their involvement in Aβ aggregation, and their potential involvement in AD-specific intercellular communication in the brain.

Introduction: Extracellular vesicles (EVs) are associated with inflammation and cardiovascular diseases (CVD). However, the role of EVs in rheumatoid arthritis (RA) and their impact on CVD in RA, as well as EVs response to treatment is far less studied. Therefore, we investigated whether qualitative and quantitative characteristics of circulating EVs differ between RA patients and healthy controls (HC), and whether they are influenced by antirheumatic therapy. Methods: We assessed 20 RA patients before and after using methotrexate alone (MTX, n=10) or Adalimumab with MTX comedication (ADA+MTX, n=10) for six weeks, in addition to 8 age-and sex-matched HC. All patients starting with ADA had been previously unsuccessfully treated with MTX. Plasma EVs were isolated by size exclusion chromatography (0.5 mL fractions, fractions 7-9), and characterized by nanoparticle tracking analysis, NanoDrop One, western blot, transmission electron microscopy, and label-free quantitative proteomics combined with Ingenuity Pathway Analysis of the proteomics data. Results: Plasma EV numbers, sizes and total protein concentrations did not differ between patients and HC, and were unaffected by antirheumatic therapy. Proteomic analysis identified 1513 proteins, wherein 169 proteins had significantly different baseline levels between patients and HC (FDR p < 0.05). These proteins were mostly related to cardiometabolic disease and upstream regulation of inflammatory pathways linked to RA, such as integrins, interleukin-8, actin cytoskeleton signaling. From patient samples, 317 proteins were significantly affected after 6 weeks on antirheumatic treatment. 115 proteins changed in response to MTX, whereas ADA+MTX had no significant impact on these proteins. Among them there were several upstream regulator proteins involved in pathogenesis of RA, including downregulation of vascular endothelial growth factor (p=0.012) and MYC (p=0.0003), and upregulation of CD437 (p < 0.0001). Summary/Conclusion: EV proteins differed between RA patients and HC, and were affected by antirheumatic treatment within six weeks. Surprisingly ADA+MTX had no significant impact on EV proteins compared to MTX monotherapy. EVs proteomic content may indicate a contributing role in pathogenesis of RA and CVD development.

Nicholy Lozano 1 ; SOFIA OLIVEIRA MIGUEL 2 ; Ana Claudia Torrecilhas 3  UNIFESP, Sao Paulo, Brazil;  UNIFESP, São Paulo, Brazil;  Federal University of Sao Paulo, Sao Paulo, Brazil Introduction: Trypomastigotes of T. cruzi release EVs enriched in gp85/Trans-sialidase superfamily and alpha-galactosylcontaining glycoconjugates. EVs induce potent inflammatory immune response and host cell invasion of macrophages in vitro The aim of our research project is analysis the effect of EVs in animal infection with Chagas Disease Methods: BALB/c female mice (8-10 week-old) were obtained from the animal facility at the UNIFESP. BALB/c mice pretreated with EVs (108 particles/ animal) followed by trypomastigote (500 parasites/ animal via intra peritoneal). The heart, spleen, bladder, lung, skeletal muscle and intestine were obtained from the animal pretreatment with EVs and infection with T. cruzi and controls (T.cruzi infection). One half was fixed in neutral buffered formalin, embedded by routine technique in paraffin wax and sectioned at 5 um for hemmatoxylin and eosin (H&E) staining Results: All organs of infected mice submitted to previous treatment with PBS or EVs were removed 15 days after infection, fixed, embedded in paraffin, and sectioned for hemmatoxylin and eosin (H&E) staining. The heart, spleen, bladder, lung, skeletal muscle and intestine of the animals pre-inoculated with EVs showed an intense parasitism with an extensive inflammatory reaction, when compared to the control. Approximately 10 or more amastigote nests were found in the infected animal preinoculated with EVs. Summary/Conclusion: The results indicate that vesicles released by trypomastigotes of T. cruzi can modulate the infection in vivo by inducing cytokines, and might play a role during the acute phase of the disease. These findings suggest that preinoculation of EVs produces an exacerbated inflammatory reaction in the animal all organs maybe by modulation cytokine production Funding: FAPESP, CAPES and CNPq

Neetu Tyagi; Jyotirmaya Behera; Yu-Ting Zheng; Rubens P. Homme UNIVERSITY OF LOUISVILLE, Louisville, USA Introduction: Recent evidence suggests that physical exercise released exosomes have been identified as novel players to mediate cell-to-cell communication in promoting skeletal development. However, the impact of EX on the progression of bone loss and deterioration of mechanical strength/quality in diabetic mice, induced by a high-fat diet and streptozotocin (HFD/STZ), remains unexplained. Hypothesis: In the present study, we investigate the effect of physical exercise released exosomes (Ex-EVs) on bone mass and mechanical quality using a diabetes mice model. Micro-CT scans and mechanical testing revealed that trabecular bone microarchitecture and bone mechanical properties were improved upon EX in diabetic mice. Methods: Circulating exosomes were isolated from the plasma of with or without exercise training 10-12 weeks old mice subjected to 15-weeks treadmill exercise. Using the standard differential ultracentrifugation method and characterized by transmission electron microscopy, NanoSight & western blot analyses. Micro-CT scans and mechanical testing were used to test trabecular bone microarchitecture and bone mechanical properties. Results: Treatment of EX-EVs restored the bone mechanical quality in diabetic mice by preventing inflammasome-associated pyroptosis response and promoting osteogenesis. Using miScript miRNA array-based screening, we discovered a particular miRNA, miR-218, as a novel target of EX-EVs induced Fndc5/irisin expression. Mechanistically, the data found that diabetes upregulated miR-218 via oxidative stress-dependent action and suppressed the Fndc5/irisin expression by binding to its 3'-UTR. Indeed, the decreased level of irisin expression further triggers the expression of pyroptosis-associated proteins-NLRP3, Caspase-1, and GSDMD in diabetic bone tissue. Summary/Conclusion: This study demonstrates an understanding of the previously undefined role of exercise-induced skeletal irisin in ameliorating diabetes-associated bone loss via inhibiting the miR-129-FNDC5/pyroptosis axis. It possibly provides a mechanism for a therapeutic effect on metabolic osteoporosis. Funding: National Institute of Health (NIH) gran: t AR-067667

Oumaima Stambouli 1 ; Robin Dittrich 1 ; Fabiola Nardi Bauer 1 ; Tobias Tertel 1 ; Peter A A. Horn 2 ; Bernd Giebel 1  Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany, Essen, Germany;  University Hospital Essen, Essen, Germany Introduction: Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) are increasingly considered as therapeutic agents. Remarkably, as monitored in a multi-donor mixed lymphocyte reaction (mdMLR) assay, and in different animal models, including ischemic stroke and acute Graft-versus-Host Disease models, only a proportion of our MSC-EV products, all of which have similar EV and protein concentrations, reveal immunomodulatory activities. Coupled to our experience that some MSC stocks reproducibly allow manufacturing of EV products with and others without detectable immunomodulatory capabilities, we hypothesize the differences derive from heterogeneities in parental MSC populations. Aiming to understand the heterogeneity of MSC-EV products, we explored whether initial human bone marrow (BM) seeding procedures have any impact on obtained MSCs or their EV products, respectively. Methods: MSCs were raised from unprocessed BM aspirates and from mononuclear cells (MNCs) harvested thereof, originally either seeded into human platelet lysate (hPL) supplemented DMEM low media or in cytokine and hPL supplemented EBM media. After 24h, non-adherent cells were removed and remaining cells continuously cultured in hPL supplemented DMEM low media in all four settings. Growth rates of obtained MSCs including their senescence status, their cell surface phenotypes and osteogenic and adipogenic differentiation capabilities were analyzed. Furthermore, their EVs were prepared, phenotypically characterized and investigated for their immunomodulatory and proangiogenic activities. Results: Overall, aspirate-derived MSCs, especially those from the DMEM seedings, appear more mature then MNC-derived MSCs. They grew slower and reached senescence quicker than MNC-derived MSCs. Furthermore, MSCs obtained from EBM seedings revealed higher expansion capacities than those obtained from DMEM seedings. Despite these differences, all EV products obtained from early MSC passages revealed comparable immunomodulatory activities in the mdMLR assay. In contrast, EV preparations obtained from the aspirate-EBM seeding promoted tube formation of endothelial cells in Matrigel more efficiently than those obtained from the other MSCs. Summary/Conclusion: Our data imply, initial seeding strategies impact the characteristics of obtained MSCs as well as that of their EVs. Introduction: Extracellular vesicles (EVs) can modulate a myriad of biological phenomenon as well as disease development through cell-cell communication, and both the naïve and the modified EVs have showed prospects as therapeutic interventions. However, the EV mediated modulation of normal aging has not been studies detailed yet.

In this study, we tested the regulation of aging process by human placenta derived mesenchymal stem cells (hpMSCs), which already showed the recovery potentials of hpMSCs in the degenerative disease models in our studies, and the human placenta derived EVs. We first intravenously infused hpMSCs into the aged mice from18-19 month old and repeated three times at every six weeks followed by assessment of diverse behavioral and molecular analyses. Results: Compared to the age matched controls, we observed in the multiply treated mice significant improvement of the cognitive and locomotive activities, enhancement of genes related neuronal activities, and suppression of aging associated genes including p16INK4a in their hippocampi. In addition, the hpMSCs delayed the cellular senescence of the cocultured human cells, such as primary fetal neural progenitor cells (hfNPCs) and lung fibroblast cells in a transwell system, indicating that the hpMSC mediated effects are systemic and consequences of secreted materials including EVs. Through the comprehensive analysis with the total RNAs from hippocampi of the treated mice and the placenta EV containing microRNA, we identified the TLR4 signlaing targeting several microRNAs, such as miR 92a, miR-150, miR -22 and miR-122, that are able to modulate normal aging and aging related decline of cognitive function. Both the EVs and the target miRNA mimics partially delayed the cellular senescence and protected the hfNPCs against Aβ42 toxicity. Summary/Conclusion: Here we first report that the hpMSCs are able to modulate aging process systemically in aged mice and the outcomes are at least consequences of the placenta EV associated modulation of Toll-like Receptor 4 (TLR4) pathway. 

Charmi Dedhia 1 ; Paolo Neviani 1 ; John js. Cadwell 2 ; Sargis Sedrakyan 3 ; Laura Perin 4  Children's Hospital Los Angeles, Los Angeles, USA;  Fibercellsystem, New Market, USA;  Children's Hospital Los Angeles -University of Southern California, Los Angeles, USA;  Children's Hospital Los Angeles-University of Southern California, Los Angeles, USA Introduction: EV clinical translation is inhibited by limitations in the scale-up of EVs production. Hollow fiber bioreactors (HFBR) support culture of large numbers of cells, at high densities, with concentrated EVs. Culture conditions may affect EV composition and potency. Here we compare the production, potency, identity, and therapeutic potential of EVs collected from cells grown in culture dish (2D) vs a small and medium-sized HFBR (3D). Methods: 1 × 10e6 human clonal stem cells from amniotic fluid (hAFSC from consented donors) were seeded in 2D (145cm2), 1.6 × 10e7 hAFSC were seeded on a small cartridge (FiberCell C2025D; 450cm2), and 1.8 × 10e8 hAFSC on medium cartridge (FiberCell C2011; 4,000cm2) with fibronectin coating. All cultures used Chang medium with 20% of ES-FBS, starved for 24hr and then EVs collected. The effect of harvest frequency was tested (8hr, 24hr, 72hr). 2D-EVs and 3D-EVs were compared by Nanosight, potency assay (by WB), identity (by Exoview), and therapeutic effect (in vivo injections in an animal model of chronic kidney disease, Alport Syndrome). Results: 2D production was ∼5.5 × 10e9EV/ml/24hrs while 3D was ∼2.8 × 10e10EV/ml (first four 24hrs) and ∼4.4 × 10e10EV/ml (two days of hourly harvests). The medium cartridge produced similar concentrations of EVs but at 10X harvest volume indicating linear scalability. Very little difference in EV concentration was observed during harvest intervals, with a very similar size distribution. This could indicate either significant EV re-uptake or inhibition of EV secretion dependent upon free EV in the supernatant. Both 3D-EVs trapped VEGF in vitro (an in vitro established potency assay) and expressed CD9, CD81, CD63, CD80, CD86, and VEGFR1 as 2D-EVs. 3D-EVs ameliorated proteinuria and histology when injected into Alport mice and also trapped VEGF in vivo as 2D-EVs. Summary/Conclusion: 3D-EVs had comparable properties and bio-activity to 2D-EVs, but the HFBR produced 10x to 100X more EVs. Cell culture conditions for hAFSC still need optimization in the HFBR, however, a currently available 1.2m2 cartridge provides a 50X scale-up potential. The HFBR is a closed system that can be cGMP compliant. In conclusion, the HFBR can produce a sufficient number of EVs to support pre-clinical and clinical applications of EVs with at least similar properties to EVs produced in 2D methods. Introduction: Beside conventional radiotherapy using X-rays dedicated to localized tumors or oligometastasis, targeted radionuclide therapy (TRT) allows to specifically irradiate diffuse and metastatic tumors. TRT consists of the administration of radiopharmaceuticals made of monoclonal antibodies or peptides coupled to a radionuclide emitting alpha, beta and Auger particles. We already demonstrated that TRT induces anti-tumor immunity at low activity. Here, we investigate the role of small extracellular vesicles (sEVs) released by irradiated cells in triggering anti-tumor immunity. Methods: B16F10 melanoma cells were subcutaneously injected in C57BL/6J and athymic mice, or in cGAS-/-and in STING-/-mice. Mice received intraperitoneal injections of TA99 mAb targeting TYRP-1/gp75 tumor antigen radiolabeled with 5MBq 177Lu-TA99 (beta TRT), or intratumor injection of sEVs purified from cells exposed to 2MBq/ml 177Lu-TA99 (TRT-sEVs) or from non-treated cells (NT-sEVs). Macrophages and DC were exposed in vitro to sEVs purified from B16F10 CRISPR ctrl, B16F10 cGAS-/-and B16F10 STING-/-exposed to beta TRT. Results: In vivo, Beta TRT efficacy was shown to require T-cells for adaptive immunity, with a factor 3 in tumor growth delay (**p = 0.001) compared to non-treated (NT), and no difference between TRT and NT was observed in athymic nude mice. Then, we focused on sEVs as a second messenger released by cancer cells that may activate an antitumor immune response through the STING pathway. EVs purified from beta-TRT exposed cells demonstrated a strong tumor growth delay and survival (***p = 0.0007). The dsDNA content between NT-sEVs and TRT-sEVs was comparable, which suggest that dsDNA was not responsible for the observed in vivo effect. Also, we demonstrated using TCGA database analysis in vivo that cGAS gene expression is required in cancer cells to obtain a significant therapeutic efficacy of radiotherapy, while STING is not. In addition, knocked-out cGAS genes in host immune cells did not affect TRT response, while knocked-out STING genes did, suggesting that cGAMP is an extracellular messenger involved in activation of immune cells. Finally, compared to TRT purified sEVs-CTRL, sEVs-cGAS-/do not activate macrophages nor DC in vitro, demonstrating that sEVS may carry cGAMP. Summary/Conclusion: sEVs contribute to TRT efficacy by mediating an antitumor immune response in vivo. Introduction: The low yields of production of extracellular vesicles (EVs) seriously challenge research and clinical application of successful products (Paganini et al, Biotech. J., 1800528, 2019). Until now, few efforts have been done to increase upstream yields of EVs. Examples include switching the cell cultivation from flasks to hollow fiber bioreactors and stirred tank bioreactors operated in batch mode. Moreover, little is known about how these changes in the upstream operations impact the rate of EV production and the properties of the final EV mixtures. Here, we simulate perfusion cultures with batch-refeed cultures, evaluate their productivity and compare them with batch cultures. Methods: 293F cells are cultured in shake flasks and maintained at two different cell densities for 14 days. The medium is collected and replaced daily. EVs are separated by immuno-magnetic isolation, quantified and the productivities of the two batch-refeed cultures and of a batch culture are measured. Introduction: Diabetic foot ulcer (DFU) is a chronic disease that can lead to subsequent limb amputation. The standard treatment for diabetic foot ulcer includes wound debridement, wound dressing, management of infection, control of ischemia, revascularization, and off-loading pressure to promote healing. Even though DFU requires a long-term treatment for successful healing, most of its sufferers fail to recover resulting in limb amputation. An alternative and effective treatment is required to accelerate the healing process of diabetic foot ulcer (DFU). Stem cell therapy is one of the regenerative medicines that has emerged as a promising treatment for DFU. It can repair and even replace damaged tissues or organ. In this study, mesenchymal stem cells (MSCs) are chosen over other stem cell populations due to their outstanding advantages such as ability to differentiate into various cell types (multipotent), cytokines and growth factor secretion, homing ability, and modulation of anti-inflammatory response. Methods: A 65-years-old male patient presented with diabetic ulcer on the left foot since September 2020. Diagnosed with type 2 diabetes mellitus in the last 5 years, the patient has been controlling his blood sugar levels. The patient has undergone 3 times surgery, 2 times wound debridement, and defect closure through skin graft and flap. The surface areas of the back lower calf and outer ankle ulcers were 7 cm x 1 cm and 2 cm x 2 cm, respectively. The depth of the outer ankle ulcers was 3 cm with exudation and pus around the area, swelling and pain of the tissue were also observed. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) and their Secretome was given as a treatment. Ten million of UC-MSCs was injected directly into the ulcers followed by Secretome spraying all over the area once in 3 days when changing wound dressing. Results: Based on the Picture 1 & 2, the ulcer size was reduced within a few weeks. The ulcer size on back lower calf (Picture 1) were gradually reduced from A) 7x1cm into B) 5x1cm, C) 5x1cm and D) 3x1cm. On the outer ankle, the size of ulcer is also gradually decreased during the observation (Picture 2). The size become smaller from the baseline were 2x2 cm into 0.5x0.5 cm at the last observation. As shown on Table 1 , the yellowish pigmentation or slough around the ulcer also improved. This is also supported by the moisture of the skin which became moist at the last observation. The presence of epithelialization and necrotic tissue was examined clinically. Necrotic tissue can be seen as blackening of the ulcer, whereas the epithelialization seen as red pigmentation. Another improvement shown from this therapy was the depth of ulcer is significantly reduced from 3 cm to 0.5 cm. Summary/Conclusion: In conclusion, this current report is one of the available evidence that confirmed the use of UC-MSCs and Secretome as a promising treatment for diabetic foot ulcer patients.

Haggai Kaspi 1 ; Chen Dekel 1 ; Ralph Kern 2 ; Stacy Lindborg 2 ; Chaim Lebovits 2 ; Revital Aricha 1  Brainstorm cell therapeutics, Petach Tikva, Israel;  Brainstorm cell therapeutics, New York, USA Introduction: Mesenchymal stem cell (MSC)-derived small extracellular vesicles (sEVs) have been proposed as a potential therapy for pulmonary disorders associated with inflammation and fibrosis, due to their significant immunomodulatory and regenerative properties. We have previously demonstrated that in LPS-induced mice ALI/ARDS model, sEVs from MSCs induced to secrete increased levels of regenerative and immunomodulatory factors (Exo MSC-NTF), had a superior effect over treatment with naïve MSC-derived sEVs (Exo MSC). In this study we aimed to examine the advantage of Exo MSC-NTF over Exo MSC focusing on different lung injury. Methods: Using the bleomycin model, we compared the effect of the two types of sEVs: Exo MSC and Exo MSC-NTF. Treatment was administered intratracheally 1 and 5 days after bleomycin instillation, and experimental observations were made through day 14. Analysis of mRNA in the lungs was performed using NanoString technology. EVs cargo analysis was performed by mass spectrometry and validated by ELISA. Results: Exo MSC-NTF showed greater improvements compared to Exo MSC in multiple parameters, including increased blood oxygen saturation, reduction in lung pathology including fibrosis and inflammatory cell infiltrates, regulation of proinflammatory cytokines in bronchoalveolar lavage fluid, as well as regulation of gene expression related to multiple pathways in the lung. Cargo analysis of proteins in Exo MSC and Exo MSC-NTF demonstrated higher expression of regenerative factors, including amphiregulin (AREG) and leukemia inhibitory factor (LIF) in Exo MSC-NTF. Summary/Conclusion: We observed positive preclinical results suggesting that intratracheal administration of Exo MSC-NTF may have potential as a clinical therapy for pulmonary pathologies associated with inflammation and fibrosis and may be more effective at modifying physiological, pathological, and biochemical outcomes than sEVs isolated from naïve MSCs.

Introduction: In the past, our research group and others have shown promising results on the use of mesenchymal stromal cell-derived small extracellular vesicles (MSC-sEV) as a therapeutic approach for neuronal injuries. MSC-sEV carry small noncoding RNAs such as microRNAs (miRNAs), which are predicted to target mRNAs encoding for proteins that are involved in premature birth-related white matter injury (WMI). We hypothesize that miRNAs, released by sEV upon uptake in their target cells have a key function in the observed beneficial effects of MSC-sEV. Methods: We isolated MSC from the connective tissue of human umbilical cords, the Wharton's jelly. The cells were stained for typical MSC-markers by immunohistochemistry. sEV were purified from the conditioned cell medium by serial ultracentrifugation followed by size exclusion chromatography (SEC). The protein and RNA contents of each SEC fraction were measured with a NanoVue Plus™. The fractions with the highest protein content were characterised by proteomics, western blot, ImageStream and ZetaView analyses. The miRNA content of the sEV was measured by quantitative PCR. Pathway enrichment of the miRNAs was analysed following Next Generation Sequencing. Results: The SEC fractions were positive for the sEV markers CD81, CD63 and syntenin-1, and the MSC markers CD73, CD90 and CD105. Further, they contained high amounts of miRNAs, such as miR-21-5p, miR-22-5p, miR-27b-3p, and members of the let-7 family. The targets of the highly abundant miRNAs in the sEV fractions are involved in apoptotic and inflammatory processes and drive oligodendrocyte differentiation. Summary/Conclusion: The miRNAs released by MSC-SEV might influence WMI outcomes. The regulatory potential of miR-NAs in gene expression are currently analysed using agomir / antagomir assays in an in vitro model of WMI, as well as dual luciferase assays. To analyse the integrity of the sEV we will check for the CD73 enzyme activity.

Veronica Tilotta 1 ; Cicione Claudia 2 ; Giuseppina Di Giacomo 2 ; Luca Ambrosio 2 ; Fabrizio Russo 2 ; Rocco Papalia 2 ; Gianluca Vadalà 2 ; Vincenzo Denaro 2  Campus Bio-Medico University(UCBM), Rome, Italy;  Campus Bio-Medico University of Rome, Rome, Italy Introduction: Intervertebral disc degeneration (IDD) affects more than 80% of the population. Current approaches to treat IDD are based on conservative or surgical procedures relieving the pain. Studies reported as paracrine factors as extracellular vesicles released by mesenchymal stem cells (MSCs) may regenerate IVD. The aim of this study is to investigate the therapeutic effects of Wharton's Jelly MSCs derived exosomes on human nucleus pulposus cells (hNPCs) in an in vitro. Methods: Exosomes were isolated by tangent filtration of Wharton's Jelly derived MSCs conditioned media. The exosomes were quantified by bicinchoninic acid assay, exosomal morphology was characterized by transmission electron microscope, western blot analysis was performed for markers expression and nanoparticles tracking analysis for vesicular size and quantification. hNPCs were treated with growth medium (control) and MSCs-exosomes at 10 ug/ml, 50 ug/ml and 100 ug/ml. At different time points each group was analyzed for: cell proliferation [flow cytometry]; nitrate [Griess] and glycosaminoglycan (GAG) production [DMBB]; histological staining for extracellular matrix (ECM) analysis; gene expression levels of catabolic and anabolic genes [real time-polymerase chain reaction, qPCR]. Furthermore, exosomes were labeled with PKH26 and the uptake was detected by confocal microscopy. Results: An increase of hNPCs proliferation was reported in exos 10 μg/ml sample group. Nitrate production was significantly reduced at 100 μg/ml. GAG content was enhanced in a dose dependent-manner by all exos concentrations under study. Histological analysis suggested that there is not a significant difference between treated and untreated hNPCs' ECM synthesis. Gene expression levels were modulated by exosomes compared to the control. Summary/Conclusion: Our preliminary results supported the potential use of exosomes as cell-free treatment of IDD. MSCexosomes ameliorate hNPCs growth, attenuate ECM degradation and oxidative stress-related IDD progression. These findings offer new opportunities for the potential use of exosomes as an attractive alternative strategy to the effects of cell-therapy. compromised by the harsh microenvironment of the disc. Funding: Financial support was received from the Horizon 2020 research and innovation program "iPSpine" # 825925 and "RESPINE"ID:NCT03737461.ClinicaTrials.gov

Rocío Mato-Basalo 1 ; Sergio Lucio-Gallego 1 ; Marta Sacristán-Santos 1 ; María Del Pilar Miranda-Quintana 1 ; Miriam Morente-López 1 ; María C. Arufe 2 ; Juan Antonio Fafián Labora 1

Introduction: Higher life expectancy of population brings health problems associated with age. Enhance our knowledge on the aging process mechanisms is essential to develop new therapies to palliate this health problems and improve quality life in people. Mesenchymal stem cells (MSCs) have a high therapeutic potential facing age-related diseases. Senescent MSCs enter in a stage characterized by loss of proliferation and pluripotency capacities, related with the deterious of the tissues and organs. These senescent MSCs produce a secretoma called senescence-associated secretory phenotype (SASP) formed by chemokines, interleukins, lipid mediator and extracellular vesicles. Our group, it was discovery that the intercellular communication through small extracellular vesicles (sEV) influence on the propagation of senescence and a proinflammatory message, main characteristics of inflamm-aging by activation of non-classical SASP through sEV. The senomorphics are drugs with interesting tool to modulate functionality senescent sEV to paracrine senescence transmission. Because of that, we wanted to know how three pharmacological inhibitors of p65 pathway (JSH-23, MG132 and curcumin) could modulate the functionality of non-classical SASP mediated by sEV. Methods: For that, we induced senescence into mesenchymal stem cells. After that, the cells were treated with the pharmacological inhibitor of p65 (JSH-23, MG-132 and Curcumin). The sEV were isolated by ultracentrifugation. The size and production were evaluated using NTA. With respect to the functionality, the isolated sEV from senescent MSC-treated with pharmacological inhibitors of p65, were used to treat non-senescent and senescent MSCs using the same concentration of particles for 6 days. Later, it was evaluated the paracrine transmission and the rejuvenation capacities by evaluation of cell proliferation and senescence phenotype using crystal violet staining and b-galactosidase assay. Results: Our data shown that inhibition of p65 pathway decreased the production of sEV in the MSC-treated with the three compounds. Besides, the sEV from MSC-treated with JSH-23, MG-132 and Curcumin loss the capacity paracrine senescence transmission on non-senescent MSCs. Otherwise, they can rejuvenate senescent MSCs. Summary/Conclusion: The pharmacological inhibitor of p65 have a high potential to change the functionality of non-classical SASP mediated by sEV. Because of that, we proposed JSH-23, MG-132 and Curcumin as senomorphics drugs to rejuvenated senescent MSCs. These three senomorphics could use to treat age-related diseases and help to improve elderly people's quality of life. Funding: JFL was funded Xunta de Galicia (ED481D-2021-020). MCA was granted by the Spanish National Health ISCIII(PI20/00497) Introduction: Mesenchymal stromal cell-derived extracellular vesicles (MSC-EV) are extensively studied as therapeutic tools in pulmonary diseases, including COVID-19. Local MSC-EV administration, intratracheal (IT) or by aerosol, is being used in ongoing clinical trials. Even if EV biodistribution is a critical determinant of therapeutic activity, it has never been evaluated following local vs. systemic administration for the treatment of lung diseases. L1 (TsEVs) exert immunomodulatory properties on human monocyte derived dendritic cells. Now, we aim to investigate whether TsEVs can also exert beneficial immunomodulatory effects in murine model of ovalbumin (OVA)-induced allergy. Methods: TsEVs were enriched from ES L1 by differential centrifugation and ultrafiltration. Experimental allergic airway inflammation was induced in BALB/c mice by intraperitoneal injection of OVA in alum on days 1 and 14. On days 21-24 mice were challenged with intranasal application of OVA, 30 min after intranasal administration of TsEVs or PBS and sacrificed two days later. Blood samples were taken for serum IgE determination. Lungs and spleens were extracted for the isolation of immune cells. Phenotype of immune cells was determined by flow cytometry and their cytokine production by ELISA assays. Results: TsEVs treatment of allergic mice lead to diminished numbers of alveolar macrophage and CD103+ dendritic cells (DC) in lungs compared to allergic control while numbers of CD11b+ DCs and their Ly6C+ subset was increased, along with CD8+ and CD19+ T cells. Upon restimulation with OVA, splenocytes and lung immune cells of TsEVs-treated mice produced lower levels of Th2 cytokines, while the production of IFN-g was elevated only in lung immune cells. Lower IgE levels were found in TsEVs-treated mice compared to sham-treated controls. Summary/Conclusion: TsEVs exert immunomodulatory properties in murine model of allergic airway inflammation by diminishing inflammation and thus they may represent the basis for novel allergen-independent therapeutics in treatment of respiratory allergies.

Introduction: Mesenchymal stem cell (MSC) extracellular vesicles (EVs) show great promise for repair and regeneration of injured tissues and their immunomodulatory effect makes them strong potential therapeutics for inflammatory bowel disease (IBD), which was the focus of this work. Methods: Conditioned medium (CM) was harvested from cultured human bone marrow MSCs to isolate EVs. CM was precleared of dead cells and cellular debris by differential centrifugation at 4 • C at 500g for 5 min twice then 2000g for 15 min. The recovered supernatant was filtered through 0.22 μm filters and subjected to ultracentrifugation onto a 25% (w/w) sucrose cushion prepared in deuterium oxide. After centrifugation at 100,000g at 4 • C for 1.5 h, the sucrose layer was resuspended in phosphate-buffered saline (PBS) and washed by ultracentrifugation at 100,000g at 4 • C for 1.5 h to pellet the EVs. The EVs were then resuspended in PBS and stored at − 80 • C for further use. Isolated MSC EVs were characterised for yield, size and protein marker expression using bicinchoninic acid assay, nanoparticle tracking analysis and the Exo-Check antibody array kit, respectively. A wound healing (scratch) assay evaluated the effect of MSC EVs on the proliferation of intestinal epithelial Caco-2 cells, while the anti-inflammatory activity of MSC EVs was determined in LPS-activated macrophages via nitrite quantitation. Results: MSC EVs were < 200 nm in diameter and EV protein marker expression was confirmed by the presence of tetraspanins CD63 and CD81 and cytosolic proteins TSG101 and ALIX. The scratch assay showed that Caco-2 cells treated with MSC EVs demonstrated accelerated wound closure over time compared to control. Furthermore, an effect of MSC EVs on nitrite concentrations in activated macrophages was apparent. Summary/Conclusion: In conclusion, MSC EVs exhibit wound repair and anti-inflammatory activity which could have potential therapeutic applications in IBD. Introduction: Osteoarthritis (OA) is the most prevalent rheumatic disease characterized by progressive loss of cartilage and alterations in all compartments within joints. The highest risk factor in this degenerative disease is age and an accumulation of senescent cells in cartilage and synovium has been shown to contribute to the functional decline of joints. We previously demonstrated that extracellular vesicles (EVs) from mesenchymal stromal cells (MSCs) largely mediate the therapeutic effect of parental cells in OA. Here, we investigated whether EVs from adipose tissue-derived MSCs (ASC-EVs) possess senoprotective effects in a new model of induced senescence in OA chondrocytes. Methods: Human chondrocytes isolated from OA patients and ASCs from healthy donors were induced to senescence using 25μM etoposide for 24 hours. Senescence was assessed by quantifying proliferation rate, SA-βGal activity, nuclear γH2AX foci Introduction: Ischemic stroke is a major cause of death and disability, intensely demanding innovative and accessible therapeutic strategies. To propose an approach, using extracellular vesicles (EV) secreted from human mesenchymal stem cells isolated from adipose tissue (hAT-MSC), with a prolonged period for therapeutic intervention and a non-invasive route for administering the treatment of stroke in normotensive and hipertensive rats. Methods: Wistar rats (90-120 days) were subjected to focal permanent ischemic stroke (IS), 24 hours after, were treated intranasally with EV (200 μg/kg) secreted by hAT-MSC. We analyzed front paws symmetry (Cylinder Task), short-and longterm memory (Open Field and Novel Object Recognition Task) and angiogenesis. Evaluation of the interaction of EVs with cells in the peri-infarct region was performed 18h after intranasal administration of EVs labeled with fluorescence, cells were labeled with antibody NeuN, GFAP and to label Hoescht nucleus. Wistar Kyoto Spontaneously hypertensive rats (SHR) (90-120 days) were subjected to IS and we analyzed front paws symmetry (Cylinder Task). Results: In Wistar rats, EV treatment recovered front paws symmetry and short-and long-term memory induced by ischemic stroke. Additionally, we observed stimulation of angiogenesis in the peri-infarction region in animals treated with EVs. In the peri-infarct region there is a greater number of EVs and in this region the EVs seem to have a preference for neurons (preliminary results). For SHR rats, we have preliminary results, where we can observe that the outcome of ischemia induction is very different and with a chronic effect and without spontaneous recovery in the symmetry of the forepaws. We are performing the EV dose and time curve for SHR rats. Summary/Conclusion: In line with these findings, our work highlights hAT-MSC-derived EVs as a promising therapeutic strategy for stroke. Introduction: Allogeneic extracellular vesicles (EVs) loaded with ovalbumin (Ova) can induce an anti-tumor immune response in mice with Ova-expressing melanoma tumors. This anti-tumor immunity of Ova-loaded EVs relies on the activation of T cells, a response that is dependent on B cells. Additionally, previous research showed that EBV-derived GP350-containing EVs target human CD21 present on B cells. We hypothesized that EVs targeting B cells would induce stronger T cell responses to EVs. Therefore, we aim to examine if decorating Ova-loaded allogeneic EVs with a fusion protein that binds CD21 target the EVs to B cells and thereby improve antigen-specific immune responses. Methods: A fusion protein containing the phosphatidylserine-binding domain (C1C2) of lactadherin and CD21-binding domain (D123) of GP350 was designed. Ultracentrifugation-isolated macrophage-like RAW264.7 EVs were dyed with cell tracker deep

Introduction: Immunotherapy has garnered increasing importance in cancer therapy, leading to substantial improvements in patient care and survival. However, while patients generally respond well to immunotherapy such as anti-PD-1/PD-L1, a proportion of patients present tumors that resist these treatments. Exosomes, small nanovesicles secreted by tumor cells, could be key actors in this resistance. We identified immunosuppressive molecules (IM) expressed by tumor-derived exosomes (TEX) in different types of cancer (melanoma, lymphoma, lung and colon cancer). Methods: Exosomes were isolated by ultracentrifugation and evaluated by nanoparticle tracking analysis (NTA technology) and TEM. Isolated exosomes were tested for the expression of exosomal markers (TSG101, CD9, CD63, CD81, Alix, Grp94). IM concentration in exosomes were measured using an ELISA. T-cell and MDSC activation/ proliferation were observed by flow cytometry. Results: First, we explored in vitro and in vivo the action of exosomal molecules on the immune system. We found that TEX have immunosuppressive properties like the cancer cell from which they are derived at inhibiting T-cell activation determined by the expression of PD-1, Ki67, granzyme B and IFNϒ. In addition, we observe that TEX can also activate myeloid-derived suppressor cells (MDSCs). MDSCs activation leads to global immunosuppression of immune cells (T-cell, macrophages, and dendritic cells) independent of the PD-1/PD-L1 pathway. We have developed an inhibitor of this TEX/ MDSC pathway and demonstrate in mouse models that the decrease in MDSC was associated with tumor regression and with intratumor infiltration of immune cells (T cells, dendritic cells and macrophages). Finally, we conducted a clinical trial to observe these immunosuppressive TEX in cancerous patients. Summary/Conclusion: Altogether, these results show that exosomes derived from cancer cells are able to mediate immunosuppression. Further study of TEX is needed to better understand resistance under anti-PD-1/PD-L1 therapy. Introduction: Detection of tumor progression in glioblastoma patients remains a major challenge for clinicians due to equivocal MRI results. Extracellular vesicles (EVs) are potential biomarkers and can be detected in the blood of tumor patients. In this study, we evaluated the potential of serum-derived EVs from glioblastoma patients to serve as a marker for tumor progression in adjunction with MRI assessment. Methods: Glioblastoma patients from two independent cohorts, one from the multicenter Phase III CeTeG/NOA-09 trial (n=36) and the other from patients treated at the University of Bonn (n=31), were included in this study. EVs from serum of GB patients and healthy volunteers were separated by size exclusion chromatography and ultracentrifugation. EVs were characterized by multiple methods in accordance with MISEV2018. (EV Track ID: EV200097). Putative glioblastoma EV markers were defined by using a proximity-extension assay and bead-based flow cytometry. Tumor progression was defined according to modified RANO criteria. Results: EVs from the serum of glioblastoma patients (n=67) showed an upregulation of CD29 (p=0.08), CD44 (p< 0.0001), CD81 (p< 0.0001), CD146 (p< 0.0001), C1QA (p=0.003), and histone H3 (p< 0.0001) as compared to serum EVs from healthy volunteers. For both independent cohorts of glioblastoma patients, we noted upregulation of C1QA, CD44, and histone H3 upon tumor progression, but not in patients with stable disease. Notably, six patients with worse survival compared to the median survival of the cohort did not fulfill RANO criteria at the time of suspected progression, yet showed an elevation of at least one out of these three markers. In a multivariable logistic regression analysis, a combination of CD29, CD44, CD81, C1QA, and histone H3 correlated with RANO-defined tumor progression with an AUC of 0.76. Summary/Conclusion: Measurement of CD29, CD44, CD81, C1QA, and histone H3 in serum-derived EVs of glioblastoma patients, along with standard MRI assessment, could improve detection of true tumor progression and thus be a useful tool for clinical decision making. The production of therapeutic EVs requires well-characterized, robust and reproducible host cells. Telomerase-immortalized MSCs have the advantage to fulfill these criteria by continuous growth and maintenance of cell type specific characteristics. Small ncRNAs including miRNAs control gene expression and are required to maintain cell differentiation status. Small RNAs are loaded into EVs resulting in distinct EV-RNA profiles representative of their cellular origin and status. It is unclear whether telomerase immortalization elicits significant changes in EV-RNA profiles indicating divergent cellular or EV status. Here, we used a novel small RNA-seq assay for genome-wide characterization of the EV small RNA cargo of 2D cultivated Wharton´s Jelly derived primary and the corresponding telomerized MSCs (WJ-MSC/TERT273). Methods: MSCs (n=3 per group) were cultivated in MesencultTM medium on ACF coating (StemCell Technologies) and EV sized particles were harvested after 48 hours followed by TFF enrichment. Exactly 109 particles were used for total RNA extraction. Small RNA-seq libraries were generated from 8.5 μl RNA to which a set of 7 spike-in oligonucleotides harboring a unique 13mer core and 4N-randomized ends was added at defined attomolar concentrations (0.01 -20 amol) to enable extensive quality control and absolute normalization. Results: On average 25 Mio reads were generated per MSC EV-sized particle sample. Read classification identified similar EV-RNA profiles for primary and hTERT MSCs (∼1% miRNA, ∼10% tRNA, ∼2% piRNA, ∼20% rRNA, ∼1% lncRNA, ∼3% mRNA, ∼8% other small RNAs). On average 500 distinct miRNAs were detected per sample and copy numbers per 1000 particles were calculated using the spike-in calibrator. A similar distribution of miRNA copy numbers was observed in primary and hTERT MSC EV-sized particles (p>0.05). Differential expression analysis (edgeR, FDR< 0.05) was performed on 214 miRNAs that passed independent filtering (DESeq2). Eight up-regulated miRNAs were identified in hTERT MSC EVs, including miR-138 and miR-424-5p, which target hTERT and act as tumor suppressors. Summary/Conclusion: Using small RNA-seq we established that the EV-RNA profiles between primary and hTERT-MSCs are highly similar suggesting comparable cellular state and EV identity. We identified two miRNAs to be elevated in hTERT-MSC EVs that are capable of silencing hTERT expression. Introduction: Spaceflight-Associated Neuro-ocular Syndrome (SANS) consists of a set of ocular structural and visual manifestations associated with prolonged exposure to microgravity. While it has been hypothesized that elevation of intracranial pressure (ICP) may be associated with SANS, ICP has not been fully examined in astronauts. We performed a transcriptome-wide profiling of exosomal RNA obtained from high-ICP patients to uncover the underlying processes possibly associated with SANS.

Hyfa Alzahrani; munitta Muthana university of sheffield, Sheffield, United Kingdom Introduction: Breast cancer (BC) is the most common type of cancer in females in the UK. Novel treatments are required to treat radio-/chemo-resistant BC as well as advanced disease. Oncolytic viruses (OV) are naturally cytotoxic and infect and kill tumour cells whilst sparing healthy tissues. The full mechanism by which this occurs remains to be elucidated, but it may in part be mediated by extracellular vesicles (EVs). EVs are nanosized, membrane-enclosed vesicles that contain molecular cargo. EVs can be taken up by cells, for instance immune cells, at local or distant sites, causing phenotypic changes in the recipient cells. Methods: The breast cancer cells lines MCF-7 and MDA-MB-231 were infected with the herpes simplex virus (HSV1716). EVs were isolated from the OV conditioned medium of infected cells and control cells by differential centrifugation. Nanoparticle tracking analysis (NTA) was used to detect the overall size and concentration of EVs. Transmission electron microscopy (TEM) was used to confirm the size and shape of the EVs and western blotting to detect well known EV protein markers as well as oncolytic viral cargo. Results: So far, our study revealed that exposure of the BC cell lines to HSV1716 resulted in increased release of EVs in comparison to the control untreated cells as detected by NTA. Released EVs were typically 50-150nm in diameter and appeared to be typically cup-shaped structures as revealed by TEM. Western blotting showed the presence of the EV biomarkers, CD9 and CD63 in both the untreated and HSV1716 treated cells, whilst the presence of HSV released antigens was only detected in the EVs from infected cells. Summary/Conclusion: In summary, HSV1716 induced increased EV release from infected breast cancer cells suggesting OV influences EV shuttling. Current studies are aimed at characterising the contents of the EVs by real time PCR and mass spectrometry. This will help identify the presence of immunological and viral cargo. Future studies will aim to investigate the antitumour properties of EVs of from infected cells.

Malika Singh; Ghazaleh Mazaheri-Tehrani; Ignacio Martin-Fabiani; Mark P. P. Lewis; Owen G. Davies Loughborough University, Loughborough, United Kingdom EVs with high drug loading capacity of MSNs. Here, we propose construction of EV-coated MSNs as drug carriers for ovarian cancer therapy. Methods: EVs were derived from SKOV-3 cell lines using differential ultracentrifugation. SKOV-3 cells were subjected to serum starvation and the serum-free cell conditioned media was collected after 24 hours. The cell conditioned media was subjected to differential centrifugation at increasing speeds for prolonged duration, i.e. 2000xg for 20 minutes to remove cell debris, 10000xg for 40 minutes to remove larger vesicles and apoptotic bodies, and finally at 100000xg for 90 minutes to pellet the EVs. Amino-functionalized mesoporous silica nanoparticles (MSNs); in three different sizes (50 nm, 85 nm , and 100 nm); were synthesized using previously established methods. The MSNs were loaded into the EVs using three different methods, double extrusion, extrusion followed by incubation: and sonication. The morphology of the EV-coated MSNs was observed with transmission electron microscopy (TEM) and the physical properties were characterized using Nanoparticle tracking analysis (NTA), zeta potential measurement. Biomarker analysis of EVs, MSNs and EV-coated MSNs was performed using Exoview. Results: EV-coated amino functionalized MSNs were successfully synthesized using the stated methods. Double extrusion improved the association of EVs with NPs compared to sonication of extrusion followed by incubation, resulting in more evenly sized EV-associated amino functionalised silica NPs, as evidenced from NTA measurements. TEM images showed complete association of the EVs around the nanoparticles. Zeta potential measurements demonstrated the reversal of positive charge of amino-functionalized MSNs to a negative charge imparted by the associated EV membrane. Exoview analysis showed that the EV-coated MSNs were CD63 positive indicating EV association around the MSNs. Summary/Conclusion: We report the successful synthesis of EV-coated amino-functionalized mesoporous silica nanoparticles using three different methods. These biomimetic particles could significantly enhance the delivery of therapeutics to ovarian cancer tumours owing to their low immunogenicity, biocompatibility, and target homing capacity. Future studies will assess their gene delivery potential and efficacy of the particles in vitro. Introduction: Most central nervous system (CNS) affections are unmet medical needs, one reason being the limited accumulation of drugs in the CNS. Nose-to-Brain administration provides a direct access to the brain while encapsulation in nanomedicines protects drugs and increases their half-live. As drug delivery systems, EV are able to cross epithelial barriers, are non-immunogenic and have an intrinsic activity. In this work, our objective was to isolate and characterize EV from human dental mesenchymal stem cells (SCAP) to later use them as nanomedicines in the scope of multiple sclerosis. Methods: EV were isolated from SCAP culture medium by centrifugation, ultrafiltration and size exclusion chromatography (SEC). Impact of ultrafiltration unit cut-off (30 and 100 kDa) and SEC (Izon qEV 70 and 35 nm) on EV yield (NTA, ZetaView) and separation from proteins (DELFIA immunoassay) was evaluated. MiRNA content (small RNAseq) and lipid composition (HPLC-MS) of EV produced by SCAP in pro-inflammatory condition (activated SCAP) vs steady-state were compared. Finally, the influence of EV on pro-inflammatory marker gene expression of microglial cells (BV2 cells) was evaluated (RT-qPCR). Results: Using a cut-off of 30 kDa and the qEV 35 nm SEC provided the highest number of EV while eliminating most of the contaminating proteins. EV were negative for calnexin and positive for CD9, CD63, CD81 and flotillin. 236 miRNAs, associated mainly with MAPK, neurotrophins and cancer pathways, were identified in steady-state EV while 44 miRNAs were significantly affected in EV produced by activated SCAP. Lipidomic analysis is ongoing. EV obtained from activated SCAP induced a slight reduction of IL-6 and iNOS in LPS-treated BV2 cells. Incorporation of bioactive drugs in SCAP EV is ongoing. Summary/Conclusion: We optimized the isolation of EV from SCAP, analyzed how their composition was influenced by the activation state of SCAP, and studied their impact on neuroinflammation. Then, bioactive molecules will be encapsulated in these EV and delivered to the CNS. Funding: This work has been funded by the FNRS, the Fondation Charcot Stichting and by the Belgian French Community (ARC; EViMS). Introduction: In addition to prophylaxis of infectious diseases, vaccines could play a role in cancer immunotherapy. Our work proposes the design of mRNA therapeutic cancer vaccines based on extracellular vesicles (EVs) from monocytes. EVs have advantages over synthetic carriers: they occur naturally; transfer their cargo to recipient cells; and are naturally loaded with biological molecules. Methods: EVs were isolated from human and murine monocyte cell lines, by differential centrifugation, and characterized by transmission electron microscopy (TEM), Western blot (WB), and nanoparticle tracking analysis (NTA). EVs were loaded with two model mRNAs (GFP as a reporter or with a model antigen) by passive diffusion. The mRNAs loading and stability, after EVs incubation with RNases, were assessed by fluorescence measurement of labelled mRNA. Finally, in vitro preliminary studies to evaluate the uptake of EVs and mRNA expression by recipient cells were performed, by flow cytometry and fluorescence microscopy. Results: EVs were characterized using three techniques. NTA and TEM confirmed EVs sizes below 200 nm. TEM showed an EVs homogeneous population, with the expected morphology. WB confirmed the presence of EV-related proteins. Importantly, mRNA loading efficiencies into EVs higher than 75% were found, which enabled stable mRNA levels for longer than 6h, in the Introduction: Mesenchymal stromal cells (MSC) are the most professional source of extracellular vesicles in addition to their regenerative potency. The properties of MSC-derived exosomes are considered as their parent cells. Exosomes (Exos), the natural nanovesicles, have gained tremendous interest recently due to their ability to deliver drugs. On the other hand, chemotherapeutic agents for breast cancer, such as sorafenib, have significant negative side effects on other organs. Methods: MSC cells were isolated from Wharton's jelly. MSC exos were isolated and their morphology, particle size, zeta potential, and expression of specific markers were evaluated. Sor-MSC exos were prepared by incubation. The cytotoxic effect of exos in human breast cancer spheroids (3D culture) was monitored by MTT, AO /EtBr, DAPI, Annexin/ PI, scratch and migration assays, colony formation and real-time PCR for P53, VEGF-A and caspase 3. Results: The loading efficiency was 35.6% and the release rate was 14.7% after 24 hours. Uptake of PKH-67-labeled Sor-MSC exos was detected by flow cytometry at 74.6%. All viability assays confirmed the inhibitory effect of Sor-MSC-Exos compared with free sorafenib. The results indicate that Sor-MSC-Exos selectively reduces tumor cell viability and spare fibroblasts and MCF -10A as non-cancerous cells. The remarkable effect of Sor-MSC-Exos was maintained for a long time after spheroid treatment. Summary/Conclusion: Sor-MSC-Exos showed a potent inhibitory effect on proliferation and induced apoptosis in human breast cancer mass (tumor mimic) compared to the same dose of free sorafenib. Thus, the exosomes containing the cargo showed significantly higher uptake than the free drug in both 2D and 3D. Thus, they can be used as drug carriers with selective toxicity to the normal cells to reduce the side effects of chemotherapeutic drugs. Introduction: Despite the proof of concept of their efficiency as drug delivery systems (DDS) compared to synthetic nanoparticles, the rationale of using extracellular vesicles (EVs) in therapy still requires improvements. Among them, their plasma instability (t1/2=4 min) when intravenous administration in a non-autologous host is a major issue avoiding reaching targeted organs by passive accumulation. In this context, our team aims at overcoming this hurdle by transiently functionalizing EVs surface to increase their plasma stability while maintaining their cell internalization capacity. Our strategy relies on the post-insertion of fine tuned bio-inspired polymers: the poly(2-oxazoline)s (POx). Known for their excellent biocompatible properties, POx also constituted an excellent alternative to poly(ethylene glycol) (PEG) as clinical awareness has risen around its overuse (e.g. anti-PEG Abs). Therefore, we designed amphiphilic POx with different lipid anchors to insert in EVs membrane: cholesterol hemisuccinate (CHEMS) and hexadecane (C16). Methods: EVs were produced from murine MSC, isolated and characterized by nanoparticle tracking analysis. EVs were extruded through a membrane of 50 nm to obtain narrower sizes, as a reproducible basis for evaluation of surface modification. The two POx were synthesized by cationic ring-opening polymerization, purified by membrane dialysis and fully characterized as CHEMS(POx)52 and C16(POx)54. Different conditions (time, buffer, temperature) were screened to obtain both POx association onto extruded EVs. Non associated chains were discarded by ultrafiltration. Thus obtained EVs were thereafter characterized at the physico-chemical (size, charge, SPR-AFM) and biological level (macrophage uptake, complement activation, internalization in complex organoids composed of pancreatic cancer cells, in vivo administration in mice). Results: The conditions of post-insertion were first determined on liposomes as synthetic model and then transposed to EVs. Overall, the grafting density was determined of respectively 0.5 for C16(POx) and 1 POx/nm 2 for CHEMS(POx) and confirmed by SPR and AFM measurements. The macrophages uptake of EVs was evaluated on primary mice cells and we demonstrated a decrease of 50% with post-inserted species compared to non modified species. To verify the transient functionalization, both EVs species were tested on organoids cells to measure their penetration capacity. The preliminary results indicated no difference in cell internalization and identical penetration to the organoids core from non-extruded EVs to extruded one and with or without POxylation and significantly higher than with liposomes. Summary/Conclusion: With these promising results, we are currently evaluating the behavior of the post-inserted EVs in vivo after IV injection in mice. We propose here to expose our latest results concerning the interest of EV surface functionalization.

rui zhang 1 ; zhiwei Wu 2  NanJing university, Nanjing, China (People's Republic);  Nanjing university, Nanjing, China (People's Republic) Introduction: Zika virus (ZIKV), a flavivirus associated with neurological disorders, constitutes a global health threat. During pregnancy, ZIKV traverses the placenta and causes congenital diseases such as microcephaly and Guillain-Barré syndrome in newborns. To develop a specific antiviral therapy against ZIKV-induced microcephaly that could cross placental and blood-brain barriers, we designed targeted small extracellular vesicles (EVs) encapsulating antiviral siRNA (small interfering RNA) to inhibit ZIKV. The neuro-specific targeting was achieved by engineering EVs membrane protein lamp2b fused with a neuron-specific rabies virus glycoprotein derived peptide (RVG). Intravenous administration of the RVG-engineered EVs loaded with siRNA (ZIKV-specific siRNA) protected pregnant AG6 mice against vertical transmission of ZIKV. Particularly, sEVsRVG-siRNA traversed placental and blood-brain barriers and suppressed ZIKV infection in fetal brains. Moreover, sEVsRVG-siRNA alleviated the neuroinflammation and neurological damage caused by ZIKV in the fetal mouse model. In general, we developed a EVs-based targeted system of antiviral therapy for brain and fetal brain infections Methods: Isolation of sEVs EVs encapsulation of siRNA by electroporation Quantification of siRNA loading into EVs by electroporation Flow cytometry Western blot analysis Nanoparticle tracking analysis Results: By expressing a neurotropic RVG peptide sequence on the surface of EVs, the current study demonstrated that the antiviral siRNA-loaded sEVsRVG was selectively targeted to brain tissues and inhibited virus in fetuses. This therapeutic approach expanded the application of EVs to treat a viral infection of brains by intravenous injection of sEVsRVG-siRNA Summary/Conclusion: we engineered EVs expressing RVG on the surface and demonstrated that intravenous administration of RVG-modified EVs loaded with siRNA (ZIKV-specific siRNA) protected pregnant AG6 mice against the vertical transmission of ZIKV. Moreover, sEVsRVG-siRNA protected the fetuses from ZIKV infection and alleviated the neuroinflammation and neurological damage caused by ZIKV. Therefore, sEVsRVG-encapsulated siRNA can provide an ideal method to enhance the delivery of cargo such as nucleic acids, achieving targeted treatment of brain viral infection and control of related neurological damage Introduction: Cell-derived extracellular vesicles(EVs) contain miRNA, mRNA, and various proteins, and many studies have been conducted on the therapeutic potential due to their intracellular communication capabilities. However, for commercial use, it is necessary to improve the production efficiency and quality control standards of existing EV production technology. Methods: Here, we have developed EV production technology using cell aggregates/spheroids to improve EV production efficiency. Since the EV production environment changes according to the size of the cell spheroid used for EV production, we developed cell spheroid production automation technology to produce uniform cell spheroids. The production of EVs derived from cell spheroids significantly increased the production index of number of EVs per cell(# of EVs/cell) compared to the conventional 2D cell culture.

Introduction: Extracellular vesicles (EVs) have come into focus of extensive research during the last decade, because of their broad biological functions and roles in various diseases. Myocardial infarction is a leading cause of death worldwide. Following injury, the organ function is impaired, and the adult human heart is lacking regenerative capability. EVs offer an exciting, novel therapeutic approach for treatment of heart failure. Methods: In our studies as a first step we are developing gene engineering technologies to engineer the surface composition of EVs to guide these nanomedicines to the infarcted heart tissues. Results: We have developed methods to put antibodies against cardiac target candidates on the surface of EVs. We have screened of range of anchor proteins and identified: nanobody against kappa chain of IgG antibody, Protein A and Protein G as successful candidates for antibodies EV display. Additionally, we developed EV based nanomedicines decorated with cardiac targeting peptides. We have optimized the fusion protein for functional display of these peptides at the surface of EV in the presence of blood plasma. Summary/Conclusion: Our data demonstrate that we can display both antibodies and peptides on EVs surface, that these are available for interaction with their binding partners in presence of the blood serum. This opens the possibilities to use targeting potential of the designed EVs for delivery of therapeutic molecules to the heart tissues.

Julien Branchu 1 ; Camille Simon 1 ; Shony Lemieux 1 ; Max Piffoux 1 ; Jeanne Volatron 1 ; Amanda Silva 2  EVerZom, Paris, France;  Laboratoire Matière et Systèmes Complexes, Paris, France Introduction: Extracellular Vesicles (EVs) are one of the most interesting alternatives to cell therapy in regenerative medicine. However, large scale EV manufacturing methods are costly and time consuming. We discovered that shear stress induces massive EV production and transformed this unexpected discovery in an industry-compatible and easy to scale-up technology to achieve large scale EV production. Shear stress is exerted on cells after their 3D culture in bioreactors thanks to well controlled turbulent flows. This method was proven to be effective for all tested cell types (>10) with both adherent and suspended cells. Methods: As an example, adherent cells like human adipose derived stem cell (hASC) may be cultivated in bioreactors on microcarriers (MC), 200 μm polymer beads that have a high surface/volume ratio and can be handled easily. This old and well-described technology allows to culture adherent cells just like if they were in suspension. Bioreactors may be viewed as bottles of cell culture media agitated by an impeller where parameters like temperature, pH, metabolic activity or gas input are controlled. When the desired cell-confluence on MC is reached, a precise mechanical stimulation in a serum-free media is applied for a few hours to induce high yield vesiculation. Results: On overall, this process achieves a ≈10-fold increase in yield in 4 hours compared to classical production by starvation in 2D our 3D. Using this technology, hASC in a bioreactor produce approximately 20,000 EVs/cell in 4 hours compared to about 4,000 EVs/cell in 2D starvation for 72 hours. This method preserves a good cell viability at the end of the process (>90%) when monitored by both Nucleocounter and immunostaining. EVs are produced within a culture media free of any contaminating human or animal protein, limiting the need for complex purification. The batches may be clarified by centrifugation or filtration and concentrated by tangential flow filtration with a global harvesting yield above 70%. Furthermore, the turbulence-generated EVs conserve the regenerative properties of their parental cells and have been shown to be effective in various small and large animal models of regenerative medicine or drug delivery. Summary/Conclusion: We describe a GMP/clinical grade EV production and purification platform to bring EV therapeutics to patients and are open to collaboration on this subject. Introduction: Extracellular vesicles (EV) are emerging as a safe alternative to cell therapeutics. Full realization of their potential requires a new paradigm where technology development and future large-scale manufacturing are considered already during the earliest stages of research. Our approach aims to combine optimization of culturing conditions for efficient EV production, purification, quality control and design of the manufacturing process. During our study, we focus on 1) use of cell lines and iPSC-derived cells for EV production and 2) detailed quality control analysis of purified vesicles. Methods: Expi293 and different iPSC derived cells were used for EV production. We developed optimal cell culture protocols and differentiation protocols for cell types of interest. Tangential flow filtration (TFF) in combination with differential ultracentrifugation were used for EV purification. EVs were characterized by nanoparticle flow cytometry (NanoFCM), Exoview, transmission electron microscopy, western blotting and mass spectrometry (MS). EV functionality was analyzed in Incucyte-based cellular assays (e.g. viability, proliferation and functional uptake) Results: We found GMP compatible cultivation conditions for efficient EV production for all cell types analyzed. During the development of our set of EV characterization procedures, we found that a combination of "state-of-the-art" methods, such as NanoFCM, TEM, MS in combination with cell based assays provides extensive and reproducible information about quality and functionality of EV batches. Summary/Conclusion: Overall, using EVs produced by Expi293 and iPSC-derived cells, we could generate proof of principle data to demonstrate that our established EV platform supports identification of optimal EV production conditions along with in depth qualitative, quantitative and functional characterization of EV production batches. We envision that this platform will be useful for a variety of therapeutic applications.

Bastien Thauvin 1 ; Camille Simon 2 ; Julien Branchu 2 ; Latifa Chebil 3 ; Eric Olmos 3  EVerZom, Nancy, France;  EVerZom, Paris, France;  Laboratoire Réactions et Génie des Procédés, Nancy, France Introduction: Extracellular vesicles (EVs) are nano-sized subcellar particles involved in intercellular communication. Because they reflect their parental cell phenotype, EVs produced by mesenchymal stem cells (MSC) have regenerative properties and are promising as cell-free therapy to repair damaged tissues. However, lack of large-scale GMP compatible EVs production processes limits clinical translation. EV production yield can be improved by mechanical stress induced by turbulence but a large number of cells is still required. Conventional 2-Dimensional systems are time consuming and allows insufficient control of cell culture parameters. Development of 3D culture systems tend to give interesting solutions to these issues. We designed a microcarrierbased (MC) cell culture process in stirred tank bioreactors using cell bead-to-bead transfer (Btb, i.e. adding fresh MC in the media) as a scale-up method for EV production without the need for enzymatic detachment step. Methods: The effect of MC addition on Human adipose derived stem cells (hADSC) cultivated on MCs in spinner flasks was evaluated based on daily cell count and metabolites analysis. After each addition, an intermittent agitation was applied to promote cell migration. Results: Cells can migrate on newly added MC thanks to intermittent agitation. Btb lead to prolong cell expansion, delay aggregate apparition and increase final harvest compared to control. Nevertheless, drop of mean cell/bead ratio below 3 inhibit cell growth and late addition of MC when cell/bead ratio is above 7 leads to aggregate formation which cannot be dissociated. Summary/Conclusion: BtB is a promising scale-up method for MSC large-scale production but the optimal cell per bead ratio and time addition need to be precised to maintain a high expansion rate. Funding: Private, European, ANRT fundings.

Michele Hamrick; Megan K. Livingston; Coby Carlson; Jacquelyn Wong; Nisa Renault Fujifilm Cellular Dynamics, Madison, USA Introduction: To advance novel extracellular vesicle (EV)-based therapeutics, there is a need for screening tools and functional release assays that are small-scale, reproducible, human-relevant, and have a meaningful product dosing strategy with appropriate controls. We have developed a cell survival assay using human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) to assess the potency of therapeutic candidates containing small EV-enriched (sEV) compositions. Methods: Cardio-protective sEV candidates were isolated by ultracentrifugation from primary and iPSC-derived cell types. Media without cells were "cultured" and ultracentrifuged for "virgin media" (MV) negative controls. CM were plated, chemically stressed, and treated with sEV or MV in standard or serum-free media. Dosing strategies based on particle number (nanoparticle tracking analysis) or the number of secreting cells producing the conditioned media were assessed. Assay controls were unstressed or stressed CM, each treated with DPBS only (vehicle control). Cell survival was assessed by quantifying viable cell number (imaging / microscopy), ATP (chemiluminescence), and cell adherence (electrical impedance). Results: Our assay consistently displayed a dose-dependent improvement in survival of CM with the addition of therapeutic sEV over controls for all readout methods. Potency results with CM paralleled data from human endothelial cells in scratch wound healing assays when using the same dosing strategies. Dosing by mother cell number was a superior strategy for product development for both assays, in part due to highly variable particle counts in sEV compositions from different donors or culture methods. Summary/Conclusion: We have developed a useful dosing strategy for product screening and development of sEV-containing compositions featuring a reproducible, human cell-based, CM-survival assay relevant to the testing, development, and final product release of sEV-containing compositions for the treatment of cardiac diseases. Funding: FUJIFILM Cellular Dynamics, Inc. FUJIFILM Introduction: Extracellular vesicles (EVs) are nanoparticles found in all biological fluids, capable of transporting biological material around the body. Research into the physiological role of EVs has led to the MISEV framework in 2018 guiding the standardisation of protocols in the EV field. To date, these guidelines have focused on EVs of human origin. As the importance of comparative medicine progresses, there has been a drive to study similarities between diseases in humans and animals. To research EVs in felines with pathologies, we must initially validate the application of the MISEV guidelines in this group. Methods: EVs were isolated from the plasma of healthy humans and healthy felines by size exclusion chromatography and characterised according to the MISEV guidelines. Nanoparticle tracking analysis, total protein concentration, western blot analysis of known EV markers and transmission electron microscopy were carried out. Further EV characterisation by mass spectrometry, asymmetrical flow field blow fractionation and metabolomic profiling was also undertaken. Results: Human and feline plasma showed a similar concentration of EVs, comparable expression of EV markers and analogous particle to protein ratios. Mass spectrometry analyses showed a similar proteomic signature of EVs from humans and felines. Asymmetrical-flow field flow fractionation showed two distinct subpopulations of EVs isolated from human plasma, whereas one subpopulation was isolated from feline plasma. The EVs from humans and felines were metabolically similar. Summary/Conclusion: Isolation and characterisation of EVs from humans and felines show that MISEV2018 guidelines may also be applied to EVs from felines. Potential comparative medicine studies of EVs provide a model for studying naturally occurring diseases in both species. Funding: Work was supported by the UCD PhD Advance Core Funding Scheme, and the Mater Foundation, Mater Misericordiae University Hospital (MUUH) Dublin, Ireland.

Olesia Gololobova 1 ; Wyatt Vreeland 2 ; Kenneth W. Witwer 1  Johns Hopkins University School of Medicine, Department of Molecular and Comparative Pathobiology, Baltimore, USA;  NIST, Gaithersburg, USA Introduction: Circulating extracellular vesicles (EVs) are potentially promising biomarkers for many diseases and pathological conditions. However, EVs are in very low abundance compared with non-vesicular extracellular particles (EPs) such as lipoprotein particles (LPP) and protein complexes, and some of these non-EV components overlap in size and density with EVs. Therefore, the separation of vesicles from blood is challenging. Currently, the most used plasma EV separation methods are ultracentrifugation (UC, with or without density gradient), size exclusion chromatography (SEC), and combinations thereof. However, alone, these methods do not fully purify EVs. Here, we apply SEC and asymmetric flow field-flow fractionation (AF4) to plasma EV separation. Methods: Gel size separation with IZON qEV70 original columns was used to deplete abundant plasma proteins such as albumin and the very small high-density lipoprotein particles (HDL) from 0.5 mL of platelet-depleted plasma. EV-containing fractions were pooled together and concentrated with Amicon 10 kDa filters. Concentrated fractions were separated using asymmetric flow field flow fractionation (AF4) with the long channel which allows separating large particles at low crossflow to minimize membrane (10 kDa) interaction. Collected fractions were probed by Western blot (WB) for lipoprotein markers ApoB100, ApoE, Introduction: Extracellular vesicles (EVs) are ubiquitously secreted by almost every cell type and present in all body fluids. The blood-derived EVs can be used as a promising source for biomarker monitoring in disease. Current development in EVs proteomics have analyzed in clinical subjects. To date, researcher have developed the EV isolation methods, including differential centrifugation, sucrose gradient ultracentrifugation, size exclusion chromatography, affinity capture and asymmetric-flow fieldflow fractionation. However, their isolation methods are limited in throughput for human subjects. Here, we introduced a novel automated EV isolation and sample preparation method for EV proteomics analysis that can be started with low volume of multiple clinical samples. Methods: EVs were automatically separated from both EDTA plasma and serum of six healthy subjects (n=3) by an affinity capture isolation method using Magcapture isolation kit, and we applied them in Mass spectrometry, data-independent acquisition. In addition, the sample preparation for EV proteomics performed using combination single-pot, solid-phase-enhanced sample-preparation (SP3) technology with Flex system in 96 well format. Results: The automation of EV purification and protein digestion made it possible to complete the pretreatment of 96 samples within one day. In particular, the automation of EV purification reduced the variability of protein identification by 70% compared to manual purification, enabling the quantification of 1400 proteins in one hour of MS analysis. Summary/Conclusion: We have successfully isolated EVs from blood using an automated isolation method and developed an automated method for EV proteomic sample preparation. This method is attractive for processing large cohort samples for biomarker development, validation and routine testing.

Reetta Pusa 1 ; Heikki Saari 2 ; Heli Marttila 3 ; Petra Ilvonen 1 ; Kai Härkönen 2 ; Ulla Impola 2 ; Hanna Oksanen 3 ; Minna Poranen 3 ; Saara Laitinen 2  Finnish Red Cross Blood Service, University of Helsinki, Helsinki, Finland;  Finnish Red Cross Blood Service, Helsinki, Finland;  University of Helsinki, Helsinki, Finland Introduction: The requirements for the EV isolation methods for materials poor or rich in contaminating particles such as protein complexes or lipoproteins are not the same. Well-accepted isolation method, size exclusion chromatography (SEC) separates impurities smaller in size by porous matrix. This is not an optimal choice to separate pure EVs from donated whole blood rich in EV-sized lipoproteins: chylomicrons, VLDL and LDL. Hence development of alternative or combinatory chromatographic techniques are needed to isolate large quantities of pure EVs. Here we report results from different chromatographic techniques: SEC and ion exchange chromatography with material derived from platelet concentrates (PC). Methods: Standard leukocyte-reduced PCs were derived from buffy coats of four anonymous ABO RhD-matched whole blood donations, were obtained from the FRC Blood Service as accepted by the Finnish Supervisory Authority for Welfare and Health. Isolations were done using selected anionic/cationic ion exchange and size exclusion chromatographic techniques. Yield and purity were estimated by NTA, imaging flowcytometry, SDS PAGE, WB, Agarose electrophoresis of lipoproteins and EM.

is known about the effect of HI on the cells and the released EVs. The aim of this study was therefore to evaluate the effect of HI on EV purity. Methods: To determine the effect of heat inactivation, three different protocols were applied based on different combinations of: 1) UC at 118,000 × g for 18h and 2) HI at 56 • C for 30 min. The three conditions tested were: FBS ultracentrifuged but not heat inactivated (no-HI), FBS heat inactivated before UC (HI-before EV-dep), and FBS heat inactivated after EV depletion (HI-after EV-dep). The FBS was add to the media of three melanoma cell lines (MML1, UM22Ctr and UM22BAP1) at a final concentration of 10%. After 72h, large and small EVs were isolated by differential UC. The EV purity was determined by protein quantity, electron microscopy (EM) and nanoparticle tracking analysis (NTA). Results: The protein quantity (μg/μl) of large EVs was similar in the three conditions analyzed. On the contrary for small EVs, the protein amount was higher when the HI was performed after EV depletion as compared to HI before the UC and UC alone. However, significantly more particles were not detected in the HI-after EV-dep which resulted in a lower purity of small EVs in HI-after EV-dep illustrated by calculating the ratio of number of particles/μg proteins. Presence of contaminants (indicated by strong background) was observed in EM pictures of small EVs isolated in HI-after EV-dep condition differently from large EV samples. Summary/Conclusion: The HI of FBS induces release of contaminating elements that end up in small EV pellets if not previously removed.

Roberto Frigerio 1 ; Alessandro Strada 2 ; Greta Bergamaschi 3 ; Lucio Barile 4 ; Gabriella Andriolo 4 ; Alfonso Gautieri 5 ; Alessandro Gori 2 ; Marina Cretich 2  Consiglio Nazionale delle Ricerche, Istituto di Scienze e Tecnologie Chimiche "Giulio Natta" (SCITEC), Milan, Italy, Milano, Italy;  Consiglio Nazionale delle Ricerche, Istituto di Scienze e Tecnologie Chimiche "Giulio Natta" (SCITEC), Milan, Italy, Milano, Italy;  SCITEC-CNR, milano, Italy;  Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale, Lugano, Switzerland;  Politecnico di Milano, Milano, Italy Introduction: Small extracellular vesicles (sEV) present distinctive lipid membrane features in the extracellular environment. These include high curvature, lipid-packing defects and lipids composition. sEV membrane could be then considered as a "universal" marker, alternative to traditional surface proteins. Our recent work has identified a class of membrane-sensing peptide, derived from Bradykinin protein, as a class of molecular ligands for integrated small EV. The membrane recognition and binding mechanisms are based on complementary electrostatic interactions between the peptide and the phospholipids on the outer membrane leaflet, that subsequently can lead to the insertion of hydrophobic residues into the membrane defects. Methods: In the present work, we investigate the role of hydrophobic aminoacids on the membrane recognition efficiency. To achieve this aim, we synthetized a small library of membrane sensing peptide derived from Bradykinin protein by replacing phenylalanine with residues with different hydrophobicity. Phenylalanine residues were replaced by halogen-aminoacids (Phe-I, Phe-Br, Phe-Cl), isoleucine and alanine; to evaluate the contribute of hydrophobic effects of Phe in membrane sensing peptides. To assess peptide binding capacity, we used peptide microarray for capturing EV-mimicking liposomes and EVs isolated by cell culture. Results: These substitutions showed how the hydrophobic component of peptide sequence play an important role in membrane sensing. In addition, the same profile of binding for both types of particles demonstrated the possibility to use synthetic liposomes as model for biological extracellular vesicles. Finally, a preliminary computational approach was used to determine the freeenergy profile of these peptides in order to assess the interaction with the phospholipid bilayer. Summary/Conclusion: These results represent a step forward to the development of a new generation of membrane-sensing peptides for EV isolation and analysis. 

Johanna Puutio 1 ; Petteri Parkkila 2 ; Saara Laitinen 3 ; Tapani Viitala 4 ; Mari Palviainen 4 ; Pia Siljander 4  University of Helsinki, helsinki, Finland;  Chalmers University, Gothenburg, Sweden;  Finnish Red Cross Blood Service, Helsinki, Finland;  University of Helsinki, Helsinki, Finland Introduction: Human cells release heterogenous extracellular vesicles (EVs) into the bloodstream as a response to pathophysiological stimuli. Due to a lack of technologies capable of detecting all EVs, accurate quantification of the total EVome (size and surface markers) remains a challenge. We selected a panel of current technologies and measured the total platelet EVome to investigate their strengths and limitations in EV detection. Methods: Human platelets were collected from clinical grade platelet concentrates and EVs were generated and fractionated with differential ultracentrifugation, ultrafiltration and asymmetric flow field-flow fractionation (AF4). The presence of EVs was confirmed by electron microscopy (EM) and subpopulation enrichment by western blotting and Single particle interferometric reflectance imaging sensing (SP-IRIS). EV sizing was carried out with light scatter (LS, e.g. UV, MALS, DLS and NTA), capture methods (surface plasmon resonance (SPR) and SP-IRIS), and resistive pulse sensing (RPS) to determine their effective detection ranges. Results: Western blotting and SP-IRIS showed that fractions enriched in large EVs (>150 nm) and small EVs (sEVs, 100-150 nm) displayed EV markers (Hsp-70, Alix, CD9, CD63) and platelet marker CD41, whereas those enriched with very small EVs (< 80 nm) carried markers associated with exomeres. The reference size range and morphology of the total EVome was confirmed with EM and multi-detector AF4. LS-based techniques failed to find EVs below 100 nm, whereas capture methods became saturated with sEVs, leaving EVs above 150 nm underrepresented. Combining "bridge methods", such as EM and AF4, with LS, RPS and capture techniques improved the characterization and quantification of the total EVome. Summary/Conclusion: A systematic combination of the current technologies comprising LS, RPS and surface-capture methods is essential for capturing the total EVome. Knowledge of the detection limits and analytical range of each technology needs to be established. Funding: This project is funded by the EVE consortium of Business Finland.

Introduction: Introduction. EVs released from cells are heterogeneous and this can challenge conventional bulk biochemical analysis. Immunofractionation is useful to enrich (or deplete) particular EV sub-sets, but most protocols lack quantitative rigor, and even qualitative assessments of reproducibility are rare. We aim to develop EV immunocapture protocols that are both quantitatively rigorous and reproducible by incorporating single vesicle flow cytometry and bead-based flow cytometry into an optimized immunocapture protocol. Methods: Methods. EVs preparations were obtained from cell culture supernatants or citrated plasma by pelleting cells (2x 2500xg, 15') and concentration by ultrafiltration where necessary. EV concentration, size and surface cargo expression were measured by single vesicle flow cytometry (vFC). Magnetic beads bearing validated capture antibodies were used to capture EVs bearing specific surface markers. Bead and vesicle fluorescence was measured using qualified and calibrated commercial flow cytometers. Results: Results. EVs in plasma were measured using vFC to determine the concentration of PLT-derived (CD41 positive) and RBC-derived (CD235ab positive) EVs. Magnetic beads bearing anti-CD41 were used to capture PLT-derived EVs from plasma, and vFC was used to confirm the quantitative depletion of CD41-positive EVs from the plasma. The beads bearing the captured EVs are analyzed directly measured by flow cytometry after staining for CD41 and additional surface markers, provide additional characterization of the immuno-enriched fraction. The combination of pre-and post-immunocapture vFC analysis and direct analysis of the captured EVs on-bead allows the input to downstream molecular or biochemical analyses to be quantitatively defined. Summary/Conclusion: Conclusions. EV immunocapture can be a useful approach to simplify sample composition for bulk biochemical or molecular analysis, but demonstrating quantitative and reproducible EV capture is generally lacking. Here, we combine immunomagnetic beads with quantitative single vesicle and single bead measurements to provide efficient and scalable EV immunoisolation of EV sub-sets that is both rigorously quantitative and reproducible. Current efforts are directed towards release and recovery of EVs from immunocapture supports in an unperturbed form to downstream functional studies. Introduction: Current method of isolating extracellular vesicles, such as ultracentrifugation, can be time-consuming, requiring specialised equipment, labour intensive, and quality control can be challenging to achieve. Here we describe the development of a reproducible, automated method to isolate extracellular vesicles using high-performance liquid chromatography (HPLC) without human intervention, targeted for clinical use for which high-throughput and reproducibility are essential. Methods: A modular HPLC system with a fraction collector coupled with either a multimodal chromatography or size-exclusion column was used for this study. To optimise the HPLC method, multiple replicates of fetal bovine serum or human serum were processed (200 μL), with multiple elution fractions collected from each starting sample. Reproducibility was calculated based on Nanoparticle Tracking Analysis (NTA) and protein quantification data. To further demonstrate the utility of this isolation platform, the isolated EVs from human plasma were subjected to both next-generation sequencing (NGS) and mass-spectrometry proteomic analysis. Results: A highly reproducible method of isolating extracellular vesicles from bovine and human serum was achieved. The average inter CV for among the technical replicate was below 10% for the particle mean (3.75 %), mode (7.68 %) and protein concentration (8.45 %). This suggests that this method is reproducible with low technical variations. In addition, from one biological sample, using the optimised automated method, most of the total vesicles can be purified into only one tube. Furthermore, the purity of the isolated vesicles was comparable to current isolation techniques and can be used for downstream analyses such as NGS and proteomics. Summary/Conclusion: The use of automated systems to isolate extracellular vesicles could have important implications in the field of diagnostics, where high-throughput and reproducibility is essential. Introduction: Extracellular vesicles (EVs) are recognized as next generation diagnostic biomarkers due to their importance in cell-cell communications and the presence of disease-specific biomolecular cargoes. A major bottleneck in EV sample preparation is the inefficient and laborious isolation of EVs from biological samples. Herein, we report an automatable microfluidic platform for EVs purification from human plasma based on the principle of size exclusion chromatography (SEC). Methods: Using a novel rapid (∼20 min) replica molding technique, an fritless microfluidic SEC device (μSEC) with on-chip sample injector was fabricated using commercial UV glue (NOA81) and resin extracted from commercial SEC columns (IZON, cutoff: 75 nm). Sample and sheath flow were introduced into μSEC using two syringe pumps and a simple 3-way valve for sample plug generation. Results: We first demonstrated on-chip nanoliter sample plug injection (600 nL) with rapid response time ( < 1.5 s) using a modified T-junction sample injector. Device performance was validated using fluorescent nanoparticles (50, 200, 500 nm), FITCalbumin, and breast cancer cells (MCF-7)-derived EVs. As a proof-of-concept for clinical applications, we directly isolated EVs (∼50 to 200 nm) from undiluted human platelet-poor plasma using μSEC, and showed distinct elution profiles between EVs and proteins based on nanoparticle particle analysis, western blot, bicinchoninic acid protein assays and flow cytometry analysis. The miniaturised μSEC reduced sample volume in commercial SEC from ∼100 μL to ∼5 μL, and EV elution volume from ∼600 μL to ∼10 μL. Summary/Conclusion: We have developed a novel μSEC device for EVs isolation from human plasma. With its modular design and continuous-flow processing, the μSEC device enables easy coupling with downstream EV-based assays for "sample in-answer out" EV profiling or real-time EV screening applications (e.g. EV cargo). Introduction: Extracellular vesicles (EVs) carry cancer biomarkers and are central to emerging liquid biopsy technologies; however, current EV isolation methods balance purity against efficiency. We investigated an alternating current electrokinetic (ACE) method to isolate EVs directly from plasma. We compared the ACE method against differential ultracentrifugation (DU) to study yield, purity and utility. EVs isolated by ACE were then used for detection of early-stage pancreatic, ovarian, and bladder cancers. Methods: Plasma from control donors (N=532, 54.5% female) and stage I-II cancer patients (N=224, 55.3% female, pancreatic N=78, ovarian N=70, bladder N=76) were obtained from a biorepository. For ACE, EVs were captured onto microarray chips, plasma contaminants washed away, then the current stopped to release purified EVs. For UC, plasma underwent 3 low-speed, then 2 high-speed spins (100,000 g, 70 min). EV particle size and concentration (nanoparticle tracking analysis) and total protein content (bioanalyzer, qubit) were compared. Concentrations of cancer-related proteins were measured using bead-based immunoassay kits, and EV expression patterns analyzed using a machine learning approach for selection of relevant biomarkers that can distinguish cases from controls. Results: DU had similar particle characteristics (138 nm, ACE; DU, 120 nm), but ACE-purified EVs had less plasma protein contamination. Analysis of biomarker levels (CA19-9, CA125) for ACE EVs showed discrimination between a subset of cancers and controls unlike DU EVs. Expression patterns for ACE EVs were analyzed for all samples, and the multi-cancer classifier had an overall area under the curve (AUC) of 0.93 (95% CI: 0.89-0.97) with an overall sensitivity of 52.2% at a 99.1% specificity. The stage I sensitivity was 42.9% and the stage II sensitivity was 62.9% at the same specificity. Summary/Conclusion: ACE isolation of EVs is streamlined and provides greater separation from blood proteins than DU. EVs isolated via ACE may facilitate detection of biomarkers and enhance their utility for liquid biopsy assays. Funding: Funding provided by Biological Dynamics Introduction: Analysis of cargo from blood circulating extracellular vesicles (EVs) originating from the central nervous system (CNS) has recently gained great interest for biomarker discovery for neurodegenerative diseases (NDD). Here, we present methods for purifying (1) total EVs and (2) neuron-, astrocyte-and oligodendrocyte-derived EVs from plasma and preview their clinical utility as disease biomarker for Alzheimer's (AD) and Parkinson's disease (PD). Methods: We developed and validated (1) a workflow for total EV purification from human plasma using size-exclusion chromatography (SEC) followed by EV marker detection, particle size and concentration measurement (TRPS), and lipoprotein contamination assessment. Detection of CNS-enriched or NDD-related protein cargo were achieved by high-throughput, multiplex immunoassays (Olink® Explore) in AD and PD. Deeper analysis was done on α-synuclein (αSyn) in PD to characterize its inside or outside location by protease protection assay, and separation by ultrafiltration (UF) and ultracentrifugation (UC). We also developed (2) a workflow combining in-silico and mass spectrometry analysis of CSF EVs for the identification of membrane CNS-enriched protein present on EV surface for their immunoprecipitation (IP). Results: We show (1) that targeted proteomics identified potential biomarker candidates only detectable in EV fraction and not in crude plasma and that a small fraction of αSyn in human plasma mirror EV markers profile elution during SEC. Protease protection assay indicated that the majority of αSyn in the plasma EV fraction is present outside of EVs. UC as well as UF of SEC-purified plasma EVs separated αSyn from EV markers suggesting no or transient interaction between αSyn and plasma EVs. Quantification of αSyn levels in the plasma EV fraction showed no significant difference between 25 PD and 25 healthy control individuals, even after normalization to different EV markers. Finally, αSyn was significantly correlated with CD9 and Flotillin-1 and with αSyn in raw plasma.

In parallel, we also show (2) that our workflow identified two new promising candidates for IP of plasma EVs of neuron-and astrocyte-and one of oligodendrocyte-origin. Summary/Conclusion: EVs may have great potential as NDD biomarkers but careful characterization is required. Moreover, by the identification and validation of CNS-enriched membrane protein candidates, IP of cell-type specific EVs appears as promising and feasible strategy. Funding: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

Vi Tran 1 ; Takamasa Ishidome 2 ; Yuki Marutani 2 ; Masayuki Yamane 2 ; Sadamu Ozaki 3 ; Kodai Sasamoto 4 ; Naoko Imawaka 2 ; Ryo Ukekawa 2 ; Takahiro Nishibu 2  FUJIFILM Wako Chemicals Europe GmbH, Neuss, Germany;  FUJIFILM Corporation, Amagasaki-shi, Japan;  FUJIFILM Corporation, Minato-ku, Japan;  FUJIFILM Wako Pure Chemical Corporation, Amagasaki-shi, Japan Introduction: Extracellular vesicles (EVs) released from mesenchymal stem cells (MSCs) are expected to be used for the treatment of various diseases. Ultracentrifugation and size exclusion chromatography (SEC) are commonly used for EV purification. However, these methods are not suitable for mass purification of EVs. The phosphatidylserine (PS)-Tim4 affinity method enables to purify PS-positive EVs with high purity and minimum damage. In this study, we verified the usefulness of the PS affinity method for the isolation of EVs from MSCs by using the PS affinity method and evaluated their biological activities. We also developed a Tim4 column-based mass purification method. Methods: Bone marrow MSC-derived EVs were recovered with Tim4 protein-immobilized magnetic beads and then eluted with buffer containing EDTA. The amount of EVs was evaluated by using ELISA capturing and detecting the EV markers CD9, CD63, or CD81. The anti-inflammatory and anti-fibrotic activities of the EVs were evaluated by the in vitro system. Tim4 column was prepared by immobilizing Tim4 on the resin and packed in a column. Results: More than 80% of EVs secreted in the medium of the MSCs was reactive to Tim4 beads and were isolated in high purity. Only a smaller portion (c.a. 10%) of the EVs were detected in the Tim4 non-reactive fraction. The anti-inflammatory and antifibrotic activities of the Tim4 reactive EVs fraction was higher than those of the EVs obtained by the ultracentrifugation method. The column purification technique also recovered EVs with a similar efficiency as the magnetic bead-based technique. Summary/Conclusion: The Tim4 protein based PS affinity method was a convenient and effective technique to isolate biologically active EVs from MSC culture supernatant. We are aiming to apply it to the purification of EVs derived from therapeutic MSCs in a larger scale. Introduction: The most severe outcome of SARS-CoV-2 infection causing COVID-19 lies in its ability to trigger a cytokine storm leading to acute respiratory distress syndrome (ARDS) with high mortality. Timely detection of the onset of this syndrome could significantly impact the treatment and clinical outcome of critically ill patients. Cytokines can be released in soluble form and/or associated with extracellular vesicles (EVs), which might interfere with accurate detection. In this in vitro study, we aimed to mimic a time-kinetic cytokine release response induced by COVID-19, and evaluate the detection of relevant cytokines that are present in different formats. Methods: We grew a 3D culture of the human Mono Mac 6 cell line in a FiberCell hollow-fiber bioreactor and stimulated these monocytes with SARS-CoV-2 S1 spike protein. Spike protein is responsible for cell entry and inflammatory response of the SARS-CoV-2 virus via ACE2 and TLR4 receptors, which were confirmed to be highly expressed on the cell membrane using flow cytometry. After stimulation, conditioned cell medium (CCM) was harvested at regular intervals for 48 hours, and partially Introduction: Extracellular vesicles (EVs) are membranous structures derived from cells and play an essential role in intercellular communication via transfer of bioactive proteins, lipids and RNAs. Both, normal and pathological cells are able to release various types of EVs with different physiological properties, functions and composition. In general, EVs are classified based on their biogenesis to the three main classes -apoptotic bodies, microvesicles and exosomes, which have different size and biogenesis. The proper definition and characterization of the subtypes of EVs varies in the literature, therefore in this study the collective term EVs is used. One of the great sources of EVs is peripheral blood plasma, as it is easily collected and does not usually cause excessive discomfort for patients. However, blood plasma contains large amounts of soluble proteins and aggregates which may cause a problem concerning of clinical grade EVs production in sufficient quantity, high efficiency and without impurities. This work is focused on the isolation of EVs from human plasma from healthy donors and osteoarthritis patients. Osteoarthritis (OA) is a disease of the entire joint including structural changes in articular cartilage, subchondral bone, synovial membrane and muscles around the joint. It is known that EVs reflect the physiological and pathological condition of cells and may operate as an effective tool for biomarkers diagnosis of OA. Methods: qEV Izon columns based on size exclusion chromatography were used for EVs isolation. Isolated exosomes were further identified by vesicle size using NTA analysis. Subsequent qualitative and quantitative analysis using flow cytometry, western blotting and ELISA methods were applied for identification of typically surface markers (CD63, CD9, CD81, TSG101, HSP70) expression. Results: Results showed the successful preparation and characterization of EVs from human plasma which were confirmed in terms of their size and protein components. NTA analysis showed isolated particles in the size range of 30-150 nm. The presence of EVs markers was different between studied groups. Summary/Conclusion: Obtained results suggested potential usage of EVs in the OA diagnostic. Introduction: There is a great need for reference materials for EV analyses i.e for calibration purposes and comparison of data from different laboratories. One of the special needs is in clinical flow cytometry in EV therapy development. Most artificial nanoparticles used for calibration are made from synthetic material such as polystyrene or latex, having a different refractive index than natural EVs. As red blood cells (RBC) possess a rich source of microparticles RBC concentrates offers an alternative to current reference materials. Methods: RBCs are pelleted and washed from leukocyte reduced erythrocyte concentrates with gentle centrifugation steps. RBC degradation is performed either with sonication or extrusion with syringe-driven single-use polycarbonate extruders with pore sizes of 1000 nm to 200 nm. Ghosting of RBCs is done with repeated freeze-thaw treatment followed by washing of RBC membranes with centrifugation. CD235a intra-and extracellular fluorescent antibodies have been used for immunostainings of RBCs and RBC nanoparticles. Results: RBC degradation with sonication seems to produce particles with more variable size and morphology than extrusion. Production of nanoparticles from ghosted RBCs may result in a purer end product, but it seems that cytoskeletal proteins, such as spectrin, may support forming of nanoparticles during degradation. However, the mass of non-encapsulated proteins should be removed from the sample afterwards to ensure high purity of reference material. Summary/Conclusion: As only a small amount of RBC material from collected whole blood is required for abundant reference material production, artificial RBC-EVs may offer a new product for blood services. The use of RBCs will not only allow the production of EV-like particles with natural lipid composition but also offers the possibility to use RBC specific marker (CD235a) as an internal standard. Funding: Business Finland ecosystem funded project EVE Academy of Finland -GeneCellNano Flagship

Giulia Silvestrelli 1 ; Markus Rimann 2 ; Susanne E. Ulbrich 1 ; Mara D. Saenz-de-Juano 1  ETH Zurich, Zurich, Switzerland;  Zurich University of Applied Sciences (ZHAW), Wädenswil, Switzerland Introduction: Extracellular vesicles (EVs) in the bovine mammary gland are involved in physiology and pathology. To date, a 3D culture model to investigate their specific functions and properties in vitro is missing. We cultured bioprinted primary bovine mammary epithelial cells (pbMEC) grown at the air liquid interface (ALI) to closely resemble the real tissue and characterize the secreted EV. Methods: A layer of Geltrex™ followed by a layer of pbMEC were jetted by a 3DDiscovery bioprinter on top of a Transwell®. Cells were then kept under ALI conditions. Apical fluid was collected and EVs isolated by differential centrifugation, size exclusion chromatography and ultracentrifugation. The EVs isolation method was validated by electron microscopy and western blot. We analysed the EVs by tunable resistive pulse sensing (TRPS), isolated the EVs RNA and evaluated the RNA fragments length. Finally, we performed an RT-qPCR analysis for the mammary epithelial cell-specific miRNAs bta-miR-200c-3p and let7a-5p. Results: Despite the low initial volume of 20-30 μL of apical fluid, the number of particles measured by TRPS was 2.6x10ˆ6 ± 1.6 x10ˆ6. The RNA length of apical EVs was on the small RNAs range. Furthermore, we detected both miRNAs let7a-5p and bta-miR-200c-3p. Summary/Conclusion: Printed pbMEC cultured under ALI conditions produced EVs. Despite the low input material, the amount of EV in the apical fluid was sufficient for the detection of mammary epithelial cell-specific miRNAs. Our results suggest that this model could be suitable for studying mammary glands EVs in vitro Funding: ETH Research Grant ETH-53 16-1.

Fruits enriched in polyphenols, such as pomegranates, have gained attention due to their therapeutic properties and promising biological activities. Here, we report the presence of vesicle-like nanoparticles in pomegranate juice (PgEVs), confirm their biomedical potential and test whether pasteurization and lyophilization treatments affect PgEVs yield and biological functions. Methods: Juice was obtained manually and stored at -80 • C. Pasteurization was carried out at 62 • C for 30 min, and lyophilization was carried out for 24 h. PgEVs were isolated by differential centrifugation followed by SEC, and characterized using NTA and TEM, and LC-MS/MS (EV-Track ID EV220000). Wound-healing assays were performed in CACO-2 human intestinal epithelial cell line (ATCC®, HTB-37TM), and anti-inflammatory assays were performed on THP-1-XBlue™-CD14 human macrophages (InvivoGen), using the Quanti-blueTM reagent (Invivogen). PgEVs citotoxicity was assessed by MTT assays. All assays were performed on FBS-free culture media. Results: Vesicle-like nanoparticles were isolated and characterized. PgEVs isolated from non-industrial treated juice showed significant higher particle concentrations than Pasteurized and Lyophilized PgEVs, as confirmed by NTA and TEM. Pasteurization affected PgEVs integrity, while Lyophilized PgEVs maintained similar morphologies than non-treated PgEVs. Proteomic analyses identified 130 proteins in PgEVs, including suggested Plant-EV markers such as TET-8, Vsp proteins and Patellins. PgEVs showed no citotoxicity and powerful anti-inflammatory properties in LPS-stimulated macrophages. In addition, PgEVs showed significant wound-healing properties. Both Pasteurized and Lyophilized treated PgEVs maintained those properties, but the required higher concentration to achieve those effects. Summary/Conclusion: Vesicle-like nanoparticles derived from Pomegranate have anti-inflammatory properties and promote wound healing in vitro, demonstrating their important role in the biological effects related to this fruit juice. Industrial processing of the juice could affect the yield and integrity of the isolated PgEVs, as well as their functional properties. The bioactive properties can be maintained increasing the dosage. Investigations are underway to identify molecules and effectors responsible for those actions. Introduction: Extracellular vesicles (EVs) secreted by antigen presenting cells (APCs) carry accessory proteins like ICAM and major histocompatibility complex (MHC) molecules loaded with antigenic peptides. Such EVs can trigger humoral and cellular immune responses against pathogens and can be considered as natural vaccines. On the basis of these EV properties, combined to a technology to engineer EVs in vivo with native membrane proteins, we developed several candidate vaccines against different enveloped viruses, of which, retroviruses, arboviruses and coronaviruses. Methods: EVs were engineered to present the major membrane antigens of HIV, Chikungunya and SARS-CoV-2 viruses. In order to benefit from the adjuvant properties of EVs, we used an immunization protocol that combines DNA prime injections with an exosome boost. The DNAs encode viral membrane proteins that, when injected, are targeted in situ to the EVs, in order that the viral antigens are presented in an autologous immune environment. The exosomes of the boost, harbor the viral membrane antigens and are produced using cell cultures. Results: Using immunochemical and biochemical tools, we showed that the viral membrane proteins presented by these EVs are in fully native and mature conformation; thus, the engineered EVs mimic the shape of the different original viruses. Mice injected with Chikungunya and Coronavirus candidate vaccines and rabbits injected with HIV candidate vaccine developed strong humoral and cell mediated immunity with high levels of neutralizing antibodies. Summary/Conclusion: Engineered EVs will be the basis for a vaccine platform that will help fighting future viral pandemics: a simple exchange of the viral membrane protein antigen for that of the emerging virus will lead to an adapted vaccine. Funding: French government FUI grants

Hyungtaek Jeon; Kyung Youn Hong; Byong Seung Cho ExoCoBio Inc., Seoul, Republic of Korea Introduction: The development of a variety of drug delivery system has been active to deliver drugs to specific targets to reduce side effects of drugs and maximize efficacy to deliver the necessary amounts of drugs. EVs that load therapeutic cargos and deliver them to target cells can be a perfect drug delivery platform. Targeted anticancer drugs have been developed to adjust specifically on cancer cells, but treatment effects can be maximized if they can act more selectively on cancer tissues to be treated when administered in human body. We have developed an innovative ATEI™ platform technology of EVs engineered with a Fc binding domain which can interact to any kinds of antibodies and were able to confirm specific delivery to target tumor cells through co-administration with antibodies. In addition, it was confirmed that it plays an effective role as and anti cancer agent by loading drugs into EVs equipped with Fc domains or fusing various proteins including cytokines to directly target tumor cells or active immune cells to control the tumor microenvironment. Although the delivery of EV-mediated drugs is still in its infancy, it has low toxicity, low immunogenicity, and high engineering ability, which accelerate the development of targeted immuno-cancer drugs using EVs. Methods: Cells were prepared by transfecting HEK293 cells with a plasmid DNA including and Fc-binding domain. Stable cell lines were prepared through monoclonal isolation. After that, EVs were isolated and purified by our methods and then separated again through UC. A certain amount of the durgs was loaded into the EVs through incubation with the target antibody. After that, the results were confirmed through in vitro and in vivo experiments. Results: A novel scaffold protein possesses two functions simultaneously. 1) Function capable of binding to Fc binding region such as antibody(Universal Targeting). 2) To enable presentation by engineering an protein of interest to the N-terminal(Protein Delivery). In addition, it is possible to load chemical anticancer drugs into the EVs(Drug loading). By combining these three core technologies, it was shown that up to 44 times more efficient chemical or protein drug delivery become possible. Summary/Conclusion: Our ATEI™ technology is the world's first to utilize dual function based on EV delivery system. This platform enables the delivery of specific proteins, and when combined with antibodies, targeting to specific cells or tissues is possible. This novel engineered EVs are new concept of universal targeting because it can bind to proteins and antibodies having all FC-binding domains. In addition to efficient delivery using and antibody, additional protein drugs can be engineered to develop novel immuno-cancer durgs or immune-enhancing agents.

Etienne Lourdin -De Filippis; Noémie Greze; Maëlle Lachat; Katarzyna Polak; Delphine Merle; Robert Z. Mamoun; Bernadette Trentin Ciloa SAS, Montpellier, France Introduction: Extracellular Vesicles (EVs) can potentially deliver a variety of cargos to any type of organs. To achieve that, there is a need for EVs customized with chosen proteins on their surface, for specific organ targeting, and/or with a chosen therapeutic cargo. However, developing reliable and robust technologies to customize EVs for their use as therapeutic vectors in vivo is still a challenge. Methods: Our aim was to evaluate an existing technology (Patent WO2011/036416) that addresses naturally any protein cargo into the EV lumen. Originally, this technology targets a protein under the EV membrane. We adapted this technology for the reversible release of the protein into the EV lumen. To reach this goal, we use two proteins that interact together in a reversible way. The first one is a carrier protein (CP, patent in process), the second is the protein of interest. The CP was inserted in between two peptides i) a Src myristylated peptide for membrane anchoring and ii) a Pilot Peptide (CilPP) that interacts with ESCRT machinery and sorts the protein into EVs. Next, the cargo protein of interest, was fused to a CP interacting peptide (CPIP, patent in process), that allows its encapsulation inside the EVs. Results: NanoLuc (nLuc), Antares2 (a fusion of CyOFP1 and nLuc), and Oct4 proteins were used as cargos. Their targeting inside the EVs was assessed by Western blots, luminescence, BRET assays and by protection from Proteinase K digestion. The reversibility of the interaction between CP and CPIP to release the cargo was also tested. Summary/Conclusion: In conclusion, we demonstrated the robustness of a technology that addresses efficiently any protein cargo into EVs, either by direct interaction with EV membrane or by a potentially reversible interaction with a CP protein addressed under the EV membrane. The next step of this work will be to combine the cargo loading with specific cell targeting in order to develop a powerful tool for the EV-mediated delivery-based therapeutics.

Chair: Pascale Zimmermann -K. U. Leuven Introduction: Individually, cancer derived EVs as well as neutrophils and their release of NETs have been shown to aid in cancer progression and metastasis. Our study aimed to characterize EV and neutrophil interactions by quantifying neutrophil uptake of EVs and the subsequent effects EVs had on NETosis across two neutrophil subpopulations. Methods: Five cell lines were used in our study: non-transformed mouse mammary epithelial cells (NMuMG), non-metastatic (67NR) and metastatic 4T1 TNBC cell lines (parental (4T1-P), liver (2776), and lung (526) metastatic 4T1). EVs were isolated from conditioned media (CM) by ultracentrifugation and the integrity and size was confirmed by NTA. Immature low-density (iLDNs) and high-density neutrophils (HDNs) were isolated from tumor-bearing Balb/c mice by density centrifugation. An optimized concentration of isolated EVs were co-cultured with either iLDNs or HDNs prior to fixation and immunofluorescent staining for NETs. NETosis was quantified based on the resulting microscopy images. Results: In agreement with previous findings, iLDNs showed increased NETosis compared to HDNs. Indeed, iLDNs cultured with EV-depleted CM from metastatic cell lines (4T1-P, 2776, 526) underwent more NETosis than HDNs under the same conditions. For both iLDNs and HDNs, the highest NETosis incidence was when the neutrophils were primed with EVs derived from the 2776 and 526 metastatic cell lines. In comparison, CM and EVs from non-tumorigenic NMuMG cells did not induce NETosis beyond background levels. Summary/Conclusion: Our findings suggest metastatic cancer derived EVs can promote NETosis to a greater extent than EVs from non-metastatic cells and that iLDNs are more prone to NETosis in response to metastatic cancer derived factors. Ongoing experiments aim to quantify neutrophil uptake of EVs derived from cell lines expressing fluorescent membrane-localizing proteins.

Introduction: Helium inhalation induces cardioprotection against ischemia/reperfusion injury, of which cellular mechanism is not fully elucidated. Extracellular vesicles (EVs) mediates cardioprotective mechanisms, but their function in heliumconditioning (HeC) is not elucidated yet. Fibroblasts are key regulators of post-ischemic remodelling, therefore they may take part in HeC-induced cardioprotection, as well. Therefore, we aimed to test, how HeC affects cardiac fibroblasts and if their HeCinduced EVs or other secreted factors mediates remodelling of the cardiac tissue. Methods: Neonatal rat cardiac fibroblasts (NRCF) were exposed to glucose deprivation and HeC rendered by four cycles of 95% helium + 5% CO2 for one hour, followed by one hour of normal culturing conditions. 40 hours later, NRCF migration was analyzed and Western Blot and quantitative PCR were used to analyze the expression of fibroblast to myofibroblast transformation markers. From serum-free cell supernatant, medium-sized extracellular vesicles (mEVs) were isolated with differential centrifugation and characterised with WB, transmission electron microscopy and nanoparticle tracking analysis. The supernatant of HeC-treated NRCF was transferred to naïve NRCF or immortalized human umbilical vein endothelial cells (HUVEC/TERT2) and migration and in vitro angiogenesis assay was performed. Results: HeC accelerated the migration of NRCF. Meanwhile, HeC did not increase the expression of myofibroblast markers. HeC tended to decrease mEV secretion of NRCFs, but supernatant of HeC-NRCF neither accelerate the migration of naïve NRCF, nor affect the angiogenic potential of HUVEC/TERT2. Summary/Conclusion: Since HeC increased the migration of NRCF but HeC-NRCF mEVs did not affect the function of remote cells, HeC may exert its cardioprotective effect via NRCFs, but does not affect cardiac remodelling remotely via NRCF mEVs.

Introduction: Physical activity is important for prevention and treatment of life style diseases. During exercise, contracting skeletal muscles release bioactive factors including extracellular vesicles (EVs) that can communicate with other cells and mediate beneficial effects. In the present study, we have examined how electrical pulse stimulation (EPS) of primary human myotubes, a model of exercise, affects the microRNA-and protein-cargo of secreted EVs. Methods: Muscle biopsies from obese patients with type 2 diabetes (n=6) were used to isolate, in vitro cultivate, and differentiate satellite cells into mature myotubes. Low frequency EPS was applied to myotubes for 24 h, and EVs were collected from serum free media the next 24 h. EVs were isolated, separated and concentrated into microvesicles (MV) and exosomes by combined centrifugation (17 000g) and filtration (0,22 um and 100 kDa filters). EV size and concentration were revealed by nanoparticle tracking analysis, tetraspanins analysed by flow cytometry, Hsc70/Hsp70 and calnexin by Western blotting and morphology visualized by transmission electron microscopy. EV content was studied by high resolution proteomics (LS-MS/MS) and transcriptomics (Affymetrix microarray) combined with bioinformatics using Ingenuity Pathway Analysis (IPA). Results: Size, concentration and CD63-and CD81-levels of MV and exosomes were unaffected by EPS. The protein content of MV and exosomes was clearly different, and they both showed significantly changed protein levels after EPS. Whereas the microRNA content of MV was basically unchanged by EPS, the exosomes appeared with a significantly altered microRNA pattern. IPA revealed that pathways involved in cell growth and protein synthesis were the most affected by EPS.

Introduction: Hepatocellular carcinoma (HCC) is the most common primary liver cancer; it represents the 6th most frequent tumor in the world and the 4th leading cause of cancer-related death. Due to the lack of efficient therapies and the difficulty to detect the pathology during early stages, HCC has a poor prognosis. Today, the immunotherapy represents the first-line treatment for advanced HCC with beneficial effect on overall survival. Despite this therapeutic advance, clinical data suggest that immunotherapy could be less effective in patients with β-catenin-mutated HCC. These tumors are characterized by an environment devoid of immune infiltrates, leading to resistant-immunotherapy tumors. However, the role of ß-catenin promoting this immune escape and leading to tumor cells trigger immunosuppressive cascades is not yet fully understood. Our project focuses on the role of ß-catenin signaling in tumor/immune cells communication through extracellular vesicles (EVs). Methods: Using transcriptomic analysis, we showed an alteration of gene expression involved in EVs machinery in HepG2 cells upon knock-down of mutated ß-catenin. We also revealed by nanoparticle tracking analysis and flow cytometry, a modulation of EVs secretion when β-catenin is mutated. Results: We further identified two target genes of the EVs machinery, RAB27A and SDC4, whose expression is ß-catenindependent. Thus, these results suggest that ß-catenin mutations inhibit EVs formation and/or secretion in liver tumor cells. As EVs and their contents are main factors for intercellular communications, we now hypothesized that the decrease of EVs production could lead to defective recruitment of leukocytes, making these tumors poor in immune infiltrates and resistant to immunotherapy. Summary/Conclusion: Our results provide a new knowledge on the impact of ß-catenin mutations on EVs biogenesis. This may allow the development of a new tool from liquid biopsies to stratify HCC patients for immunotherapy response.

Chanel Mosby-Haundrup; Melissa Jones University of Florida, Gainesville, USA Introduction: Human noroviruses (HNoVs) are the leading cause of gastroenteritis. An incomplete understanding of HNoV pathogenesis has hindered development of effective antivirals. Due to difficulty in culturing HNoV in vitro, murine norovirus (MNV) is used as a surrogate virus. It was recently shown that the interaction of norovirus and Enterobacter cloacae (E. cloacae) results in gene expression changes and increased production of bacterial extracellular vesicles (bEVs). Proteomic analysis of E. cloacae bEVs saw significant differences in protein content between bEVs produced in the presence and absence of MNV. Based on these findings, our hypothesis is that the presence of MNV will result in changes to the bEV lipidomic and metabolomic profiles. Methods: E. cloacae was incubated with MNV and grown to produce bEVs. Low speed spins pelleted bacterial cells and the supernatant underwent sequential ultracentrifugation steps and 0.2μm filtration to recover bEVs. The bEVs were sent off for global lipidomics and metabolomics. Results: Significant differences were found in the lipidomic and metabolomic profiles of the bEVs in the presence of MNV compared to controls. An increased abundance of lipids in the phosphatidylethanolamine (PE) class are of particular interest as increased PE lipids have been seen in hypervesiculation mutants and are thus consistent with previously published data demonstrating that MNV increases vesicle production. Summary/Conclusion: The changes in the bEVs lipidomic and metabolomic profiles support changes previously seen in the protein cargo of the bEVs. The data also suggests that the presence of MNV alters the biogenesis pathway for E. cloacae bEVs whereby bEV formation shifts from explosive cell lysis to membrane blebbing. These findings will help to further the understanding of how norovirus interacts with commensal bacteria and lead to a better understanding of norovirus pathogenesis as well as how commensal bacteria respond to external stressors through bEV production. Funding: R21 NIH grant# R21AI140012 Pilot Grant funded by the Southeastern Center for Integrative Metabolomics

Apolonija Bedina-Zavec 1 ; Rebeka Podgrajšek 2 ; Ana Špilak 2 ; Maja Jamnik 2 ; Matic Kisovec 1 ; Gašper Šolnič 2 ; Nejc Petrišič 2 ; Veronika Kralj Igič 3 ; Gregor Anderluh 2 ; Marjetka Podobnik 1  National Institute of Chemistry, Department of Molecular Biology and Nanobiotechnology, Ljubljana, Slovenia, Ljubljana, Slovenia;  Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, SI- Ljubljana, Slovenia, Ljubljana, Slovenia;  Biomedical Research Group, Faculty of Health Sciences, University of Ljubljana, SI- Ljubljana, Slovenia, Ljubljana, Slovenia Introduction: Listeriolysin O (LLO) is a toxin of the intracellular pathogen Listeria monocytogenes, that forms pores in cholesterol-rich lipid membranes of host cells. Its action depends on pH. The pH optimum of LLO is pH 5.5, a condition found in late endosomes, while it can bind to the membrane,form pores and damage cells even at neutral pH. Our group generated the LLO mutant Y406A whose activity at room temperature is even more dependent on pH than the activity of the wild type (LLO wt). We compared the effect of LLO wt and LLO Y406A on various cancer blood cells with the effect on healthy cells isolated from blood. Methods: The cell lines K562 and Raji were used. PBMCs were obtained from the blood of healthy donors (with informed consent and according to the study protocol approved by the National Medical Ethics Committee (0120-21/2020/4)). Cells were incubated with LLO or its mutant Y406A for 30 min at 37 • C. Cytotoxicity was measured by quantification of cellular membrane integrity (propidium iodide and trypan blue staining) and by cell viability assay (Presto Blue). ROS (reactive oxygen species)-generation activity and the amount of ATP in cells were also measured. EVs were isolated by differential centrifugation. The concentration of larger EVs in the samples was measured by flow cytometry using Annexin-FITC. The concentration of smaller EVs in the samples was measured by DLS.

The results showed that the toxicity of LLO Y406A to cancer and healthy blood cells was very low at physiological pH (7.4). Treatment with LLO Y406A resulted in reduced cell viability by 20% at concentrations 300 to 7000 times higher than treatment with LLO wt. In addition, LLO wt and LLO Y406A were found to be more toxic to cancer blood cells than to healthy blood cells under physiological conditions. Concentration of LLO wt needed to kill 20 % of cells is 2 to 20 times higher for healthy cells than for cancer cells. Concentration of LLO Y406A needed to kill 20 % of cells is 2 times higher for healthy cells than for cancer cells. As pH decreases, the activity of both proteins continues to increase. At pH 6.5, concentration of LLO wt needed to kill 20 % of cells is 4 times higher for healthy cells than for cancer cells; and concentration of LLO Y406A needed to kill 20 % of cells is 3 to 15 times higher for healthy cells than for cancer cells. Increased extracellular vesiculation in response to LLO wt and LLO Y406A was detected at protein concentrations 10 to 100 times lower than initial cell death. LLO wt and LLO Y406A did not cause an increase in ROS, but LLO wt caused an increase in ATP levels. Summary/Conclusion: The differences in the toxicity of LLO wt and LLO Y406A to cancer and healthy cells, as well as the increased activity of proteins at pH 6.5, suggest that both LLO wt and LLO Y406A are of potential interest for stimuli responsive applications and cancer treatment. Funding: Slovenian research agency, P1-0391. Inserm, CNRS, Sorbonne Université, CIMI-Paris,  Paris, France, paris, France;  Centre de Recherche en Cancérologie de Marseille, Marseille, France;  K. U. Leuven, Leuven, Belgium Introduction: Exosomes, small extracellular vesicles of endosomal origin, are master regulators of cell-to-cell signaling in physiology and disease. Exosomes are highly enriched in tetraspanins and syndecans, the latter occurring mainly in proteolyticallycleaved form. While both protein families are membrane scaffolds appreciated for their role in exosome formation, composition and activity, we currently ignore whether tetraspanins and syndecans work together to control exosome composition. Syndecans can regulate the compartmentalization of tetraspanins (and associated cargos) into exosome [1] . Methods: Small extracellular vesicles from MCF7 breast cancer cells were isolated by serial ultracentifugation and analyzed by proteomics and nanoparticle tracking analysis. The expression of proteins of interest were modulated using loss-and gain-of function experiments and the effects were further analyzed by immunoblots, biochemical analysis and confocal microscopy. Results: We showed that Tetraspanin-6 associates with syndecan-4 and acts as a negative regulator of exosome release by supporting the lysosomal degradation of syndecan-4 and syntenin [2] . Strikingly, our recent data show that syndecans control the balance between cellular and exosomal ADAM10, a sheddase regulating a plethora of signaling receptors implicated in neurodegenerative diseases, inflammation and cancer. Summary/Conclusion: Our work highlights various mechanisms by which syndecans control the biology of exosomes. These findings clarify our understanding of the molecular determinants governing exosome formation, composition and heterogeneity. This would help to rationalize their use as source of biomarkers and therapeutic tools.

Introduction: Secretion of RNAs in extracellular vesicles (EV) is a recently recognized form of cell-to-cell communication in which RNAs can change gene expression of recipient cell. Some RNAs are preferentially enriched into EVs, but mechanisms controlling this specific sorting remain poorly understood. Here, we investigated the impact of mutations on NRAS, BRAF, and TP53 genes on the non-coding RNA content of melanoma small EV (sEV). Methods: Melanoma cell lines established from local patients with different mutational status were cultured in EV-depleted media for 24h. sEV were isolated from culture supernatant using differential ultracentrifugation and characterized for size and concentration by transmission electronic microscopy and Tunable Resistive Pulse Sensing experiments. The expression of sEV marker proteins were assessed by western blot. Profiling of non-coding RNAs (ncRNAs) were performed by next-generation sequencing for microRNAs (miRNAs), and by micro-array for long non-coding RNAs (lncRNAs). Enriched RNA motifs in sEV and sequences recognized by RNA binding proteins were identified by bioinformatic analyses. Sam68 protein quantification was performed by western blot. Results: We showed that TP53-mutated cells release lncRNAs and miRNAs that are distinct from those secreted by TP53 wildtype melanoma cells, whereas BRAF and NRAS mutations did not modulate the RNA cargo of sEV. Furthermore, we found that long and small ncRNAs enriched in TP53-mutated sEV share a common sequence motif, highly similar to the RNA binding motif of Sam68, a protein interacting with hnRNP proteins. Finally, we found that Sam68 protein is enriched in TP53-mutated melanoma cell lines, leading probably to this selective sorting. Summary/Conclusion: Our results support the existence of cellular mechanisms for selective export of different RNA classes into small EVs depending on the TP53 mutational status of the cell. sEV-derived nucleic acids could provide clinically relevant information on cancer progression or other pathophysiological features. "Giulio Natta" (SCITEC), Milan, Italy, Milano, Italy Introduction: Extracellular Vesicles (EVs) based diagnostic practices must comply with the putative clinical value of low to very low abundant EV sub-types within complex media. In this regard, the enormous diagnostic potential of EVs could be unleashed by the extraordinary sensitivity offered by digital biosensing platforms, namely capable of reaching the single molecule detection level. As far as immune-phenotyping of EVs coupled to digital readout, two platforms are finding their place in the spotlight: the Single Particle Interferometric Reflectance Imaging Sensing (SP-IRIS) and the Single Molecule Array technology (SiMoA). Methods: EVs from human Cardiac progenitor Cells (CPCs) isolated by Tangential Flow Filtration were characterized by Western Blotting (WB), Nanoparticle Tracking Analysis (NTA) and Cryo-Electron Microscopy. The sample was used to provide a side by side multiparametric comparison of the two platforms by studying the abundance of CD9/CD63/CD81. Monoclonal antibodies anti-CD9, anti-CD63 and anti-CD81 were selected for this work and, for a fair comparison of data, applied to both SP-IRIS and SiMoA platforms using custom functionalization protocols of chips and beads respectively. Results: Calibration curves were generated with different assay schemes. After assessing that the tetraspanin monoclonal antibodies used in this work are not cross-reactive to mouse, to promptly compare the two digital platforms, we mimicked a liquid biopsy by spiking the human CPCs -EVs into mouse serum. The lowest detectable concentration of EVs were determined leading to a limit of detection (LOD) of 10ˆ8 EV/mL for the SP-IRIS platform and 10ˆ7 EV/mL for the SiMoA instrument showing that both platforms sensitivity is affected by sample matrix effect (1 order of magnitude higher LODs compared to spiking in PBS). Summary/Conclusion: Despite the higher sensitivity demonstrated by SiMoA platform compared to SP-IRIS in this assay, careful consideration of multiple features such as reading time, multiplexing, dedicated software and additional information provided by SP-IRIS (size, biomarker co-localization) have to be considered. 

Tarlan Esalmi Arshaghi 1 ; Jerry Clifford 2 ; Stephanie J Davies 2 ; Frank Barry 3  Regenerative Medicine Institute , National University of Ireland Galway, Galway, Ireland, GALWAY, Ireland;  Valitacell Ltd., Dublin, Ireland, Dublin, Ireland;  Regenerative Medicine Institute , National University of Ireland Galway, Galway, Ireland., GALWAY, Ireland Introduction: Extracellular vesicles (EVs) have attracted wide interest in recent years due to their potential applications in regenerative medicine, as biomarkers for disease, and because of their role in cell-cell interactions. However, EV isolation, quantification, and characterisation remain challenging in terms of purity and specificity as well as time-and cost-effectiveness. This work aims to develop a novel and high-throughput EV quantification tool based on the interaction between a fluorescently labeled probe and a specific EV surface component, using fluorescence polarisation (FP) for detection. The method analyses the change in the polarisation of emitted light between unbound and bound probes, with the observed polarisation in a mixture of labeled probe and target being proportional to the fraction of bound probe. This property of FP allows its use to quantify the amount of EVs in a sample. Methods: Two distinct strategies have been investigated, with probes targeting (i) specific EV surface markers (Tetraspanins e.g., CD63) for EV sub-population quantification or (ii) the EV phospholipid bilayer membrane for total EV quantification. Commercially available fluorescently labeled CD63 binding aptamers and proteins, and lipophilic dyes have been evaluated. EVs derived from HEK and MSC cultures have been purchased (HansaBioMed) or isolated through PEG-precipitation and ultracentrifugation, and particles quantified through Nano Tracking Analysis (NTA) or Imaging Flow Cytometry. Each probe candidate was incubated with EVs, and FP was measured over time using a Spark Cyto plate reader. Results: Tetraspanin specific (Anti-CD63) aptamer probes and lipophilic dyes demonstrated increased fluorescence polarisation in response to increasing EV concentration. Summary/Conclusion: This initial proof of concept supports the use of FP as a high throughput EV detection and quantification method, with the ability to provide both total and CD63+ve particle numbers. Funding: Marie Curie Career FIT Enterprise Ireland University of Gothenburg, Gothenburg, Sweden, Gothenburg, Sweden;  Krefting Introduction: Extracellular vesicles (EVs) are secreted by virtually all cells. Depending on their origin they appear as promising biomarkers in health and disease, especially if they can be recovered in plasma samples. However, there are many parameters that affect the detectability of EVs in given samples. Amongst others the anticoagulants being used as well as required processing steps in obtaining and preparing plasma for the EV analysis and especially the EV detection method of choice influence the diagnostic usability of EVs essentially. Methods: Recently, we have qualified imaging flow cytometry (IFCM) as a valid method for the phenotypic characterization of antibody labelled EVs at the single object level. In contrast to many other novel single EV characterization methods, IFCM does not require any major pre-and post EV labelling processes. Antibodies can be simply added to EV containing biofluids and analyzed following a typically 60 minutes lasting incubation period. Using a panel of 20 different antibodies and applying our optimized IFCM analysis strategy, we now have investigated impacts of conventionally applied processing steps, i.e. the centrifugation step regularly applied for the cell and debris clearance of applied plasma samples, as well as that of the eight most frequently used anticoagulants on the detectability and the phenotypic appearance of EVs within the plasma of healthy donors. Results: Our results demonstrate that the speed of the clearance centrifugation step (2, 000, 2, 500, 3 ,800 x g centrifugation, or following Ficoll gradient centrifugation) do not recognizably affect the composition of detected EV populations. In contrast, the choice of the anticoagulant significantly impacted the composition of the different EV populations. While we hardly observed any impact on myeloid cell derived EVs, e.g. CD16+ or CD71+ EVs, the abundance of CD9+, CD41+ and CD61+ EVs, assumedly platelet-derived EVs, was significantly affected. Most CD41+ or CD61+ EVs were recovered in serum or, when Heparin was used as anticoagulant, the lowest amount was detected in EDTA containing plasma. In contrast, HLA-ABC+ and HLA-DR+ EVs had lowest abundances in serum while with all other anticoagulants no differences in quantity are detected. Summary/Conclusion: Thus, our analyses demonstrate significant impacts of anticoagulants on the detectability of certain EV types, especially on PL-derived EVs.

Introduction: Introduction. Rigor and reproducibility are major issues for science in general, and the EV field in particular. It is increasingly appreciated that the key to data interpretation lies in the details of the methods used to produce the data. Single vesicle analysis using flow cytometry is a case in point, with literally hundreds of different ways to prepare and measure a sample using a flow cytometer, and the details of those methods and validation of their performance are required for understanding the data. The recent MIFlowCyt-EV guidelines address this need by providing a checklist of issues that must be addressed to enable data interpretation and sharing. Here we illustrate the reporting an EV FC method and results in a MIFlowCyt-compliant manner. Methods: Methods. We measured individual EVs in culture media and plasma by single vesicle flow cytometry using a commercial flow cytometer and assay kit. Preanalytical steps included pelleting of cells, dilution, and/or concentration by ultrafiltration. The flow cytometers were qualified and calibrated using calibration beads to enable data reporting in standardized units. EV concentration, size, and surface cargo was measured by single vesicle flow cytometry (vFC) which incorporates validated reagents reagents, essential controls, and standardized protocols and data analysis. Results: Results. We report size, concentration, and tetraspanin number per EV in standard units on EVs from blood and cell culture. Calculation of these parameters is described within the context of the sample preparation and assay protocols. Example calibration and control data that might be included in supplementary methods as suggested by the MIFlowCyt-EV ISEV position paper is reported. Data analysis protocols with an example of how to report a gating strategy and resulting data are illustrated. A walkthrough of the data repository and best practices for use are described. Summary/Conclusion: Conclusion. Single EV analysis using flow cytometry offers great potential for understanding the diverse origins and functions of EVs, but only if performed in a way that allow assay specificity and sensitivity to be transparently documented. The MIFlowCyt-EV guidelines provide a framework that enables EV FC measurements to be reported in the context of essential experimental details, calibrations, and controls. Many of the concepts and procedures developed for EV FC are extensible to other single EV counting, sizing, and cargo analysis methods.

John P. Nolan; Erika Duggan Scintillion Institute, San Diego, USA Introduction: Introduction. EVs carry molecular cargo from their cell of origin, but the mechanisms of cargo selection and loading into EVs are not well understood. We used quantitative single cell and vesicle flow cytometry to measure membrane protein expression on cells and EVs. Methods: Methods. PC3 cells were cultured, media collected, and EVs concentrated using ultrafiltration (100K MWCO). Cell surface markers were measured by flow cytometry (FC). EV concentration, size, and cargo were measured by single vesicle flow cytometry (vFC). Instruments were calibrated and intensity reported in units of antibodies per cell or EV. Results: Results. PC3 cells express surface markers at high (>250K median copies/cell: CD71, CD29, CD44, CD54), medium (50K-250K copies: CD9, CD63, CD49f) and low (< 50K copies: CD81, EPCAM, EGFR, STEAP-1) abundances. Cell permeabilization reduced staining for CD9 and CD81 (due to disruption of the plasma membrane), and increased staining for CD63 and STEAP1 (due to accessibility of internal antigen). EVs expressed detectable (>∼10 PE MESF) CD9, CD63, CD81 and CD29, with a fraction (∼50%) also staining with AnnV. Expression was proportional to EV surface area, with surface densities ranging from a background of ∼10 molecules/um2 to >1000 molecules/um2 for high abundance targets. Several high abundance markers (CD71, CD44, CD54) were not detectable on EVs, suggesting differential packaging of cell surface cargo into released EVs. CD63 was expressed at low abundance overall, but a subset of smaller EVs (< 100 nm) expressed CD63 at high surface density (∼1000 um2). Summary/Conclusion: Conclusions. We find that the abundance and surface density of cargo on vesicles can be higher or lower than on the cell of origin. Some abundant cell surface molecules (CD71, ICAM, CD44) were undetectable on EVs, while others (CD9, CD81, CD29) were present at surface density similar to cells. CD63 was present at high density on smaller EVs, consistent with enrichment of CD63 on small exosomes formed inside the cell.

A single aliquot of diluted exosomes was measured six times in succession. The overall particle concentration was 2.63e7 p/mL with a standard deviation of 1.13e6 p/mL. This reflects a coefficient of variation of 4.31%. In these measurements, an average of 1640 particles were observed in each measurement. Concentration for each size range was obtained and the concentration uncertainty of each bin estimated from the repeats. Plotting the uncertainty as a function of number of analyzed particles shows an approximate square root dependence. Summary/Conclusion: Repeated measurements can be used to estimate measurement uncertainty for stable dilutions of EV's. The observed coefficient of variation, 4.3% is close to but somewhat higher than the expected from the number of particles counted and Poisson statistics. The predicted value is 1/sqrt(1640) or 2.5%. Repeatability for the concentration in each size class roughly followed Poisson statistics. This indicates that repeatability improves in proportion to the square root of measurement time.

Dimitri Aubert 1 ; Alice Law 1 ; rebecca J. Lees 1 ; Rob Tempest 1 ; Ben Peacock 2  NanoFCM, Nottingham, United Kingdom;  NanoFCM Co., Ltd., Nottingham, United Kingdom Introduction: Extracellular vesicles (EVs) are often co-isolated with other non-EV particles of similar density or size, especially when derived from biofluids. Common characterization of size and/or concentration may provide total particle information without specificity towards EVs. Many popular advanced techniques for EV characterization utilize fluorescent labelling to identify subpopulations of EVs. Methods: Nano-flow cytometry (nFCM) performed on a NanoAnalyzer has the benefit of elucidating both the total particle population as well as the total EV and/or specific EV subpopulations by detecting scattered and fluorescent light. Whilst antibody staining is an option for identifying specific targets, the use of EV compatible dyes allowing fast determination of EV concentrations within complex particle isolations will greatly facilitate research. Dyes used in this study include CFSE, Cell Mask, Memglow, PKH, and many more. Results: However, currently there are caveats to be aware of for some commercially available dyes. nFCM allowed for in depth comparison of the efficacy of several dyes identifying capabilities such as, high intensity fluorescence, self-quenching and low background, even distribution and correlation with size. Several limiting factors were also identified including, desorption and leeching, micelle formation, and poor specificity. Summary/Conclusion: Direct comparison of these commonly available dyes will help to inform researchers as to the most applicable dyes for specific scenarios, forming a roadmap for future EV research. University, Washington, USA;  Genotypic Technology Pvt Ltd., Bangalore, India;  King Edward Memorial Hospital, Pune, India;  Children's National Hospital, Washington, USA Introduction: Introduction: Maternal nutritional status and other in utero exposures are associated with infant adiposity, childhood obesity, and adult cardiometabolic disease. We hypothesize that maternal adipocyte-derived small extracellular vesicles (ad-sEVs) carry miRNAs during early gestation that are associated with high infant adiposity via enhanced fetal adipogenesis. Methods: Methods: Ad-sEVs were isolated from maternal blood at 8 weeks gestation from a representative subset (n=28) of a maternal-infant cohort (IndiaGDM cohort, n>330; the study was approved by the ethics committee). Newborns were classified high (n=12) or low adiposity (n=16) according to the sum of skinfolds. EV RNA libraries were prepped and sequenced on Illumina NextSeq for about 10 million reads per sample. Data were analyzed in Partek™Flow™. Sequences (16-40nt) were aligned Introduction: Adipose tissue (AT) plays a critical role in the metabolic crosstalk between organs, relayed by adipokines which participate to obesity-associated complications. Recent studies evidenced AT-derived EV (AT-EV) as important mediators of obesity-associated metabolic dysfunctions. We performed AT-EV adipokine profiling to identify EV-associated factors that could relay EV metabolic effects. Methods: AT-EV were purified from conditioned media of visceral AT from lean or obese (ob/ob) mice by differential centrifugation. We isolated large (13K) and small (100K) AT-EV that were further characterized by a combination of biochemical, microscopical and physical methods. Results: We evidenced the ability of AT to secrete both large (lEV) and small (sEV) EV subpopulations, a secretion that is enhanced in obese mice compared to lean animals. Adiponectin was the most enriched adipokine in AT-EV under its oligomeric active forms. Adiponectin mainly distributed at the EV external surface for both AT-EV or plasma EV, as a result of unspecific adsorption of soluble adiponectin. EV-associated adiponectin maintained its insulin-sensitizing properties on target cells and its injection in high fat diet-fed mice prevented the animals from the development of insulin-resistance. Summary/Conclusion: Altogether, our results highlight EV as new original carriers of metabolic forms of adiponectin, that are likely to participate to its beneficial effects. Introduction: The composition of extracellular vesicles (EVs) is altered in many pathological conditions, and their molecular content provides essential information on features of parent cells and mechanisms of crosstalk between cells and organs. Metabolic Syndrome (MetS) is a cluster of clinical manifestations including obesity, insulin resistance, dyslipidemia and hypertension that increases the risk of cardiovascular disease and type 2 diabetes mellitus. We investigated the crosstalk between liver and adipocytes by characterising EVs secreted by primary hepatocytes isolated from Zucker rat model, and studied the effect they have on 3T3-L1 adipocytes. Methods: By using differential ultracentrifugation, density gradient, NTA, cryo-TEM, Western-blotting, Raman spectroscopy, proteomics and lipidomicss we perform a deep molecular characterisation of EVs secreted by lean and obese primary hepatocytes. In addition by using radio labelling we analyse the bio distribution of these vesicles, and by using metabolomics we study the effects that these hepatocytes-secreted vesicles produce on adipocytes metabolism. expression. These EV can carry Hb, heme, damage the endothelium and alter blood vessels. In T2D, reports diverge and it remains unclear whether RBC-derived EV vary in quantity or in characteristics. We studied intravascular hemolysis and EV shedding in T2D. Methods: We collected blood from 109 T2D patients and 65 controls, purified RBC, saved plasma. We studied RBC contents and membrane fragments. In plasma, we characterized heme-related absorbance (Hb, Heme) by spectrophotometry (Soret band) and circulating EV by cryogenic transmission electron microscopy, nanoparticle tracking (NTA) and flow cytometry (CD235a). We studied the association of these biomarkers with micro-and macro-vascular complications of T2D. We stimulated RBC vesiculation in vitro by calcium influx or shear stress. We characterized RBC EV phenotypes and compared their activity using tests of thrombin activation (adapted CAT assay), and endothelial oxidant stress stimulation. Results: Heme-related absorbance was increased in T2D plasma compared to controls (+57%) and more in obese diabetic (+27%). However, CD235a+RBC EV observed by FACS were not increased. T2D plasma EV, or those shed by T2D RBC (both types of stress), were all smaller (-27%) and rich in heme-related absorbance. In T2D, the raise in absorbance (+30%) was associated specifically to peripheral nerve injury, amongst all other vascular complication. CD235a+ RBC EV were not associated to any complication. In vitro, T2D RBC shed more EV then control RBC under the same stress. T2D RBC EV stimulated oxidant stress in endothelial cultures et thrombin activation in plasma, more intensely than control EV. Annexine-A5, a PS inhibitor, and hemopexin, a heme inhibitor, blocked these effects. Summary/Conclusion: T2D was associated to low grade intravascular hemolysis, with more heme and EV in plasma. Hemerelated absorbance could represent a novel circulating biomarker of peripheral nerve injury, whereas classical flow cytometry, sensitive to the largest EV, seems ill-adapted to demonstrate smaller EV in T2D, particularly those derived from glycated RBC. Therapeutic approaches limiting hemolysis of neutralizing RBC degradation products, like EV PS-neutalising molecules, could help protect blood vessels in T2D. Introduction: Extracellular vesicles (EVs) are secreted from many cell types and contain bioactive molecules, including proteins, messenger (mRNA), and microRNA (miRNA). Notably, placental EVs from gestational diabetes mellitus (GDM) pregnancies are enriched in miRNAs that regulate glucose metabolism. GDM placental sEVs decrease insulin-stimulated glucose uptake in primary skeletal muscle cells, suggesting that placental EVs regulate insulin sensitivity during pregnancy. Here, we determine the association between EV-associated miRNAs and changes in insulin sensitivity across gestation. Methods: Samples were obtained from a multicenter randomized controlled trial conducted from 2012 to 2014 [the DALI (vitamin D and lifestyle intervention for GDM prevention) lifestyle study]. EV-associated RNA were isolated from pregnant women (n=179, four times during pregnancy (716 total samples were analysed), longitudinal study design). Insulin sensitivity was determined using by Homeostatic model assessment (HOMA) index four times during gestation. Sequencing libraries were generated using the TruSeq® SmallRNA Library Prep Kit and processed for cluster generation and sequencing using NextSeq 500 High Output kit 75 cycles and Illumina NextSeq 500 sequencing platform. Results: A total of 2822 miRNAs were analyzed using a small RNA library, and a total of 17 miRNAs that significantly correlates with changes in insulin sensitivity across gestation. Clustering analysis identified a total of 12 (hsa.let.7a. 1.3p, hsa.let.7a.3.3p, hsa.mir.486.1.3p, hsa.mir.486.2.3p, hsa.mir.185.5p, hsa.mir.30c.1.5p, hsa.mir.30c.2.5p, hsa.mir.486.2.5p, hsa.mir.486.1.5p, hsa.mir.125b.1.5p, hsa.mir.125b.2.5p, and hsa.mir.183.5p), and 5 (hsa.mir.1260b, hsa.mir.101.1.3, hsa.mir.101.2.3p, hsa.mir.423.3p, hsa.mir.23b .3p) EV-associated miRNAs that positively and negatively correlates with HOMA across gestation, respectively. Summary/Conclusion: The mechanisms associated with maternal insulin resistance are not fully elucidated. We suggest that miRNAs encapsulated in EV might be involved in this phenomenon. This new information will help us better understand the maternal metabolic adaptation to pregnancy, critical for normal fetal growth and development. Introduction: Extracellular vesicles (EVs) including exosomes and microvesicles provide a means of communication for delivering bioactive cargoes between cells. In the past decade, EVs have been shown to contribute to the transport of proteins, lipids, mRNA, and microRNA. In vivo, embryos receive nutrients and specific signals through the oviduct to enhance embryonic development. Methods: Therefore, we aimed to isolate EVs from porcine oviduct epithelial cell (pOECs) that were primed with steroid hormones to mimic the in vivo conditions of reproductive cycle and studied their effects on the in vitro produced embryos development. pOECs were treated with formulated estardiol (E2) and progesterone (P4) combinations in two treatment groups: 50 pg/ml E2 + 0.5 ng/ml P4 (group H1), and 10 pg/ml E2 + 35 ng/ml P4 (group H2). The control group was not supplemented with hormones. Embryos were prepared after in vitro maturation and parthenogenetic activation. Embryos were randomly distributed into 4 groups: control, non-primed pOECs-derived EVs treated group (EV group), and two primed pOECs-derived EVs groups (H1 and H2 groups). EVs were isolated through targeted filtration commercial kits. Data were analyzed by ANOVA test and P < 0.05 was considered statistically different. Results: pOECs-derived EVs were supplemented with embryo culture medium after measuring the protein concentration (260.6 μ/ml, 284.3 μ/ml, 283 μ/ml, 279.6 μ/ml, for EV, H1 and H2 groups, respectively) . The number and concentration of the EVs was measured through nanoparticles tracking analysis. EVs uptake by the embryos was investigated after staining of the EVs with lipophilic fluorescent dye, PKH26. Results showed that EVs from hormone primed pOECs improved the blastocyst formation rate compared to the control group (20.3 ± 0.5 %, 22.8 ± 0.7, 25.0 ± 0.6, and 25.1 ± 1.1 %, for control, EV, H1 and H2 groups, respectively, P < 0.05). TUNEL assay showed that EVs-supplemented embryos contained less apoptotic cells when compared with the control group (11.8 ± 2.7 %, 6.9 ± 2.2 %, 6.09 ± 2.04 %, and 2.4 ± 0.7 %, for control EV, H1 and H2 groups, respectively, P < 0.05). Summary/Conclusion: In conclusion, EVs derived from pOECs cultured in hormonal conditions that simulate the in vivo environment have a positive effect on porcine blastocysts formation and reduced the embryonic cell apoptosis, which would improve the porcine in vitro embryo production such as cloned animals.

Introduction: A key challenge in editing the male germline is the delivery of gene editing tools into sperm's tightly packed DNA. To this end, biocompatibility and engineerability make extracellular vesicles (EVs) potential delivery vehicles. While EV-sperm interactions have been studied using dye-labeled EVs, results have been inconclusive. We thus investigated if a GFP-transgenic bovine fibroblast produced GFP-loaded EVs and whether the contents were delivered to sperm. Methods: EVs were collected from conditioned media of bovine GFP-transgenic fibroblast or WT-fibroblast by ultrafiltration using an Amicon Ultra Filter (100kDa). EV isolation was validated by: (1) nanoparticle tracking analysis; (2) CD9 protein expression; (3) transmission electron microscopy; and (4) protein concentration. GFP cargo of EVs was evaluated by plate reader and ELISA. 10μg/mL of WT and GFP EVs were incubated with sperm and motility and viability were then measured over 90 min. At 90 min sperm were washed, fixed for confocal imaging, and lysed for ELISA. Data were analysed in R via linear or mixed-effects models. Results: 6.5x109 EVs/mL enriched in CD9 were isolated from WT and GFP cells. We observed 37±19 and 42±33 ng of proteins in 1x106 EVs in GFP (n=11) and WT (n=6), respectively. GFP fluorescence in 1x106 EVs was higher in GFP (n=11) than WT (n=10) EVs (0.01±0.007 vs 0.006±0.001; p=0.006), which was confirmed by ELISA: 6±0.14 vs 2±0.07 pg/mL GFP in 1x106 EVs in GFP (n=9) and WT (n=6), respectively (p=0.005). Sperm viability and motility did not change with incubation nor time. GFP was detected in sperm (20±9.5 pg/mL of GFP in 0.5x106 sperm) incubated with GFP EVs, and not in WT (n=3, p=0.03). At 90 min all sperm incubated with GFP EVs showed GFP fluorescence by confocal analysis. Summary/Conclusion: GFP-transgenic fibroblasts produced EVs with GFP cargo. These EVs delivered GFP to sperm with no measurable impact on viability and motility. This work demonstrates the potential of EVs as non-invasive delivery tools for sperm. We are now validating methods to load EVs with Cas9-RNP complexes for testing sperm gene editing. Introduction: Extracellular vesicles (EV) are released from the prepubertal testis but potential roles in the development of the spermatogonial stem cell (SSC) niche are poorly characterised. Damage to the SSCs from gonadotoxic compounds, eg the chemotherapeutic cisplatin, can lead to aberrant spermatogenesis and male infertility. This scenario would typically occur in a prepubertal boy with cancer, for whom there are no viable options for fertility preservation, as they do not produce sperm. We hypothesise cisplatin alters EVs in the testis and these EVs negatively impact the remaining SSCs and the supporting Sertoli cells. Methods: EV characterisation from GC1-spg (mouse prepubertal SSC) and TM4 cells (mouse prepubertal Sertoli) was undertaken, growing cells in EV-depleted media, comparing cisplatin-treated cells to controls. Change in EV number was quantified using nanoparticle tracking analysis and impact on treatment-naïve recipient cells was assessed using in vivo cell imaging and detection of cleaved caspase-3. EV uptake and movement within target cells was examined using super-resolution microscopy. Results: Mouse SSCs and Sertoli cells treated with cisplatin release twice as many EVs as control cells (p=0.004). EVs released by cisplatin-treated cells have greater uptake in treatment-naïve cells (p=0.002) and show peri-nuclear localisation. We identified EVs released by cisplatin-treated Sertoli cells induced higher rates of apoptosis in treatment-naïve Sertoli cells vs control EVs. However, when incubated with treatment-naïve SSCs, they resulted in lower rates of apoptosis vs control EVs (p= 0.02). Summary/Conclusion: EVs released from prepubertal SSCs and Sertoli cells were significantly altered when incubated with cisplatin. Altered release, uptake and impact on treatment-naïve cells, are novel findings in the prepubertal testis. Damage to these cells in the testis may be a contributing factor to future male infertility and represent previously unidentified off-target effects of cisplatin. Funding: MPR is funded by a UK Medical Research Council Centre for Reproductive Health Grant No: MR/N022556/1.

Diabetes mellitus (DM) is one of the most common metabolic disorders. By 2030, it is expected that more than 300 million individuals will be affected by diabetes. Glucose transporter type 4 (GLUT4) is a transmembrane protein that is involved in the elimination of glucose from the bloodstream. In addition to GLUT4, several investigations have shown that interleukin-6 (IL-6) plays an important role in DM. Recent evidence has indicated that IL-6 is a key regulator that affects glucose homeostasis. Interleukin-6 (IL-6) is a pleiotropic cytokine that can be discovered in the secretome of MSCs. The aim of this study is to examine whether IL-6 that was found in the Mesenchymal Stem Cells (MSC)-derived secretome could increase the GLUT4 translocation through AMPK activation in the HepG2-insulin resistance model. Methods: MSC-derived secretome was produced by culturing the umbilical cord mesenchymal stem cells in a normoxic conditioned until it reach 80% confluency using DMEM High Glucose and supplemented with 5% Human Platelet Lysate. Upon it reached the confluency, the growth medium was discarded and changed with serum-free media, and incubated for 24 hours. The medium was collected and centrifuged for 500xg for 5 minutes and filtered using a 0.2μm filter membrane. The IL-6 concentration was confirmed using the MACSPlex Cytokine 12 kit. The insulin resistance model of HepG2 cells is established by adding 100nM of insulin for 24 hours. To detect the GLUT4 translocation. The insulin resistance model was fasted for 12 hours prior to the treatment using MSC-derived secretome for 24 hours. GLUT4 translocation was detected using immunofluorescence. HepG2 insulin-resistant model was fixed using methanol and cells were incubated with GLUT4 antibody-conjugated with Alexa488. Results: The treatment of MSC-derived secretome increased the GLUT4 translocation in the HepG2 insulin model compared to the control (p < 0.05). This result corresponds with previous studies that show the expression of the GLUT4 gene is downregulated in T2DM. The expression of GLUT4 is lowered in diabetic patients due to blood glucose buildup. IL-6 plays an essential role in regulating glucose homeostasis by activating the GLUT4 translocation through AMPK activation. Given the critical roles of IL-6 that were found in the MSC-derived secretome, it could be a treatment strategy against diabetes. Introduction: Prostate cancer (PCa) represents a major global health problem. PCa cells present metastatic organotropism primarily to lymph nodes (LN) and bones. Metastatic spread accounts for the deadliest phase of cancer progression. Previous studies from our laboratory have demonstrated a crucial role for melanoma-derived sEVs in LN and lung premetastatic niche (PMN) formation. In PCa, the mechanisms underlying early metastatic dissemination are still poorly understood. We hypothesize that PCa-derived sEVs distribute through the lymphatic vasculature promoting the re-education and reprogramming of the microenvironment in the LNs and bones, thus favoring early metastatic dissemination. Methods: To identify protein signatures associated with metastatic progression, mass spectrometry analysis was performed on tumor-derived sEVs from a panel of PCa models. We performed a bioinformatic study to select those candidates that were significantly enriched and were related to the PMN formation process. We have analyzed the biodistribution of PCa-derived sEVs in mice after footpad and orthotopic injection. SEVs were purified, fluorescently labeled, and then injected in mice. Biodistribution of sEVs was tracked and measured by multispectral imaging. We studied the main cell types taking up PCa-derived sEVs in the LNs by flow cytometry and immunofluorescence. In order to define the mechanisms involved in PCa early metastatic dissemination, we are analyzing the extracellular matrix and immune cell composition of LNs derived from patients and mouse PCa models by immunohistochemistry and flow cytometry. Results: We found that PCa-derived sEVs can reach the sentinel LN and the pelvic bone. Interestingly, sEVs derived from the LN metastatic model (C4-2B) reached more efficiently LNs, while those derived from bone metastasis (PC3) were significantly increased in the bone. We found that lymphatic endothelial cells (LECs), fibroblastic reticular cells (FRCs), and macrophages were the main cell types taking up sEVs in the sentinel LNs.

Proteomic analysis showed around 600 proteins differentially expressed when comparing sEVs derived from LNCaP and C4-2B lymph node metastatic models. We are currently analyzing the signatures associated to the highly metastatic model C4-2B. Summary/Conclusion: Our data show that sEVs derived from LN and bone metastatic models home more efficiently to lymph nodes and bone respectively, suggesting their involvement in PMN formation in these organs. Detailed analysis showed that LECs, FRCs, and macrophages are the main cell types in the sentinel LNs taking up sEVs. Our results support that PCa-derived sEVs play a role in PCa lymph node PMN formation. We are currently analyzing the main signatures associated with LN metastasis and the main changes in metastatic sentinel LNs. Introduction: Colorectal cancer (CRC) is the third most common tumor in the world frequently associated with liver metastasis causing unfavorable prognosis. A recent study performed in our laboratory has demonstrated that CRC small extracellular vesicles (SEVs) induce an epithelial to mesenchymal transition (EMT) of hepatocytes (heps) driving them to actively participate in the pre-metastatic niche formation, probably contributing to form a liver fibrotic microenvironment. Since 2D cell cultures partially reflect the structural complexity of the in vivo microenvironment to give more power to our functional model, we switched to use hepatocyte spheroids (HeSPHs), which can give us more proper information on the consequences of CRC_SEV-induced hepatocytes EMT on cell-cell interactions and extracellular matrix remodelling which can drive and support the invasion of tumor cells. Methods: We isolated SEVs from SW480 CRC cells, through differential centrifugation followed by ultracentrifugation. HeSPHs were obtained by seeding normal human liver cells (THEL-2) in ultra-low attachment 96 well plates. After treating HeSPHs with CRC_SEVs, we analyzed the modulation of expression of structural and functional hepatocyte markers. By co-culturing HeSPHs with SW620-GFP cells, we evaluated the ability of CRC_SEV to increase the invasion potential of tumor cells. Results: Our data on HeSPHs confirmed the ability of CRC_SEVs to alter the expression of hepatocyte structural and functional markers (ApoE, albumin and cytokeratins 8/18) observed in the 2D model. Moreover, we found that in CRC-SEVs-treated HeS-PHs the invasive capability of tumor cells increased, indicating that injured-SEV heps can have driving and supporting tumor liver colonization. Summary/Conclusion: Overall, the HeSPHs represent a promising model to study the role that tumor-derived SEVs can have in rendering heps able to actively drive the formation of an environment conducive to metastasis.

Walther Traberg-Christensen; Roisin Owens University of Cambridge, Cambridge, United Kingdom Introduction: Tumor cell-derived EVs (TDEs) have recently been reported to play an active role in tumorigenesis and metastasis owing to their ability to transmit oncogenes. One hypothesis is that TDEs contribute to metastasis by inducing the epithelial-tomesenchymal transition (EMT), characterized by the loss of barrier function in barrier tissue-forming cells. Methods: This study aims to elucidate the role and mechanisms of TDE uptake by investigating the process by which TDEs "infect" normal cells inducing EMT prior to metastasis. Current strategies for investigating the spatial and temporal aspects of EV uptake, often optical, lack the ability to obtain quantitative data in real time. We have shown that organic electronic devices based on the conducting polymer poly(3,4-ethylenedioxythiophene): poly(styrenesulfonate) (PEDOT:PSS) can be interfaced with biological systems of varying complexity allowing for quantitative real-time monitoring of biological interactions. The optical transparency of our PEDOT:PSS-based devices provides the unique advantage of dual transduction increasing the credibility of our platform and enriching the biological information obtained. Results: We have shown that CD63-positive TDEs derived from the triple-negative breast cancer cell line MDA-MB-231 can induce an EMT-like process in MCF-10A non-tumorogenic breast epithelial cells as demonstrated by changes to cell morphology, loss of apico-basal polarity, reorganization of filamentus actin, and gain of mesenchymal proteins, such as vimentin and Ncadherin. An integral feature of EMT is the dissolution of tight junctions and the consequential loss of lateral cell-cell adhesion leading to a phenotype with increased motility and invasiveness. This decrease in barrier integrity of the cell monolayer was monitored electrically using organic electrochemical transistors, gaining of truly quantitative insight into TDE-induced EMT with higher temporal resolution than conventional, orthogonal methods. Summary/Conclusion: These combinatorial optical and electrical measurements of TDE interactions with cell monolayers provide invaluable information for developing strategies that may inhibit TDE interactions by e.g. blocking specific surface markers, thereby preventing TDE-induced EMT with implications for preventing cancer metastasis. Moreover, the compatibility of electronic devices with microfabrication methods allows for high throughput studies. Funding: This work is funded by a research grant from King Abdullah University of Science and Technology and The Cambridge Trust.

Shelly Loewenstein 1 ; Fabian Gerstenhaber 2 ; Talya Kalfon 2 ; Stav Leibou 2 ; Osnat Sher 2 ; Guy Lahat 2  Tel Aviv Sourasky Medical Center, Tel-Aviv, Israel;  Tel Aviv Sourasky Medical Center, Tel Aviv, Israel Introduction: Peritoneal carcinomatosis is orchestrated by a reciprocal interplay between invading cancer cells and resident normal peritoneal cells. Within that process, angiogenesis constitutes an important control point of cancer progression. We investigated the role of mesothelial cells derived extracellular vesicles (EVs) in angiogenesis. Methods: Endothelial, mesothelial, and gastric cancer cell lines were used for in vitro experiments. Gastric cancer and mesothelial extracellular vesicles (EVs) were produced from cell cultures. Proliferation, migration, and invasion assays were used to evaluate the phenotypic behavior of treated endothelial cells. Matrigel tube-and plug-formation assays were used to demonstrate angiogenesis in vitro and in vivo. Angiogenesis protein array was used to identify proangiogenic proteins in mesothelial EVs. Finally, Lenti viral shRNA were used for gain and loss of function experiments to investigate specific signaling pathways. Results: Initially, we demonstrate a robust uptake of mesothelial and gastric cancer EVs by endothelial cells. We show that these vesicles enhance endothelial cell proliferation, migration and invasion and that mesothelial-derived EVs are more effective when compared with gastric cancer-derived EVs. We also demonstrate that mesothelial EVs significantly augment in vitro and in vivo tube formation. Angiogenesis array identified 43 differently expressed regulators of angiogenesis within mesothelial EVs. Based on the results of the array, we specifically investigated Angiopoietin-2 (Ang2) -Tie2 pathways. We show that the above noted effects of the mesothelial EVs were mediated by Ang2 through Tie2, PI3K, and Akt activation, whereas inhibition of Ang2 significantly decreased the pro-angiogenic effect of mesothelial EVs. Summary/Conclusion: We demonstrate for the first time uptake of mesothelial EVs by endothelial cells and the induction of angiogenesis by these EVs. We also demonstrate that these effects are mediated by Ang2 trough the PI3K/AKT pathway. Taken together, our data imply that mesothelial EVs are important regulators of angiogenesis, thus playing an active role in the progression of peritoneal metastasis.

Irene Bertolini; Michela Perego; Jagadish Ghosh; Dario Altieri The Wistar Institute, Philadelphia, USA Introduction: Small extracellular vesicles (sEV) are produced by all types of cells and have emerging roles in cell-to-cell communication. Cancer cells produce higher levels of EV compared to normal cells, and this response is further augmented by stress conditions, such as hypoxia. Intra-tumoral hypoxia is a common occurrence in advanced breast cancer, correlating with increased risk of metastasis and poor prognosis. We have recently demonstrated that sEV produced by cancer cells in hypoxic, but not normoxic conditions, induce oncogenic changes in normal epithelial cells in vitro. Mechanistically, hypoxic sEV deliver ILK to recipient normal mammary epithelial cells resulting in heightened mitochondrial dynamics and increased cell motility and invasion. In this study, we sought to elucidate the ILK signaling axis activated by hypoxic sEV. Methods: We isolated sEV from two different human breast cancer cells (MCF7 and MDA-231) and one normal mammary epithelial cell (MCF10A) maintained in hypoxic (sEVHYP) or normoxic (sEVNORM) conditions (1% or 5% O2 respectively for 24 h). sEV were isolated using differential centrifugation followed by size exclusion chromatography (SEC, Izon). sEV characterization was carried out according to MISEV guidelines. sEV were quantified after each isolation using the ZetaView system and the same number of sEV was applied to recipient MCF10A cells in every co-culture experiment. Results: We found that breast cancer-derived sEVHYP, but not sEVNORM, potently activate NFκB gene expression in recipient MCF10A cells. In turn, this increases the production and release of inflammatory cytokines, heightens mitochondrial dynamics and stimulates cell motility, disrupting epithelial acini morphogenesis in 3D cultures. Mechanistically, pharmacologic or genetic targeting of ILK or p65 NFkB abolishes the effect of sEVHYP on recipient mammary cells. Summary/Conclusion: sEV released by breast cancer cells under hypoxic conditions activate NFκB in normal mammary recipient cells. This pathway may drive multiple pro-tumorigenic steps of heightened inflammation, deregulated mitochondrial dynamics and aberrant 3D epithelial morphogenesis in a breast cancer microenvironment. Introduction: The spread of cancer cells from the primary tumor into surrounding tissues and metastasis to distant organs is the primary cause of cancer morbidity and mortality. Recent data suggest that EVs released by cancer cells may stimulate tissue invasion and dissemination of tumor cells to target tissues. The invasive and metastatic character of several cancer types is associated with the presence of CD133 and CD44, which predict poor outcomes of cancer patients. CD133 is among one of the molecular signatures of EVs. CD44 has been reported to be associated with EVs, but its role in signal-mediated EVs is not known. The major goal is to correlate the expression of CD133 and CD44, to EV secretion, their EV-mediated invasive and metastatic properties with a long-term goal for the development of innovative EV-based therapeutic approaches. Methods: EVs were isolated by size exclusion chromatography (SEC) and by ultracentrifugation from breast cancer (MDA-MB468, MCF7) and osteosarcoma (U20S, SAOS) cell lines and characterized by WB, NTA, and EM to evaluate particle size, EV morphology, and composition. Results: We found that all EV samples isolated by SEC represent a heterogeneous population categorized into three prominent sub-populations non-vesicular particles (< 50 nm), and two subpopulations consisting of small-EVs (50-100 nm) and large-EVs (150-250 nm). Interestingly, in osteosarcoma cell lines, the range and the abundance of EVs were lower as compared to breast cancer cells lines. EVs specific markers (CD9, CD81, CD63, Alix, Tsg101) were detected in the four cell lines. Although breast cancer and osteosarcoma cell lines produce EVs loaded with CD133 and CD44, their expression levels are higher in the triplenegative breast cancer cell line (MDA-MB468). Summary/Conclusion: Our results thus reveal that the expression of biomarkers of interest does not occur equally in all cell types, which may influence their metastatic capacity and the development of the disease.

also stimulated the macrophages phenotypic shift towards M2 polarization in vitro as noted by the increment of IL10 and CD206 levels. Importantly, 90 EV-miRNAs were detected in plasma-EVs, 5 of them differentially expressed between ESA-and ESD-EVs. Summary/Conclusion: Our findings suggested that ESD-EVs generate a pre-metastatic milieu by endothelial activation and the modulation of stromal cells phenotype. Moreover, EV-miRNAs could be used as prognostic biomarkers in early stages lung cancer patients. Funding: The study was supported by grants from Italian Ministry of Health RF-2018-12367824 to G.S.

Adnan Shafiq La Trobe University, PRESTON, Australia Introduction: pithelial-mesenchymal transition (EMT) describes an evolutionary conserved morphogenic process defined by loss of epithelial characteristics and acquisition of mesenchymal phenotype, and altered patterns of intercellular communication, leading to functional changes in cell migration and invasion. In this regard, we have previously reported that oncogenic H-Ras induced EMT in Madin-Darby Canine Kidney (MDCK) cells (21D1 cells) trigger changes in the protein distribution pattern in cells, exosomes, and soluble protein factors (secretome) which modulate the tumor microenvironment. Methods: EVs were isolated using differential centrifugation including OptiPrep density gradient and characterised by NTA and western blot analysis. Results: Proteomic analysis revealed that the protein spectra of RNA-binding proteins and mitochondrial proteins in 21D1-sMVs differ profoundly compared to those of exosomes, likewise proteins associated with suppression of anoikis. We show that 21D1-sMVs promote cell migration, confer anchorage independent growth, and induce EMT in parental MDCK cells. An unexpected and novel finding was the selective sorting of tissue transglutaminase-2 (TGM2) into 21D1sMVs; there was no evidence of TGM2 in MDCK-sMVs. Prior treatment of 21D1-sMVs with neutralizing anti-TGM2 or anti-FN1 antibodies attenuates the invasive capability of fibroblasts. These finding suggest that microvesicle-associated TGM2 may play an important contributory role in the EMT process and warrants further investigation Summary/Conclusion: Collectively, our findings suggest that MDCK-sMV cargo changes that occur following oncogenic H-Ras transformation are sufficient to confer cellular migration, anchorage-independent growth capabilities and induce EMT in recipient MDCK cells. Among the important question for the future will be to better understand how the cargo of different EV classes (microvesicles and exosomes) along with the secretome act ina coordinated fashion to mediateEMT. It's also important to explore further whatthe impacts the sMVmakeonthe cancermicroenvironments as anenablerof cancerprogression. Harnessing this knowledge will open avenues for future EV studies aimed at furthering our understanding of the role of sMVs in the EMT program and the targeting of EVs as potential druggable molecules for therapeutic application. Introduction: The formation of distant metastases is the major obstacle in treating cancer. Metastatic spread requires a cancerinduced modulation of the surrounding environment, which can be mediated via the release of tumor-derived extracellular vesicles (T-EV). Using a syngeneic mouse model for colorectal cancer (CRC), we identified two variants of the murine CRC cell line CMT93, which differed significantly in their metastatic colonization in vivo. The aim of this study was to identify the molecular mechanisms responsible for the effect. Methods: The two CMT93 variants were characterized regarding their basic characteristics using proliferation, invasion, migration and adhesion assays. T-EV released by the cells were collected via differential ultracentrifugation at 17,000 g for larger microvesicles (MV) and 143,000 g for small EV. The obtained EV were characterized by immunoblotting, nanoparticle tracking analysis, transmission electron microscopy and proteomics.

Results: Analysing the cell-intrinsic basic characteristics of the cells revealed no major difference between the two CMT93 variants. Thus, we extended our analysis to the released secretome, which revealed a major difference in the amount and composition of released T-EV. While T-EV released by the malignant CMT93 variant induced tumor invasion, this effect was not observed with T-EV from the less aggressive CMT93 variant. Intriguingly, this functional difference was more prominent with MV than with small EV. To identify the molecular characteristics responsible for this difference, the MV released by both CMT93 variants were compared by proteomic analysis. The results revealed that the MV released by the malignant CMT93 variant were enriched in proteins associated with adhesion. Using immunoblotting, we were able to validate an upregulation of the proteins Itga3 and Fascin1 on MV released by the malignant variant. Analysing microarray data, we identified that these proteins were upregulated in human primary and metastatic CRC in contrast to normal colon tissue, highlighting the translational relevance of our findings. 

Bibek Bhatta Introduction: Most cancers harbor mutations in the TP53 gene (encoding for the p53 tumor suppressor protein). Furthermore, gain-of-function (GOF) mutation in p53 imparts an aggressive traits to the cells when compared to the cancer cells harboring inactivating mutations or wild-type (WT)-p53. Notably, multiple studies have delineated the presence of GOF-mutant p53 protein in untransformed cells or in stromal compartments of tumor microenvironment (TME). In recent years, the involvement of extracellular vesicles (EVs) in cell-to-cell communication has emerged as a major route by which cancer cells can interact and educate immune, and non-immune cells in TME to become tumor supportive. To this end, we hypothesize that mutant p53 protein can be shuttled via EVs to TME cells thus shedding light on a novel non-cell autonomous role of mutant p53 cancers Methods: EVs were isolated from various cancer cell lines (pancreas, lung, colon) differing by their p53 status and the effect on neighboring cancer cells and TME cells was studied in vitro and in-vivo. We also utilized the human colorectal Colo-320DM cancer cell xenograft model, which expresses the R248W p53 mutant. FFPE sections of subcutaneous tumors derived from the Colo-320DM xenografts were stained for p53 using the DO-1 antibody that specifically recognizes human p53. Results: Our data demonstrated that mutant p53 protein can be selectively sorted into EVs; that mutant p53 in EVs can be taken up by neighboring cancer cells and macrophages that do not harbor mutant p53 protein. Evident of macrophage education was seen with the increased expression and secretion of pro-inflammatory cytokines. Notably, mutant p53 expression was also found in non-tumor cells in both human cancers, and in non-human tissues in human xenografts. Summary/Conclusion: Cancer cells harboring GOF p53 mutants, can package mutant p53 proteins in EVs, and deliver them to neighboring cancer cells and to the TME. Introduction: Prostate cancer (PCa) is a commonly diagnosed pathology. A critical role of cell-to-cell communication during tumorigenesis has been proven, allowing to re-program the tumor microenvironment to facilitate its progression. In this sense, exosomes, small extracellular vesicles released to the extracellular and their microRNAs content (exomiRNAs) has been shown regulate gene expression and PCa development. Thus, our aim was to investigate a differential exomiRNA cargo in two PCa cell lines PC-3 (representing the most aggressive stage) and LNCaP (less aggressive stage) versus non-cancerous prostate cell line RWPE-1, and to identify their putative targets and their possible role on the PCa development. Methods: PC-3, LNCaP and RWPE-1 cell lines were cultured during 24h in their respective culture medium. Exosomes were isolated from 8 ml culture medium by exoEasy Maxi Kit (Qiagen) and characterized by western blot and transmission electron microscopy. ExomiRNAs were isolated by ExoRNeasy Maxi Kit (Qiagen) and qRT-PCR was performed by using miRCURY LNA miRNA miRNome PCR Panel I+II V5 (Qiagen), that included 752 mature human cancer-related miRNAs. Pathway and functional enrichment analysis on putative targets of the selected exomiRNAs was performed by using miRNet, Reactome and STRING softwares. Results: Microarray analysis revealed a total of 38 exomiRNAs differentially express in PC-3 and LNCaP versus RWPE-1 cell line. MiRNet analysis showed 41 putative genes regulated, at least, by 20 of the 38 differentially expressed exomiRNAs. Reactome and STRING analysis demonstrated that MDM2, TNRC6A, TNRC6B and AGO1 were common targets of the putative miRNAregulated pathways with an important role on miRNA expression regulation. Summary/Conclusion: PCa secreted exomiRNAs can modulate tumor microenvironment gene expression and may be crucial to the progression of cancer. Funding: This study is funded by PI20/00418 Instituto de Salud Carlos III and Co-founded by European Union (ERDF/ESF "A way to make Europe/Investing in your future") Introduction: Recently, more and more evidence is found that extracellular vesicles (EVs) are linked to therapy resistance. The majority of malignant melanoma cases harbor oncogenic BRAF mutations. BRAF inhibitors, like vemurafenib, show remarkable initial responses, however, resistance emerges in almost all patients. Our aim was to investigate the role of EVs in the evolvement and spread of therapy resistance in an in vitro setting using sensitive-resistant syngeneic melanoma cell pairs, which were isolated before and after the vemurafenib treatment from each patient. Methods: Melanoma cells were kept in DMEM supplemented with 1% EV-depleted FBS for at least three days before collecting media. ExoQuick exosome precipitation reagent was used to isolate EVs. Total protein content was analyzed using the Qubit Protein Assay Kit. EVs' size distribution and concentration were determined using Microfluidic Resistive Pulse Sensing (MRPS). Baseline vemurafenib sensitivity and EVs capacity to transfer therapy-resistance were evaluated by SRB viability assay and videomicroscopy, cell proliferation and migration were quantified. Results: Treatment with the EVs produced by the resistant syngeneic cell line diminished the effect of vemurafenib treatment. Similarly, treatment with self-produced EVs showed a tumor-promoting effect. However, the observed effects were cell line dependent. Summary/Conclusion: Our result indicates that the effects of extracellular vesicles on vemurafenib sensitivity, proliferation and migration are cell line dependent. EVs potentially contribute to resistance against the BRAF inhibitor vemurafenib. Funding: Supported by the ÚNKP-21-3 New National Excellence Program of the Ministry for Innovation and Technology from the source of the National Research, Development and Innovation Fund.

Ana Špilak; Andreas Brachner; Ulrike Kegler; Christa Nöhammer; Winfried Neuhaus AIT Austrian Institute of Technology, Vienna, Austria Introduction: Besides potentially having a major role in cancer metastasis, small extracellular vesicles (sEVs) released by cancer cells can serve as liquid biopsy biomarkers for detection in bodily fluids such as saliva (PMID: 34087328). However, the mechanisms of how sEVs interact with biological barriers are not clarified. We investigated the effects of prostate cancer (PCa) sEVs on a human submandibular salivary gland barrier (SSGB) model in vitro. Methods: sEVs were isolated from conditioned serum-free medium after 48h collection with size exclusion chromatography from PCa DU145 cell line in normoxic and hypoxic conditions and characterized with NTA for particle count, size distribution and zeta potential, microBCA and western blots for CD81, CD9, ALIX and GM130. sEVs were applied to the basolateral (blood) side of a SSGB Transwell model based on HTB-41 B2 cells (PMID: 32842479) and barrier integrity was assessed with the transepithelial electrical resistance (TEER) measuring CellZscope device in one-hour intervals. After 40 hours apical and basolateral media as well as HTB-41 B2 lysates were collected. sEVs in media were measured with NTA, mRNA of 96 barrier relevant targets were quantified by high-throughput qPCR (Fluidigm). Results: Experiments with PCa sEV in serum-containing or serum-free SSGB medium revealed serum dependent effects on TEER over time, highlighting the importance of optimized experimental settings for sEV/barrier interaction studies. sEVs showed minor effects on TEER compared to controls, but addition of hypoxia PCa sEVs resulted in an increase of apically found sEV particles in comparison to sEVs from normoxic conditions. The found effects correlated to mRNA regulation of selected barrier targets in HTB-41 B2 cells. Summary/Conclusion: Our results indicated that the different PCa sEV fractions influenced either the sEV secretion of SSGB cells or possessed different permeation properties. Current studies aim to elucidate the origin of the apically found sEVs and the underlying mechanisms for these differences. Introduction: Extracellular vesicles (EV) are enriched in leukemia patients and were found to promote migration, invasion and chemoresistance of acute myeloid leukemia (AML) cells, and quiescence of hematopoietic stem cells. We asked if AML-EVs can also target immune cell functions to facilitate immune escape and/or disease progression. Methods: AML cell lines HL-60, KG-1, OCI-AML3 and MOLM-14 were cultured in particle-free medium under largescale culture conditions to obtain EV-conditioned medium. AML-EVs were purified and enriched using two sequential rounds of tangential flow filtration (TFF) and characterized by electron microscopy, western blotting and bead-based flow cytometry, confirming double-membrane morphology, purity and identity. AML patient plasma-derived EVs were isolated by size exclusion chromatography. Purified AML-EVs were used to modulate T cell proliferation and NK cell-dependent cytotoxicity. Results: Purified AML-EVs showed a significant dose-dependent inhibition of PHA-stimulated T cell proliferation. Furthermore, primary AML patient and cell line-derived EVs reduced NK cell-mediated lysis of K-562 target cells in a dose-dependent manner. Also, osteogenic differentiation capacity of bone marrow stromal cells was increased by AML-EVs. Analysis of EV surface marker expression by MACSPlex compared to flow cytometry for AML cells showed a comparable pattern reflecting the AML cell origin. RNA cargo analysis of AML-EVs revealed small RNA species (e.g. miRNAs) as well as longer RNA molecules, albeit to a lesser extent. Summary/Conclusion: We show that AML-EVs inhibit T cell proliferation and NK cell functionality while enhancing osteogenic differentiation capacity of bone marrow stroma. These effects could contribute to the creation of a leukemiapermissive niche and immune escape of AML mediated by their EVs. The underlying molecular mechanisms need further investigation. Funding: The presented work received funding from the European Union's Horizon 2020 research and innovation program and by Land Salzburg in the framework of EV-TT, EV-TT -Bpro, and Cancer Cluster Salzburg. genetic risk factor for sporadic AD is apolipoprotein E-e4 (APOE-e4). Female APOE-e4 carriers develop AD more frequently than age matched males, and have more brain atrophy and memory loss. Consequently, biomarkers that are sensitive to biological risk factors may improve AD diagnostics and provide insight to underlying mechanistic changes that could drive disease progression. Here we assessed the effects of sex-and APOE-e4-dependent on the miRNA cargo of cerebrospinal fluid (CSF) extracellular vesicles (EVs) in AD. Methods: Ultrafiltration combined with size exclusion chromatography was used to enrich for CSF EVs (e.g., Flotillin+, but depleted for proteins and lipoproteins not associated with EVs). CSF EVs were isolated from females and males AD or controls (CTLs) that were either APOE-e3,4 or -e3,3 positive (n=7/group, 56 total). MiRNA expression levels were quantified using a custom TaqMan array that assayed 190 miRNAs previously found in CSF, including 25 miRNAs that we previously validated as candidate AD biomarkers. Results: We identified changes in the EV miRNA cargo that were affected by both AD and sex. Four miRNAs (miR-454-3p, -409-3p, -331-3p, -16-5p) were significantly increased in AD vs. CTL and three miRNAs (miR-150-5p, -342-3p, -146b-5p) were significantly increased in females vs. males. Furthermore, we found that APOE-e4 status affects different subsets of CSF EV miRNAs in females vs. males. The predicted gene targets of the four miRNAs increased in AD identified highly relevant pathways (e.g., senescence and autophagy). Summary/Conclusion: These studies demonstrate the complexity of the biological factors associated with AD risk, and their impact on EV cargo, which may play a role in AD pathophysiology. Introduction: This investigation combined functional Magnetic Resonance Imaging (fMRI) and Neuronally Enriched (NE) Extracellular Vesicle (EV) technology to identify cellular processes underlying insula dysfunction during interoception in major depressive disorder (MDD). MicroRNA-93-5p (miR-93) is a putative depression biomarker targeting genes highly expressed in the brain that regulate neuronal axogenesis. We investigated whether: 1) MDD patients differed from healthy comparisons (HC) in NEEV miR-93 expression and 2) groups differed in associations between brain activation and NEEV miR-93 expression. Methods: The study was approved by the Western Institutional Review Board, performed in accordance with the Declaration of Helsinki, and participants provided informed consent. 41 MDD and 35 HC completed an interoceptive attention task during fMRI and provided blood. EV were separated from plasma using a polymer-based kit. NEEV were enriched by a magnetic streptavidin bead immunocapture kit against the neural adhesion marker biotinylated antibody. NEEV specificities were confirmed by flow cytometry and western blot; size and concentration were determined by nanoparticle tracking analysis. NEEV small RNAs were purified and sequenced. EV-TRACK ID EV210507. Results: MDD exhibited lower NEEV miR-93 expression than HC (p=.037, d=.48). Groups differed in bilateral insula-NEEV correlations (left: F1,71 = 6.34, p=.014; z=2.57, p=.010; right: F1,71=9.75, p=.003; z=3.49, p<.001), such that within HC but not MDD, miR-93 expression was positively correlated with left (r =.34, p=.047) and right (r =.54, p=.001) insula signal. Summary/Conclusion: MDD is associated with decreased miR-93 expression but this expression is unrelated to insula activation, raising the possibility of abnormal NEEV regulation in depression. Neuroimaging-EV integration provides an exciting opportunity to discover novel cellular disease targets for depression. Funding: This work was supported by The William K. Warren Foundation, and the National Institute of General Medical Sciences Center Grant Award (P20GM121312).

Olga The development and validation of surrogate tissue assays for non-invasive evaluation of the lung could provide a practical means for routine assessment of lung pathologies, as well as for early detection of disease pathogenesis/risk. We have developed a method for precisely evaluating and enriching deep lung tissue microRNA signals from exhaled breath condensate (EBC), which we compared to miRNA signatures from four different upper and lower airway levels, collected simultaneously during routine care, or clinically indicated (bronchoscopy) from the same patients. Methods: Eighteen individuals undergoing routine bronchoscopy for various clinical reasons had non-invasive upper airway specimens collected; namely, mouth rinse (MR), buccal brushings (BuBr), and EBC, followed by the collection of lower airway bronchial brushings (BrBr) and bronchioalveolar lavage (BAL). Bronchial brushings and lavages were accomplished at mainstem and lobar standard locations, remote from any focal pathology, such as a nodule, or an infiltrate. Small-EV isolations from both BAL and EBC samples were performed using our ultra-sensitive anti-CD63-based small-EV purification assay (EV-CATCHER). Small-RNA next-generation sequencing (NGS) was then performed on MR, BuBr, whole EBC, EBC-small-EVs, BrBr, whole BAL and BAL-small-EVs. Results: Small EV isolations from whole EBC samples increased the EBC microRNA read count depth by 10-fold (p< 0.01) when compared to whole EBC read counts. We observed the strongest correlation in miRNA signatures between EBC and deep lung specimens (BAL and BrBr) when compared with those of upper airway (MR, BuBr). Summary/Conclusion: Exhaled microRNAs encapsulated within small-EVs provide a robust representation of deep lung tissue processes. Targeted isolation of exhaled lung tissue small-EVs enhances microRNA NGS quantitation and provides a novel avenue for non-invasive biomarker identification of deep lung pathologies. Funding: NIH-R33HL156279

Kanchana K. Ayyar 1 ; Ria Shah 2 ; Jean Mendez 3 ; Sharmeel K. Wasan 2 ; Alan C. Moss 4  Boston Medical Center, East Boston, USA;  Boston Medical Center, Boston, USA;  Massachusetts General Hospital, Boston, USA;  Boston Medical Center, boston, USA Introduction: The incidence of Inflammatory Bowel Disease (IBD) is increasing since 2000, 1 in 200 individual suffers from IBD. Diagnosis of IBD involves endoscopy, colonoscopy, CT, MRI. Early detection and treatment can help patients as IBD is a progressive and destructive disease which strongly affects the quality of life. There is a need for novel measures that can assess disease severity with minimal risk and inconvenience to patients. Extracellular vesicles (EV) are tiny packets of protein and nucleic acids that are released by most cell types, including inflamed intestinal epithelial cells. EVs can be found in most biological fluids including intestinal fluid. We sought to characterize these signals in patients with IBD. Methods: Intestinal fluid was collected from healthy individuals and IBD patients during colonoscopy. EVs were isolated by differential ultracentrifugation. Isolated EVs were characterized using nanoparticle tracking analysis on the Nanosight NS300 instrument. Tetraspanin markers were identified on isolated EVs using Exoview platform. RNA was extracted from 1011 EVs and converted to cDNA. A Taqman array plate containing 96 different genes was used to quantify gene expression using qRT-PCR. Results: The mean and mode size of particles isolated from intestinal fluid was 171.9nm and 111.7nm (n=111). The average concentration of EVs isolated from intestinal fluid was 2.52x1012 particles/ml (n=111). EVs isolated from intestinal fluid express CD63 and Syntenin. CD81 and CD9 expression is moderate to low. qPCR identified 14 genes that were differentially expressed between healthy and IBD group, including CCL2, IFN-γ and TNF. Most of the differentially expressed genes are involved in the Th17 pathway and play a role in inflammation. Summary/Conclusion: Intestinal fluid contains abundant EVs, with RNA cargo. Gene expression studies illustrate differences in RNA profile between controls and patients with IBD. Further studies to identify IBD biomarkers are warranted. Funding: This study is funded by the Helmsley Charitable Trust.

Introduction: Mesenchymal stem cell-derived extracellular vesicles have significant age-dependent differences in their immune profiles and the miR-21-5p has been shown to play a key role in the regulation of immune and inflammatory responses. The senescence-associated secretory phenotype (SASP) is being seen to offer a new perspective as a marker for the progression of many age-related diseases. Being able to give us an idea of the state of inflammation and complexity of multiple diseases associated with age from multiple pathologies. For this reason, in this work we studied the paracrine function of miR-21-5p on extracellular vesicles behaviour and SASP. Understanding all these properties of extracellular vesicles makes them especially attractive for the development of new therapeutic approaches based on gene therapy Methods: Stable and transient transfections of mesenchymal stem cells were performed to inhibit or overexpress miR-21-5p respectively. Their extracellular vesicles were then extracted using ultracentrifugation. The concentration and density of EV in the transfected mesenchymal stem cells as well as the expression of different SASP markers using NTA and RT-PCR respectively.

Finally, these extracellular vesicles from the transfected cells were used to treat mesenchymal stem cells and to verify the paracrine effect of miR-21-5p through evaluation of SASP components using RT-PCR in the the recipient mesenchymal stem cells. Results: The inhibition of miR-21-5p produced a significant decrease in the amount of extracellular vesicles produced and its overexpression produced the opposite effect in cells. Inhibition of miR-21-5p significantly decreased the expression of SASP (IL-6; HMGB1; S100A4; S100A6; IL-1B). Furthermore, this effect on SASP transmission was significative increased in the mesenchymal stem cells treated with extracellular vesicles from overexpression miR-21-5p mesenchymal stem cells and the opposite effect in the extracellular vesicles from MSCs with the inhibition of miR-21-5p. Summary/Conclusion: The immunomodulatory properties of mesenchymal stem cells and their ability to release extracellular vesicles has been modified by miR-21-5p. Extracellular vesicles would be acting as a vehicle for cell-cell communication to influence cellular activities in receptor cells, regulating the expression of your target genes related with SASP. Introduction: Microparticles(MP) are plasma membrane-originated vesicles formed in conditions like cell activation, stress, and apoptosis and released into the extracellular environment. The size of MPs is 100-1000 nm globular-like structures, contains different bioactive molecules such as proteins, lipids, signaling molecules, mRNA, microRNA, long non-coding RNAs, and even DNA.MPs are released by most cell types and they can be found in all biological fluids. Urine is the source of podocyte-derived MPs, the contents and the amount of them changed in renal injury conditions. So they have been considered as biomarkers in many Kidney diseases with podocyte injury like IgA nephropathy. In this study, we have isolated and characterized the podocytederived MPs in the urine of patients with IgAN to use them as diagnostic biomarkers as a noninvasive method versus invasive renal biopsy Methods: In this study, 15 biopsy-proven IgAN patients and 15 healthy people participated. Early morning urine was collected, The samples were centrifuged by differential centrifugation. First, centrifuge at 300g for 10 minutes to remove all live cells and second the collected supernatant of first step centrifuge at 25000g for 20 minutes at 4 • C, the MP-containing pellet was resuspended in 4X PBS before characterization. To size the isolated MPs, they are visualized by scanning electron microscope (SEM). Then we use the western blot method to quantify the 6 podocyte-specific protein markers Nephrin, Podocalyxin, flotillin, CD-9, TSG101, ALiX, and HIST1H as a negative marker in both IgAN patients and healthy group. Results: the Images of SEM indicate that the mean size of MPs isolated from urine sample of IgAN patients is 125.8 nm and by western-blot, we had observed that the 5 podocyte-specific proteins Nephrin, Podocalyxin, flotillin, TSG101, and ALiX present in two groups but the density of them are higher in IgAN patients versus healthy group but the density of CD19 was almost the same in two IgAN positive IgAN negative samples. The absence of protein HIST1H as a negative marker confirms the presence of podocyte-derived MPs in samples. Summary/Conclusion: Characterization of MPs indicates that podocyte-derived MPs exist in the urine of IgAN patients. This investigation provides the study of early IgAN diagnosis with specific biomarkers as a noninvasive method in the future.

Beatrice Vilardo 1 ; Davide Raineri 2 ; Fabiola De Marchi 3 ; Letizia Mazzini 4 ; Giuseppe Cappellano 5 ; Annalisa Chiocchetti 6  Università del Piemonte Orientale -Immunomics Laboratory, Novara, Italy;  Università del Piemonte Orientale, Novara, Italy;  Ospedale Maggiore della Carità Novara, novara, Italy;  Ospedale Maggiore della Carità -Novara, Novara, Italy;  Università del Piemonte Orientale, novara, Italy;  Università del Piemone Orientale, Novara, Italy Introduction: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease whose triggering factors are still poorly understood. ALS is a ''silent'' disease compared to other neurodegenerative disorders, manifesting earlier cognitive defects Introduction: Isolating neuronal-derived extracellular vesicles (NDEV) in blood and studying their cargo may help us understand cellular processes and follow their evolution during neurological disorders. Recent publications have challenged the claims that neuronal cell adhesion molecule L1CAM is EV-associated and that targeting it with immunocapture effectively isolates NDEVs from human plasma. Here we provide evidence that some plasma EVs carry L1CAM on their surface and, therefore, L1CAM may be utilized for isolation of NDEVs from plasma. Methods: We isolated EVs from 10 ml of plasma of three healthy individuals using qEV10 70 nm size-exclusion chromatography columns and collected 13 fractions. We replicated the published SIMOA assay for L1CAM developed by Norman et. al. and measured L1CAM levels in each fraction. This assay uses L1CAM (5G3) as capture antibody and L1CAM (UJ127) as detector. We also developed a novel homebrew SIMOA assay to detect L1CAM on intact EVs (NDEV assay) in each fraction. We used L1CAM (5G3) as capture antibody, mixed biotinylated CD9, CD81 and CD63 antibodies as detectors and human iPSC-derived neuronal EVs as calibrator during assay development. Results: In agreement with findings of Norman et. al., using the L1CAM SIMOA assay, we detected L1CAM levels close to LLOQ in fractions 1-4 but detected increasing levels of L1CAM from fraction 5 to 10; L1CAM levels declined gradually between fractions 11 and 13. However, using our novel homebrew NDEV SIMOA assay in the same fractions, we found that L1CAM+ EVs were present in highest levels in fractions 1 to 5 and in declining levels in fractions 6 to 13 Summary/Conclusion: Our study provides evidence that L1CAM is a surface marker of EVs of variable size, making it plausible that L1CAM may be used for immunocapture of an EV sub-population of neuronal origin. Funding: This research was supported by the Intramural Research Program of the National Institute on Aging, NIH.

Martijn H. van Heugten; Charles J. Blijdorp; Hester van Willigenburg; Mahdi Salih; Ewout J. Hoorn Erasmus Medical Center, Rotterdam, Netherlands Introduction: Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited form of kidney disease and often leads to kidney failure. However, disease progression is highly variable and unpredictable. Here, our aim was to identify a protein biomarker in urinary extracellular vesicles (uEVs) to differentiate rapid from slow disease progression. Methods: Patients were selected from the clinical DIPAK trial (informed consent, medical ethics approval). We created a discovery (n=10) and confirmation cohort (n=10) including patients with rapid and slow disease progression (eGFR decline ≥4 or < 2 mL/min/1.73 m2/year). uEVs were isolated from 50 mL spot urines by a three-step differential ultracentrifuge protocol, and uEV-proteins were mass tag labeled before tandem mass spectrometry. A third validation cohort (n=24) was created to validate the mass spectrometry findings with immunoblotting. All patients were matched for established risk factors for disease progression, including age, sex, baseline eGFR, and genetic mutation, and had equal total kidney volume. Results: We identified 2,727 and 1,115 unique uEV-proteins with over 60% annotated for the extracellular exosome (Benjamini < 0.01). In the discovery and confirmation cohort, a significantly different uEV-protein abundance was found for 65 and 36 proteins, respectively. Matrix metalloproteinase 7 (MMP-7), a protein previously implicated in kidney disease progression, was consistently higher by 47% and 64% in patients with rapid disease progression in both cohorts (p < 0.05). Pathway analysis showed enrichment of Wnt-signaling (q-value < 0.05) of which MMP-7 is a downstream mediator. In the validation cohort, MMP-7 was also higher by 120% in uEVs of patients with rapid disease progression (p < 0.05). Summary/Conclusion: uEV-associated MMP-7 distinguishes patients with slow or rapid ADPKD progression independently of established disease progression markers. MMP-7 is a novel and biologically plausible urinary biomarker for ADPKD which we are currently validating in a larger cohort of patients using a high-throughput assay. Funding: Dutch Kidney Foundation.

Introduction: Extracellular vesicles (EVs) are involved in cell to cell communication. In vitro, JAr (human choriocarcinoma cell line) cell spheroids (3D) are used as mimics of embryo trophoblasts when studying EV mediated trophoblast-endometrial communications. However, the differences of the proteomic profile EVs from 3D JAr spheroids compared to 2D JAr monolayer cultures are unknown. Therefore, we investigated the proteomic profiles of EVs derived from 3D compared to 2D in vitro trophoblast cell culture systems. Methods: JAr cells were cultured and trophoblast spheroids (3D) were formed. JAr monolayer cultures were used as 2D cell culture system. FBS-free culture media conditioned by these JAr 2D cultures or 3D spheroids for 6 hours were collected. Following the sequential centrifugation to remove cells and cell debris, EVs were isolated by size exclusion chromatography and subsequently characterized by nanoparticle tracking analysis (NTA), electron microscopy and mass spectrometry shot-gun based quantitative proteomics. Differential enrichment of proteins in EVs from 3D spheroids compared to 2D JAr cultures and pathway enrichment analysis were performed. Results: EVs enrichment in our purified samples were verified with all the methods, including enrichment of EV markers such as CD9, CD63 and CD81 in the samples. 1509 proteins were identified. 65 of proteins, including LAMB1, LAMC1, COL4A2, LAMA1, HSPG2, LAMA5 and VWF were differentially abundant in 3D culture samples. KEGG pathway analysis showed the enrichment of Extracellular matrix receptor interaction and focal adhesion pathways in 3D JAr cell culture system. Summary/Conclusion: EVs derived from 3D JAr cell culture system provide a different proteomic profile compared to the 2D cultures. Hence, the culture system affects the biological function of these cells in vitro. 

Charisse N. Winston 1 ; Sonia Podvin 2 ; Vivian Hook 2 ; Robert Rissman 2  UC San Diego, Lemon Grove, USA;  UC San Diego, La Jolla, USA Introduction: Introduction: Recent studies suggest neuronal and astrocyte-derived exosomes may serve as the source of biomarkers for many neurodegenerative diseases, include AD. However very few studies have characterized the biomarker potential of microglial derived exosomes (MDEs). Here, we employed the use of unbiased mass spectrometry (MS) to characterize the molecular composition of MDEs derived from healthy controls and pathologically confirmed Alzheimer's disease patients. Methods: Methods: Plasma exosomes were extracted, precipitated, and enriched against a microglial source (TMEM119) using magnetic immunocapture and fluorescence-activated cell sorting (FACS) sorting. MDEs were characterized by size (Nanosight) and shape (TEM). Exosome marker profiling was done by western blot. Proteomic profiling was conducted for novel biomarker identification. Results: Result: Blood-based MDEs demonstrate similar size distributions and shape to previously reported exosome preparations. Western Blot demonstrated that MDEs were positive for exosome marker Flotilin-1; microglial markers CD68 and IBA1 and negative for astrocyte marker, GLAST. Out of 143 total proteins identified by MS, only 1 protein, thrombospondin, was unique to the control group as compared to the AD group. Summary/Conclusion: Conclusion: MDEs can be successfully isolated from human blood. MDE cargo are associated with lipid metabolism (APOE), the complement cascade (Clusterin, C3, C5), and DNA translation and repair (CDK12). Further characterization is required to determine functional protein network analyses of MDE cargo and to determine the biomarker potential of MDE cargo in AD. Introduction: Thorough examination of the molecular composition of small extracellular vesicles (sEVs) by spectroscopic methods is a promising but hitherto barely explored approach for diagnosing cancerous diseases, especially central nervous system tumors. We attempt to reveal the potential role of plasma-derived sEVs in diagnosing seven different patient groups through Raman spectroscopic analyses using a relevant number of clinical samples.

The study is conducted in accordance with the Declaration of Helsinki, informed consent forms are collected and the study was approved by a national ethics committee. Up to 490 plasma samples will be obtained from seven patient groups (glioblastoma multiforme, meningioma, melanoma and non-melanoma brain metastasis, colorectal tumors, melanoma and a control group). SEV isolation is performed through differential centrifugation. The isolates are characterized by Western Blot, transmission electron microscopy and nanoparticle tracking analysis. Principal Component Analysis-Support Vector Machine algorithm is performed on the Raman spectra for classifications. Classification accuracy, sensitivity, specificity and the Area Under the Curve (AUC) value are used to evaluate the classification performance. Results: According to preliminary results, the patient groups are distinguishable with 80-95% sensitivity and 80-90% specificity. AUC scores of 0.82-0.9 suggest excellent classification performance. Summary/Conclusion: Our results support that Raman spectroscopic analysis of sEV-enriched isolates from plasma is a promising approach for developing non-invasive, cost-effective methods for the clinical diagnosis of different cancerous diseases. 

Loïc Steiner 1 ; Rosanne E. Veerman 1 ; Maria Eldh 1 ; Amir Sherif 2 ; Susanne Gabrielsson 1  Division of Immunology and Allergy, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden, Stockholm, Sweden;  Umeå University, Umea, Sweden Introduction: Urinary Bladder Cancer (UBC) is the 4th most common cancer type in men and associated with a poor survival rate and a bad prognostic. At the time of diagnosis, one third of detected cancers are already muscle-invasive and start invading other organs. Due to their abundance in urine, Extracellular Vesicles (EVs) make ideal biomarker candidates for early diagnosis of UBC. Thus, we characterized the protein content of urinary EVs to determine an EV protein signature of UBC and link it to some clinical features. Methods: Urine was collected from 40 bladder cancer patient at different stages and 5 age-matched healthy controls. EVs were enriched from urine by centrifugation (300g, 3000g and 10 000g), followed by tangential flow filtration (100kDa column) and a final ultracentrifugation step at 120 000g. After characterization of EVs by nanoparticle tracking analysis, bead-based flow cytometry and western blot, the vesicles were lysed and their protein content was analyzed by proximity elongation assay (Olink, immuno-oncology panel). Results: The proteomic analysis highlighted important changes in the protein composition of EVs. Firstly, urinary EVs from UBC patients greatly differed from healthy controls establishing an EV protein signature of urinary bladder cancer. We also compared matched samples of urinary EVs and bulk urine (not EV-enriched) from UBC patient and discovered a set of proteins exclusively found on purified EVs, strengthening our rationale for focusing on isolated EVs for biomarker discovery. Additionally, the protein profile was correlated with clinical data such as invasiveness or response to chemotherapy and sets of proteins were identified as predictors of such features. Summary/Conclusion: Taken together, these results suggest the great potential of using isolated urinary EVs as a biomarker for UBC that can then be employed to predict clinical markers and better orient the care of the patients at an early stage of the disease. Introduction: Cancer takes the lives of hundreds of thousands of people every year. Most cancer deaths result from the metastatic propagation of drug-resistant cells, thus the biomarkers capable of predicting tumor progression and therapeutic response are required. A major cause of chemotherapy failure is attributed to subpopulation of self-renewing, drug-resistant cells suspected to originate from the stem cells. Liquid biopsy is a promising approach to characterize tumor phenotype. EVs are potential sources of tumor biomarkers since they carry the same membrane histological markers as a tumor. However, a major challenge faced while analyzing histological and particularly stem cell markers on EVs is their low quantity compared to CD81/CD9/CD63 antigens that make them difficultly detectable since the signal is often under the limit of detection of commonly used techniques. We develop a highly sensitive system for FACS profiling of low-represented EVs membrane markers. The system was validated by analysis of EpCAM, CD166 and CD117 expression on EVs isolated from cell culture supernatants and biological fluids of cancer patients. Methods: EVs were isolated from HT29 and SK-OV-3 cell cultures by ultrafiltration. EVs from clinical samples of ovarian cancer patients were isolated using the adapted procedure of ultrafiltration. Pre-purified EVs were incubated with CD9 beads followed by CD81, EpCam, CD166 and CD117 staining and flow analysis. Results: Limit of detection (LOD) of the system was determined on HT29 and SK-OV-3 derived EVs and estimated to be 1e+5 for CD81, 5e+5 for EpCam (HT29), 2e+8 for CD166 (HT29), and 1e+8 for CD117 (SK-OV-3). Signal calibration method was proposed for evaluation of tumor differentiation degree and validated using forced HT29 cells differentiation and clinical samples of cancer patients. Summary/Conclusion: A highly sensitive system for profiling of low-represented small EVs isolated from cell cultures and physiological fluids has been developed.

Lone Peijs 1 ; Naja Z Jespersen 2 ; Sarah Heywood 2 ; Diana Samodova 1 ; Bente K Pedersen 2 ; Atul S Deshmukh 1  Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Denmark, Copenhagen, Denmark;  Trygfondens Centre for Physical Activity Research, Rigshospitalet, Denmark, Copenhagen, Denmark Introduction: Exercise facilitates beneficial effects on peripheral tissues, and extracellular vesicles (EVs) may play an important role in mediating these effects. In this study, we aim to map the circulating cargo of EVs in plasma following an acute bout of exercise using a multi-omic approach. We will use liquid chromatography mass spectrometry (LC-MS/MS) for characterization of EV-associated proteins and small-RNA sequencing for analysis of miRNAs. Methods: Twenty-five healthy males undertook a 45 min acute exercise bike intervention. Blood plasma samples were collected at just prior to, and immediately after cessation of the exercise intervention. Moreover, blood samples were collected 1 hrs and 3 hrs into recovery as well as 24 hrs after the exercise intervention. As an initial test, plasma samples were pooled from all individuals at each timepoint and EVs were isolated in technical triplicates using size-exclusion chromatography (SEC) with a 70 nm pore size. The EV isolation protocol was qualitatively evaluated using transmission electron microscopy (TEM) and quantitatively evaluated using nanoparticle tracking analysis (NTA). Protein cargo analysis was performed using LC-MS/MS Results: TEM images showed successful isolation of intact rounds vesicles in size ranges ∼30-200 nm. Preliminary NTA analysis showed increased concentration of EVs in the recovery period. Mass spectrometry-based EV preliminary proteome analysis led to quantification of 834 protein, among these were known protein EV-markers such as CD9, CD63 and CD81. Summary/Conclusion: The preliminary NTA and MS-based analysis emphasizes that acute exercise triggers release of EVs that might convey important signals to multiple organs. The upcoming experiments directed at using integrative approach of multiomics data may enhance the understanding of the molecular dynamics of systemic beneficial effects mediated by exercise. Introduction: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Since young TBNC patients (< 40 years) show a shorter survival time, this age-related group could have different molecular behavior promoted by microR-NAs (miRNAs). Then extracellular vesicles (EVs) arise as a minimally invasive option for searching disease biomarkers. Herein, we compared vesicular miRNA levels from plasma of young (n=5) and elderly (n=6) Brazilian TNBC patients. Methods: All samples were retrieved from the Academic Biobank for Cancer Research at the University of São Paulo. The informed consent and an epidemiological questionnaire (Ethics Committee Approval N • 3.007.737/18) were obtained from all patients enrolled in the study. EVs were collected from plasma samples (1mL) using size-exclusion chromatography, quantified by nanoparticle tracking analysis, and characterized by transmission electron microscopy as well as the expression of CD63, CD9 and/or flotillin by western blot. EVs were treated with proteinase K and RNase A before RNA extraction using a silica column method. Finally, vesicular miRNA content was detected and quantified through a digital barcode technology. Results: We did not find statistical differences in EVs plasma concentration between young and elderly patients with TNBC (p=0.792). However, we found 37 differentially expressed miRNAs (DEmiRs) between these groups (p< 0.05 and |fold change (FC)| > 1.2). Among these DEmiRs, hsa-miR-409-3p (FC=2.2, p=0.004), hsa-miR-3180-5p (FC=1.9, p=0.002), hsa-miR-10a-5p (FC=1.9, p=0.008), hsa-miR-933 (FC=1.9, p=0.008), hsa-miR-367-3p (FC=1.7, p=0.009), and hsa-miR-133b (FC=-2.7, p=0.001) showed remarkable differences. Summary/Conclusion: Our preliminary study indicates that vesicular miRNAs show the potential to identify new biomarkers and therapeutic targets in an age-related manner in TNBC patients. 

Susana Garcia-Silva 1 ; Cristina Vico-Alonso 2 ; Lisa Meyer 3 ; Mikkel Noerholm 3 ; Johan Skog 4 ; José-Luis Rodríguez-Peralto 5 ; Pablo L. Ortiz-Romero 2 ; Héctor Peinado Selgas 6  Spanish National Cancer Research Centre (CNIO), Madrid, Spain;  Department of Dermatology, Medical school, Universidad Complutense de Madrid, Instituto i+, Hospital Universitario  de Octubre, Madrid, Spain, Madrid, Spain;  Exosome Diagnostics, a Bio-techne brand, Martinsried, Germany., Martinsried, Germany;  Exosome Diagnostics, a Bio-techne brand, Waltham, MA, Waltham, USA;  Department of Pathology, Medical school, Universidad Complutense de Madrid, Instituto i+, Hospital Universitario  de Octubre, Madrid, Spain, Madrid, Spain;  Spanish National Cancer Research Center (CNIO) , Madrid, Spain Introduction: Liquid biopsy provides a suitable option with progressively improved sensitivity and specificity to complement or to substitute the information obtained by regular biopsy. Extracellular vesicles (EVs) are emerging as powerful tools for providing Introduction: Canine leishmaniasis (CanL), caused by Leishmania infantum parasites, is a disease with high mortality and morbidity. In endemic areas, dogs are considered the main reservoir, allowing the life cycle perpetuation associated with infected vectors. CanL is a risk factor for human zoonotic leishmaniasis, therefore CanL control is essential. The management of CanL requires new tools and biomarkers to address complex epidemiological scenarios. Due to their characteristics, EVs are a resource with great potential that might provide an alternative to conventional biomarkers. Methods: EVs were separated from the plasma of healthy (n=8) and CanL (n=20) dogs by SEC. Fractions were characterized by protein quantification and bead-based flow cytometry assay, using EVs markers, such as CD5L and CD71. Selected fractions were submitted to proteomic analysis by LC-MS/MS. Results: Proteomic analysis allowed the consistent identification of EVs markers. The number of identifications in healthy dogs was significantly higher (p< 0.0001) than in CanL dogs. To increase proteome robustness, a comparative analysis was performed using the merged data from each group. This originated a core proteome of 1786 proteins: 44% were detected in both groups, 37% were only detected in healthy dogs and 19% were only identified in CanL animals. Protein network visualization models and pathway analysis revealed a distinct profile between the 2 groups. Reactome pathways associated with central carbon metabolism; signaling by the B Cell Receptor; cytokine signaling in immune system and antigen processing -ubiquitination & proteasome degradation were enriched in healthy animals. No specific pathway enrichment was associated with CanL dogs. Summary/Conclusion: EVs' characterization in CanL enabled the identification of distinct metabolic pathways that will contribute to a better understanding of disease pathophysiology and the discovery of disease-related biomarkers.

The Gram-negative bacterium Yersinia ruckeri is spread in water columns across the globe and is a threat for fish welfare as well as playing a negative financial factor for the fish farming industry. Infection with the pathogenic Y. ruckeri is causing the enteric redmouth disease (ERM), also called yersinosis and is mainly found in salmonids. Relatively little is known about the molecular and pathogenic mechanism of this bacterium. The aim of the project is the further investigation of Y. ruckeri pathogenesis, where we will mainly focus on characterization of secreted extracellular vesicles (EV). The secreted amount of bacterial EVs varies between bacteria species and they are shown to contain various components (protein, DNA, RNA, LPS, lipids) which resemble the mother bacterium from which they derive. Additionally, isolated EVs have been reported as an alternative vaccine candidate as their injection has been shown to elicit a protective immunity in a host. Methods: For bacterial EVs to be economically beneficial as an immunogenic agent for aqua-cultural purposes, an applicable amount must be produced. In this project, knockout strains were generated, lacking a membrane protein. EVs were isolated from 2L of over-night culture, using tangential flow and ultra centrifugation. Results: Mutant and wild type derived EVs were characterized by both electron microscopy and proteomics analysis. The isolated EVs will further be studied for their function in cellular uptake and cytotoxicity will be investigated in cell-based assays and initial results will be presented. Summary/Conclusion: In the future, zebrafish (Danio rerio) will be used as infection and vaccine model where the fish will be immunized with isolated Y. ruckeri EVs. Injection of EVs may enable the host to evolve an immunity against the pathogen, resulting in a vaccinated state.

Paula Meneghetti 1 ; João Paulo Ferreira Rodrigues 2 ; Nobuko Yoshida 3 ; Ana Claudia Torrecilhas 4  UNIFESP, Sao Paulo, Brazil;  UNIFESP, São Paulo, Brazil;  UNIFESP, SAo Paulo, Brazil;  Federal University of Sao Paulo, Sao Paulo, Brazil Introduction: Trypanosoma cruzi, the etiologic agent of Chagas disease. The parasite spontaneously releases EVs into the culture medium. In previous studies of our group, we showed the importance of EVs release from parasite to communication between vertebrate and invertebrate host. The aim of our research is to characterize the EVs released from metacyclic trypomastigotes to distinct T. cruzi strains (Y, G and CL). Methods: The EVs isolation and purification from metacyclic trypomastigotes (MT) of T.cruzi (Y,G and CL strains). We characterized size and concentration of the EVs from metacyclic trypomastigotes by NTA. We performed Immunoblotting and CL-ELISA used EVs isolated from MT to detected the expression of gp82, gp90, TS, alpha-GAL and total parasite membrane. In parallel, we performed functional assay (invasion assays) and NO production from monocytes supernatants. Results: We observed the expression of gp82 only in extracts of parasites and EVs from G and CL strains. The EVs isolated from MT forms of CL strain have a higher number of particle concentration than EVs from Y and G strains. CL-ELISA showed that EVs from Y strain (TM) express more glycoconjugates, mucins and TS than G and CL strains. In the Functional assays, we showed that EVs isolated from Y, G and CL increase the number of intracellular parasites and the induction of NO production in human monocytes cultures.

Alissa M. Weaver; Lizandra Jimenez; Cherie Saffold; Bahnisikha Barman Vanderbilt University School of Medicine, Nashville, USA Introduction: Extracellular vesicle (EV)-carried miRNAs can influence gene expression and phenotypes of recipient cells. Ago2 is a critical RNA silencing protein and can transport miRNAs into small EVs (SEVs). However, detection of Ago2 and RNA inside of EVs has been variable. Here, we evaluate the effects of serum and growth factors on the detection of Ago2 and miRNAs in SEVs. Methods: Colorectal cancer cells were conditioned with 4 different culture media (Serum-free DMEM, EV-depleted FBS in DMEM, DMEM+EGF, and Opti-MEM). SEVs were purified from conditioned media by cushion-density gradient ultracentrifugation and analyzed by nanoparticle tracking, Western, and dot blot analyses, and QRT-PCR in the presence or absence of detergent and RNase. Results: For all media conditions, we find both Ago2 and EV markers in gradient fractions 6 and 7. Ago2 is also present in the non-vesicular fractions for the media containing EV-depleted serum. Western blots of equal vesicle numbers revealed that Ago2 levels are reduced in SEVs purified from Opti-MEM and EV-depleted FBS conditions, compared to the serum-free DMEM condition. A detergent sensitivity analysis further showed Ago2 primarily on the inside of SEVs for all conditions. QPCR revealed that miRNAs are primarily on the inside of SEVs purified under Serum-free DMEM and Opti-MEM conditions, as they only were sensitive to RNase A treatment in the presence of Triton X-100. In contrast, the same miRNAs are primarily on the outside of SEVs purified from conditioned media containing EV-depleted serum. We also tested the effect of supplementing DMEM with EGF and found no change in Ago2 SEV levels compared to Serum-free DMEM. that IFC can serve as a powerful approach to directly follow the distribution dynamics of EV cargo components post uptake into different target cells. This could pave the way to a not just better understanding of the EV's mediated communication, but also may provide new insights on the functions of distinct cargo component. Methods: We obtained EVs from malaria-infected red blood cells, stained their RNA cargo using thiazole orange and followed the distribution dynamics of the RNA cargo following uptake to different recipient cells-monocytes and macrophages. Results: The distribution of the RNA cargo inside recipient monocytes and macrophages is different. Summary/Conclusion: Our measurements may hint different properties of the cargo distribution dynamics inside these recipient host cells. Interestingly, while the kinetic of the uptake was similar, we observed a significant difference in the signal distribution pattern, showing an opposite trend in its pixel intensity distribution and the dynamic of cellular localization over time.

Dae Chung 1 ; Khang Le 1 ; Michael Lai 1 ; Natalie Collins 1 ; Elizabeth Payne 1 ; Nihal Salem 1 ; Susan Weintraub 2 ; Rajesh Miranda 1  Texas A&M HSC, Bryan, USA;  The University of Texas Health Science Center at San Antonio, san antonio, USA Introduction: Prenatal alcohol exposure can alter the growth and maturation of neural stem cells (NSCs), leading to diminished brain growth. NSCs reside in a complex microenvironment rich in sub-200 nanometer-sized extracellular vesicles (EVs), which may traffic protein, lipid, and RNA cargo between cells, as a mode of intercellular communication. Methods: Using fetal mouse derived cortical neuroepithelium, cultured ex-vivo as non-adherent neurosphere cultures, we investigated the impact of ethanol on the proteome of NSC-EVs by employing quantitative proteomics to profile the protein expression across treatment groups of 18 EV and its 18 parent NSCs. Results: We hypothesized that the proteome of EVs differs from the proteome of their parent cells, and that alcohol alters the EV proteome differently from the cell proteome. For subcellular location categorization, we found significantly different protein expression in EV vs Cell and in Sex. In all 6 locations, there were a group of proteins that were significantly enriched in EV samples compared to cell samples, proposing a possible specific loading of proteins into EVs at the expense of cells. This broad categorization of proteins by subcellular location gives insight into the diversity and complexity of EV proteome. Furthermore, ethanol exposure of NSCs significantly altered the profile of proteins packaged within EVs. Statistical analyses showed ethanol exposure differentially regulated EV proteins, with majority of them being upregulated, while majority of differentially regulated proteins in cells were downregulated. Due to this contrast, we hypothesized that ethanol increases loading of specific proteins into EVs. For this, expression of proteins that were significantly altered in EVs by ethanol exposure were compared to the same proteins in cells. For both moderate and heavy ethanol exposures, the majority of these proteins were upregulated in EVs but downregulated in cells, meaning that increased level of certain proteins in EVs is not due to increased expression of same proteins in cells. Therefore, at the expense of their intracellular levels in NSCs, ethanol exposure results in increased loading of specific ethanol-sensitive proteins into EVs. Summary/Conclusion: In summary, our proteomic data supports that ethanol alters the proteins of EVs differently from their parent NSCs, and that EV proteome differs from their parent cell proteome. Due to the heterogeneity of NSC population, where only a subpopulation of cells expresses specific RNAs and proteins at a given time, EVs may be carrying ethanol-sensitive proteins as intercellular mediators within the NSC niche. By better understanding the effects of alcohol on EV proteins, we hope to elucidate the mechanisms that mediate PAE's effects on NSC growth and maturation. Funding: NIAAA-F31AA028446, NIAAA-R01AA024659

Silvia Picciolini; Alice Gualerzi; Francesca Rodà; Valentina Mangolini; Marzia Bedoni Laboraotry of Nanomedicine and Clinical Biophotonics, IRCCS Fondazione Don Carlo Gnocchi, Milan, Italy, Milan, Italy Introduction: The use of Surface Plasmon Resonance imaging (SPRi) for EV analysis has attracted much attention due to its ability to monitor in real time the interaction between ligands and targets. As the sensitive area of SPRi detection is within 200 nm on the chip surface, EVs are ideal targets that perfectly meet the requirement of the technology allowing to detect EV populations according to the presence of surface markers. Once detected, EVs could be further characterized analyzing the expression of other molecules; to do this the sensitivity of the SPRi method needs to be improved. Gold nanoparticles (AuNPs) are surface plasmon-assisted field amplifiers and intrinsic refractive index sensors representing a powerful tool thanks to their rich surface chemistry, low toxicity and high electron density. In this work we used spherical AuNPs for the amplification of SPRi signal intensity, in order to investigate the expression levels of antigens on the surface of multiple EV populations. Numerous investigations have demonstrated that phloem also act as a distribution route for signalling molecules since different macromolecules, including nucleic acids and proteins, change under stress situations. The participation of extracellular vesicles (EVs) in this communication has been suggested, although little is known about their role. In fact, the presence of EVs in plants has originated a great controversy in the last decade, where major concerns arose from their origin, isolation, and even nomenclature. Methods: Phloem exudates from 5-week-old melon plants, either uninfested or infested with Aphis gossypii (n=15, 4 replicates each), were collected by cutting the tip of the stem with a sterile blade. EVs were isolated from phloem sap by differential centrifugation followed by Size Exclusion Chromatography. EVs were characterized using NTA and TEM (EVtrack ID EV220007). EVs protein content was analyzed by LC-MS/MS. Confirmation of proteasome proteins in sap EVs was achieved by TEM-immunogold and activity was measured as described for total leaf extracts (1) with several modifications. The proteasome inhibitor MG132 was used as a control.

(1) Üstün S & Börnke F (2017). Ubiquitin Proteasome Activity Measurement in Total Plant Extracts, Bio-protocol 7 (17): e2532. Results: Here we confirm the presence of EVs in phloem sap in vivo and characterize them by NTA and TEM. Phloem sap was obtained from melon plants, either uninfested or infested by aphids. Changes in number and composition of those EVs were observed in response to insect feeding, revealing the presence of typical defence proteins in EVs as well as more components of the proteasome complex. EVs from infested plants showed two times more proteasome activity than those from healthy plants. In both cases, such activity was inhibited in a dose-dependent manner by MG132. Summary/Conclusion: Our findings confirm the presence of EVs in phloem sap and explain the high abundance of proteasome complex proteins usually found in proteomic studies, and open up the possibility of an evolutionary conserved mechanism of defence against pathogens/stresses by higher eukaryotic organisms. Funding: Supported by AEI-Ministerio de Ciencia e Innovación, Spain (Grant PID2019-105713GB-I00/AEI /10.13039/501100011033), and Conselleria d'Educació, Cultura i Esports, Generalitat Valenciana, Spain (Grant PROME-TEO/2020/071). C.M.S-L is the recipient of a predoctoral fellowship (PRE2020-092458) funded by AEI/10.13039/501100011033. Part of "Red Traslacional para la Aplicación Clínica de Vesículas Extracelulares, Tentacles", (RED2018-102411-T, Agencia Estatal de Investigación, Spain).

Marek Jelemenský 2

Monika Hofbauerová 4 ; Bernadett Kiss 5

Ágnes Kittel 7 ; Viktor nabil Sayour 5

Miroslav Barančík 2

Péter Ferdinandy 9

 Bratislava, Slovakia;, bra, Slovakia;  Institute of Physics, Slovak Academy of Sciences, Dúbravska cesta , SK- Bratislava, Slovakia; Centre of excellence for advanced material application, Slovak Academy of Sciences, Dúbravská cesta , SK- Bratislava

« Syndecan-syntenin-ALIX regulates the biogenesis of exosomes

« Tetraspanin-6 negatively regulates exosome production

TP mutations correlate with the non-coding RNA content of small extracellular vesicles in melanoma

Joelle Veziers 4

Results: Results: We identified 8 differentially expressed (FC≥|1.5|; P≤0.05; no FDR) ad-sEV miRNAs between mothers of babies with high vs. low adiposity. These included 1 upregulated (i.e., miR-144-3p -an upregulator of adipogenesis) and 7 downregulated miRNAs (e.g., miR-215-5p and miR-486-5p -associated with childhood obesity). These miRNAs are predicted to target 922 mRNAs. Among them, adipogenesis signaling pathway members STAT5B and FGFR3 are predicted to be upregulated

Summary/Conclusion: Conclusions: Maternal ad-sEV miRNAs potentially enhance fetal adipogenesis through several signaling pathways, including STAT3 and PTEN. The findings provide new insights toward understanding the relationship between early fetal exposures, childhood obesity, and adult cardiometabolic disease PF. Extracellular vesicles are stable carriers of adiponectin with insulin-sensitive properties

Xavier Prieur 4

Luisa Vergori 5

Cédric Dray 6

CNRS, INSERM, l'institut du thorax

Sihem Sadoudi 2

Hélène Bihan 5

Camille Baudry 5

Introduction: Type 2 diabetes (T2D) involves hyperglycemia, glycated hemoglobin (Hb), rigid red blood cells (RBC) and altered hemorheology. In T2D, circulating extracellular vesicles (EV) levels are elevated. And may come from RBC

Francesca Pontis 1 ; Ilaria Petraroia 1

Ugo Pastorino 2

Methods: Plasma-EVs were obtained by ultracentrifugation from 20 early stages patients survived at 5 years (ESA-EVs) and 20 died within two years (ESD-EVs). EV's characterization was performed by Nanoparticles tracking analysis, Flow Cytometry and TEM. For uptake experiments PKH26-labeled-EVs were used. Functional experiments were carried out in vitro in 2D and 3D-bioprinting co-cultures models. Nanostring was used to investigate the EV's miRNA cargo. Results: Patient's plasma-EVs were sized between 100 and 150 nM and possess the EVs markers (CD9, CD81, CD63). Notably, FC analysis revealed that ESD-EVs were enriched in CD31 compared to ESA-EVs. Moreover, In vitro experiments showed that plasma-EVs were mostly incorporated by stromal cells; mainly by endothelial cells followed by macrophages and fibroblasts rather than epithelial cells. Strikingly, ESD-EVs treatment increased endothelial VCAM1, CXCR4 and CXCL1 levels compared to ESA-EVs in 2D and in 3D models

Emanuela Carollo 2

A crosstalk between adipocytes and PCa has been demonstrated; however, the study of this dialog has been limited to soluble factors, although emerging evidence points to a key role of extracellular vesicles (EVs) in the control of tumor progression. Methods: After isolation by SEC, EVs were characterized by NTA, TEM and Western blot analysis (TSG101, Hsc70, Alix, calnexin and cytochrome c). In 3T3-L1 adipocytes, the effects of PCa EVs on lipolysis were assessed by flowcytometry and colorimetric assay, while changes in adipokine production were determined by ELISA assay; Akt and MAPK phosphorylation was analyzed by Western Blot. In PC3 and DU145 PCa cells, the effects of EV-treated adipocyte conditioned media on tumor proliferation and survival were evaluated by Trypan blue exclusion assay, while cell metastatic potential was investigated by scratch test and Boyden chamber assay. Results: We demonstrated that PCa EVs can promote both lipolysis and adipokine (interleukin 6, MCP-1 and TNFα) production in adipocytes

Summary/Conclusion: Overall, these data indicate that an EV-mediated crosstalk exists between PCa and adipocytes, endowing the latter with pro-tumor properties. Further studies will be performed to confirm this evidence in vivo and to identify the EV molecular cargo responsible for the modulation of the interactions between adipose tissue and PCa. Funding: Fabrizio Fontana was awarded with an AIRC Fellowship for italy

Extracellular vesicles released by differentiating colorectal cancer stem cells induce a switch from oxidative to glycolytic metabolism in cancer-associated fibroblasts

Department of Basic Biotechnological Sciences, Intensive and Perioperative Clinics, Catholic, Roma, Italy;  Department of Basic Biotechnological Sciences, Intensive and Perioperative Clinics

Reciprocal relationships between cancer cells and the surrounding non-neoplastic cells are critical important in several aspects of tumor development. It has been known,for a while, that cancer cells are metabolically distinct from other nontransformed cells. This metabolic phenotype is not peculiar to cancer cells but reflects tumor microenvironment characteristics. Recently, it has been shown that Extracellular Vesicles (EVs) are involved in the metabolic switch occurring in cancer and tumorstroma cells and sustain the growth of tumor cells. Our previous studies showed that differentiation process affects the content of EVs released by Colorectal Cancer Stem Cells (CR-CSCs). In this regard, this study aims to analyze the effects of EVs on Cancer Associated Fibroblast (CAFs) metabolism reprogramming. Methods: Proteomic profile of EVs content was performed by a classical bottom-up platform followed by high-resolution nano-HPLC-ESI-MS/MS (VelosOrbitrap-Elite). Metabolomic content was analyzed by the ion-pairing HPLC method in recipient cells (CAFs)

Stephanie Bollard 1

Extracellular Vesicles (EVs) are tiny lipid bilayer bound nanoparticles, known to play a role in melanoma progression. Their cargo can drive several specialised functions in cancer, including the control of tumour proliferation and premetastatic niche formation. In other malignancies, EVs have been shown to alter chemokine secretion in the tumour microenvironment (TME) favouring tumour progression. This study aims to assess the EV-mediated interaction between melanoma cells and fibroblasts within the TME. Methods: Metastatic melanoma (A375, RPMI-7951) and normal dermal fibroblast (Malme-3) cell lines were maintained in culture. EVs were harvested from melanoma cell lines using differential ultracentrifugation and characterised according to MISEV2018 guidelines. A co-culture experiment in which EVs derived from melanoma cell lines were applied to Malme-3 was performed, and the resulting secretome was analysed after 48 hours using a chemokine array (Abcam, ab169812). Data were analysed using GraphPad Prism v.9 for macOS. Welch t-test was used for comparison between secretion profiles, p< 0.05. Results: The secretion of several chemokines was increased significantly with the addition of EVs in comparison to Malme-3 cells alone. When Malme-3 fibroblasts were co-cultured with EV's from A375 melanoma cells there was a significant increase in the secretion of CCL5 (36.20-fold

Summary/Conclusion: The EVs derived from metastatic melanoma cell lines significantly altered the secretome of normal dermal fibroblasts when co-cultured. These results give insights into the mechanisms of paracrine signalling between cell populations in the melanoma TME and highlight potential targets for further study to investigate the mechanisms of melanoma progression within the TME. Funding: This work was performed within the Irish Clinical Academic Training (ICAT) Programme, supported by the Wellcome Trust and the Health Research Board (Grant Number 203930/B/16/Z), the Health Service Executive National Doctors Training and Planning and the Health and Social Care, Research and Development Division

Periprostatic adipose tissue exomicroRNA profile in prostate cancer patients with different degree of aggressiveness

ExomiRNAs were extracted, and expression profiling of n=4 samples of PPAT and 4 samples of ABAT tissue was performed by using miRCURY LNA miRNA miRNome PCR Panel, that included 752 mature human cancer-related miRNAs. Deregulated exomiRNAS were validated the 24 AT samples. Pathway and functional enrichment analysis on putative targets of the selected exomiRNAs was performed. Results: Western blot and TEM revealed characteristics of exosomes in AT samples. Nine miRNAs were found in the pilot study to be differentially express between PPAT and ABAT samples. Validation analysis showed that hsa-miR-18a-5p

Summary/Conclusion: PPAT microRNA content is linked with PCa and could be envisage as new possibility to be addressed for therapeutic strategies. Funding: This study is funded by PI20/00418 Instituto de Salud Carlos III and Co-founded by European Union (ERDF/ESF "A way to make Europe/Investing in your future

PS: Biomarkers: EV nucliec acids II

Alpha- acid glycoprotein and microRNA miR--p in urinary extracellular vesicles as potentials biomarkers of primary aldosteronism

Inflammatory parameters such as C-reactive protein were evaluated ultrasensitive PCR (usPCR), PAI-1, MMP9, IL-6, and also lipocalins AGP1 and NGAL by immunoassay. Urinary EVs were isolated of all subjects by ultracentrifugation and characterized by nanotracking analysis (NTA), transmission electron microscopy (TEM) and western blot (CD63). The expression of miR-21 and Let-7i in uEVs by qPCR-Taqman. Statistical analyzes and ROC curves were performed using SPSS v15 and Graphpad Prism v9. Results: We observed an increase in AGP1 levels in PA subjects with respect to the EH and CTL group (p < 0.05). We did not observe significant differences in usPCR, PAI-1, MMP9, IL-6 and NGAL. We detected significant associations of AGP1 with aldosterone

Summary/Conclusion: We observed a higher concentration of circulating AGP1 and lower levels of miR-21-5p in uEVs in PA subjects. The associations of AGP1 with aldosterone, PRA and ARR together with the discriminatory ability of AGP1 and miR-21-5p to identify the PA, suggesting a potential role as biomarkers of PA. Funding: Partially supported by grants ANID-FONDECYT 1212006 and 3200646

Science Initiative Program-IMII P09/016-F, ICN09_016

APOE genotype differentially effects the microRNA cargo of cerebrospinal fluid extracellular vesicles in females with Alzheimer's disease compared to males

Multiple biological factors including age, sex, genetics, and family history influence Alzheimer's disease (AD) risk. Of the 6.2 million Americans living with Alzheimer's dementia, 3.8 million are women and 2.4 million are men. The strongest PS. In-depth characterization of human brain pericyte-derived small extracellular vesicles and their

Ricardo Figueiredo 4

Rémi Chassagnon 6

This barrier is formed during brain development and matures after birth thanks to cell-cell communications between ECs and neighbor cells particularly brain pericytes (BPs). Indeed, they (i) induce the pro-angiogenic mechanisms in ECs and promote barriergenesis, (ii) structure the junctional complexes between the ECs, (iii) restrict aspecific vesicular transport, and (iv) are essential for the maintenance of the BBB main features after birth and throughout life. An extracellular vesicle (EV)-mediated cell-cell communication between ECs and BPs is hypothesized as observed in bone marrow where BP-derived EVs promote the ECs differentiation. The objectives of this study are to characterize in depth the composition of BPs-derived EVs and particularly the small EVs (sEV) composition, and to study their modifications once exposed to pro-inflammatory cytokines such as TNF-α, IL-1ß and IFN-γ, mimicking a neurodegenerative context. Methods: EVs features are evaluated by microscopy approaches (transmission electronic microscopy (TEM), atomic force microscope (AFM)), dynamic light scattering (DLS) and ζ potential measurements once isolated by differential ultracentrifugation (2K, 10K and 100K fractions). Protein and lipid compositions are checked by mass spectrometry, completed by a large nucleic acids sequencing (miRNAs, long non-coding RNAs, …)

Summary/Conclusion: This study opens discussions about the role of these sEV in (i) establishing and maintaining the BBB main features and (ii) their pathological impact on ECs in pro-inflammatory conditions. Funding: Camille Menaceur is a recipient of a doctoral grant from the Hauts-de-France Region and the University of Artois

Role of miR- on MSC-derived EV and their paracrine effect

Arufe 2

Medicina y Ciencias Biomédicas, Facultad de Ciencias de la Salud

Mariusz Kępczyński 4 ; AgataŻak 4

Department of Medical Physics, M. Smoluchowski Institute of Physics, Faculty of Physics

Extracellular vesicles are lipid and protein rich entities and their molecular content reflect cell metabolism, structure and membrane composition. We aimed to collect and in-depth analyze data on the quantitative and qualitative composition of proteins and lipids transported by EVs. Methods: We applied novel techniques, developed in material science and nanotechnology, to characterize EVs content: SIMS -Secondary Ion Mass Spectrometry, PALS -Positronium Annihilation Lifetime Spectroscopy with the 22Na isotope as a positron emitter, and TRAMS -time-resolved fluorescence anisotropy measurements with DPH probes. Exosomes and larger subpopulations of EVs were isolated from preconditioned media of human microvascular endothelial and beta-cells for our investigation. Results: SIMS allowed us to define lipids including sterols, prenols, and sphingomyelins, and characterize the abundance of amino acids in different subpopulations of EVs. We observed altered lipid composition of EVs under hyperglycemic conditions, in both cell lines and different EV subpopulations

Summary/Conclusion: We conclude that hyperglycemic conditions have an impact on EV formation and their molecular content. Such metabolic alterations can be characterized on the molecular level to define molecular content and structure combining complementary information obtainable by SIMS, PALS, and TRAMs methods. Funding: Study supported by the National Science Centre grant OPUS 17 to E

LCAM is associated with neuron derived extracellular vesicles

Keerthie Dissanayake 2

Kasun Godakumara 2

Subhashini Muhandiram 3

 Laboratory of Microscopic Image Analysis and Machine Learning

Hungary information about the tumor and its microenvironment. The detection of specific mutations in EV-loaded nucleic acids has shown similar or improved sensitivity and specificity compared to cell free DNA analysis. Our group has shown that detection of BRAFV600E mutation in EV-associated nucleic acids (EV-NAs) isolated from lymphatic drainage from melanoma patients has prognostic significance. In this work, we have determined BRAF status (BRAFV600E mutation or BRAF wild type) in melanoma patients using an EV-NA-based liquid biopsy test in plasma and compared it to tissue biopsy information. Methods: Plasma was collected from a cohort of 18 melanoma patients (stages II-IV). Samples were collected in EDTA tubes, inverted several times, spun at 500 xg and frozen until subsequent analysis. BRAF status in tissue samples was determined using Cobas assay (Roche). Plasma samples were analyzed using the ExoDx BRAF V600E/K test by Exosome Diagnostics. Co-isolation of EV RNA and DNA along with any cfDNA present (EV-NA) was purified using the ExoLution Plus extraction technology (Exosome Diagnostics). Samples were subjected to reverse transcription and a highly sensitive quantitative PCR for determination of BRAF wt and BRAFV600E alleles. Results: From 18 patients, 5 were detected as BRAF mutant carriers by tissue biopsy. In two of those, BRAF mutation was detected also in plasma. Additionally, in 2 more patients, BRAF mutation was present in the plasma but not detected in the original tissue. The mutant allele frequency spanned between 0.01-3.43%. Twelve patients developed distant metastasis and presence of BRAFV600E mutation

Summary/Conclusion: Combination of information from tissue and EV-NA-based liquid biopsy analyzing BRAF mutational status achieves prognostic significance whereas only tissue biopsy is not informative enough. This study also adds evidence about the relevance of the analysis of BRAF in melanoma patients in their visit to the dermatologist to improve their prognosis. Funding: LABAE19027PEIN Fundación AECC

Detection of circulating extracellular vesicle-associated mutant DNA in the plasma of metastatic colorectal cancer patients

Stephanie Petrillo 1

Anthoula Lazaris 1

EVs were isolated from plasma of metastatic colorectal cancer (mCRC) patients (n=5) and patients with no disease (n=10) using ultracentrifugation and size exclusion chromatography. EVs were further characterized using nanoparticle tracking analysis, transmission electron microscopy and western blots. EV-associated DNA (EV-DNA) was isolated using ethanol precipitation methods. We employed droplet digital PCR (ddPCR) to detect single nucleotide polymorphism in the KRAS genes. Data was processed using QuantaSoft v.1.6 (Bio-Rad). The tumour mutations were verified using next generation sequencing. Results: The total EV-DNA of mCRC patients was significantly higher compared to healthy controls (P< 0.005). In addition, we successfully detected KRAS G12D mutant copies in the plasma of one of the five mCRC patients. Interestingly, the patient who was positive for mutant KRAS EV-DNA showed tumour allele frequencies (AF) of 60%, whereas the remaining four patient had AF below 20%

Summary/Conclusion: We showed higher levels of total EV-DNA in all cancer patients compared to the control cohort. The data supports the use of EV-DNA measured by ddPCR to detect tumour-specific DNA in clinical samples

Exosomal miR- promotes rhabdomyosarcoma tumor progression through paracrine signaling

Philippe Clezardin 4 ; Bassam Badran 2

Exosomes are extracellular vesicles that carry and deliver protein and nucleic acid cargo capable of modulating recipient cell function and influencing cancer progression. Previously, we identified miR-1246 as a commonly enriched miRNAs within all RMS-derived exosomes based on microarray results. Cellular and exosomal miR-1246 have been implicated in promoting cancer metastasis and can therefore contribute to RMS progression. Methods: We downregulated miR-1246 in RMS cells and subsequently isolated their corresponding exosomes. To explore the paracrine signaling induced after cellular miR-1246 knockdown we treated normal fibroblasts with these exosomes and investigated their proliferation, invasive and metastatic ability and by Hoechst staining and transwell migration and invasion assays. Results: Consistent with our previous results, treatment with RMS-derived exosomes resulted in a significant increase in recipient fibroblasts proliferation, migration, and invasion. Knockdown of cellular miR-1246 in RMS cells and subsequent treatment of fibroblasts with the exosomes reversed these effects indicating a potentiating role of exosomal miR-1246 in promoting progression of RMS through modulating recipient cell functional behavior

Summary/Conclusion: Our results demonstrate pro-tumorigenic effects of miR-1246 in RMS cells that can be mediated through paracrine signaling via exosomes. miR-1246 is therefore a promising biomarker and possible therapeutic target in RMS that should be further studied. Funding: This work was funded by a research grant from the Lebanese University

Exosomal miRNA and mRNA signatures as biomarker for head and neck cancer (HNSCC)

Linda Hofmann 1 ; Jasmin Ezic 1

Simon Laban 1

Primary tumor cells were obtained from the same HNSCC patients. Total exosomal and tumor RNA was used for targeted profiling of 798 miRNAs and 730 mRNAs. Differential presence and discriminatory potential of exosomal RNAs between HNSCC and HD were analyzed by multiple Mann-Whitney test and unsupervised hierarchical clustering. Ingenuity pathway analysis was applied to predict downstream effects of miRNAs and identify related pathways. Results: Of all detected exosomal RNAs, 55 % of miRNAs and 31 % of mRNAs were HNSCC-exclusive, while 8 % of miRNAs and 48 % of mRNAs were HD-exclusive. 91 miRNAs and 347 mRNAs were significantly differentially present between HNSCC and HD exosomes. Both exosomal RNA signatures could successfully assign samples to "Tumor" or "Healthy". 165 miRNAs and 146 mRNAs overlapped between corresponding tumor and exosomes and were considered as tumor-originating RNAs. These were filtered to 23 miRNAs inversely targeting 17 mRNAs

Summary/Conclusion: Exosomal miRNA and mRNA signatures have high discriminatory potential between HNSCC patients and HD. Final RNA candidates are currently validated in an independent cohort. Identified RNAs were related to pathways of immune regulation, inflammatory response and cellular development which highlights their relevance for disease pathogenesis. Funding: Deutsche Forschungsgemeinschaft (DFG) grant TH

SK-Hep-1, and SNU-398) and serum samples from HCC patients were analyzed. Exosomes were isolated from cell culture medium and serum samples using ExoQuick, and it was confirmed by expression of exosome markers (CD9, CD63, and ALIX) based on immunoblotting. PD-L1 protein expression was determined by western blot analysis using anti-PD-L1 antibody. Expression of PD-L1 mRNA

The two cell lines (SK-Hep-1 and HepG2) displayed consistent PD-L1 expression status in mRNA as well as protein. In further analysis of PD-L1 related miRNAs, 3 exosomal-miRNAs (miR-15a, -16, and -203) were positively correlated and 2 exosomal-miRNAs (miR-21 and -34a) were negatively correlated with PD-L1 expression. We also found significant correlations between PD-L1 expressing exosome numbers and clinicopathological features in serum samples of HCC patients

Summary/Conclusion: We provided the evidence for the potential of exosomal-miRNAs as novel non-invasive circulating markers for prediction of treatment efficacy of anti-PD-1/anti-PD-L1 therapy in HCC patients

Profiling of exosomal lncRNAs from colorectal cancer patients by RNA sequencing

Marie Madrzyk 1

Josef Mašek 3 ; Milana Šachlová 4

Introduction: Colorectal cancer (CRC) is the third most common cause of cancer-related deaths despite advancements in the disease detection. Late diagnosis is associated with poor prognosis therefore an early-stage CRC biomarkers are acutely needed. As a class of potential biomarkers, exosomal lncRNAs are often tissues specific and can serve as signalling molecules in intercellular communication. We aimed to optimise the RNAseq method of exosomal lncRNAs from the serum of CRC patients. Methods: Exosomes were separated by size exclusion chromatography from 150 μl of serum of CRC patients and healthy donors. The particles were characterised by electron microscopy, dynamic light scattering and by vesicle-specific content analysis. Protein markers were detected by western blot. After RNA isolation, the samples were converted into libraries using NEBNext Ultra II Directional RNA Library Prep Kit

Sequencing results comparing exosomal RNA samples from CRC patients (n=20) and healthy volunteers (n=6) revealed significantly altered levels of protein coding and non-coding RNAs including 37 lncRNAs (p-value< 0.01, fold change>2). The analysis showed dysregulated levels of lncRNAs associated with CRC such as SNHG1 but also genes that were not previously reported (eg. FAM201A and AC105389.2). Moreover, the gene set enrichment analysis revealed significantly enriched gene sets

Summary/Conclusion: Initial sequencing results suggest that lncRNAs have a great potential as biomarkers for non-invasive CRC detection, however it is necessary to validate lncRNAs of interest on the larger patient cohort. Also, some functional followup experiments are needed to determine their biological role in CRC pathogenesis. Funding: This work was supported from the projects GA20-18889S

Development of exosome-based plasma RNA biomarkers for high-risk prostate cancer

Khodor 1 ; Christian Fischer 1

Radka Stoyanova 2

Matthew Abramowitz 2 ; Sanoi Punnen 2

Alan Pollack 2

Exosomes were isolated from 1mL plasma, with RNA prepared using the ExosomeDx isolation platform Exo-Lution (PMID: 29051321). A hybrid-capture-based Next Generation RNA Sequencing (RNAseq) analysis was then performed to profile and identify differentially expressed genes. Results: We detected over 10,000 protein coding genes and ∼400 lncRNAs in plasma samples. 273 genes were differentially expressed between high-risk vs control but not in low-risk vs control. We identified four potential plasma RNA biomarkers of high-risk prostate cancer. These include three mRNAs with >20-fold lower expression in plasma from high-risk in comparison to low-risk patients. TCGA data show two of these mRNA markers are downregulated in prostate cancer tissue in patients with worse survival, confirming the role of the identified markers in aggressive prostate cancer

Summary/Conclusion: Plasma exosomal long RNA provides a rich reservoir of currently untapped biomarkers for high-risk prostate cancer. Investigation in larger patient cohorts is warranted. PS: Physiology and pathology: EVs from microorganisms Chair: Mariola J

Extracellular vesicles (EVs) are small membrane-bound vesicles involved in intercellular communication and initially described in reticulocytes, the host cell of P. vivax, as a selective disposal mechanism of the transferrin receptor (CD71) in the maturation of reticulocytes to erythrocytes. We have recently reported the proteomics identification of P. vivax proteins associated to circulating EVs in P. vivax patients using size exclusion chromatography (SEC) followed by mass spectrometry (MS). Parasite proteins were detected in only 2 out of 10 patients. Methods: We have implemented the direct immuno-affinity capture (DIC) technique to enrich in EVs derived from CD71-expressing cells. Results: Parasite proteins were identified in all patients totaling 48 proteins and including several previously identified P. vivax vaccine candidate antigens (MSP1, MSP3, MSP7, MSP9, Serine-repeat antigen 1, and HSP70) as well as membrane, cytosolic and exported proteins. Notably, a member of the Plasmodium helical interspersed sub-telomeric (PHIST-c) family and a member of the Plasmodium exported proteins

Summary/Conclusion: Enrichment of EVs by CD71-DIC from plasma of patients, allows a robust identification of P. vivax immunogenic proteins. This study represents a significant advance in identifying new antigens for vaccination against this human malaria parasite. Funding: IAH is a predoctoral fellow supported by the Ministerio de Economia y Competitividad (FPI BES-2017081657)

NF-κβ signaling, and 8 proteins were down-regulated. In cells treated with FhEVs, 14 proteins were up-regulated and 9 downregulated, including fibrinogenic proteins involved in extracellular matrix remodeling. No common proteins were found up-or down

FhEVs induce the production of fibrinogenic proteins by LX-2 HSC cells, suggesting their possible role in associated fibrosis, whereas DdEVs activate pathways related to inflammation, oxidative stress and apoptosis. These differences could correspond to differences in the migration of each fluke

Extracellular vesicles of Bacillus cereus: First insights into their potential role in pathogenicity

Bacillus cereus is a Gram-positive spore forming bacterial pathogen, which is well-known for its food poisoning potential [1]. In recent years, this opportunistic pathogen is gaining increasing importance as a causative agent of non-GI related diseases, including systemic as well as local infections [2]. With our current work, we aim to gain the first insights into the role of extracellular vesicles (EVs) in pathogenicity of B. cereus. Methods: In contrast to EVs of Gram-negative bacteria, EVs in Gram-positive bacteria are far less studied and their role in bacterial physiology and pathogenicity is largely unknown. Therefore B. cereus derived EVs were thoroughly analyzed by using multiple approaches: First EVs were isolated using a size-exclusion approach, which was further simplified to an ultracentrifugation only isolation protocol. The EV morphology was investigated using resin-embedded TEM, and NTA was used to enumerate the EVs and to estimate the sizes. To analyze the EV content a combinatory approach of proteomics and FTIR spectral fingerprinting was used. To assess the biological activity of the B. cereus EVs, in-vitro cell culture models using Caco2-Cells and bone-marrow-derived macrophages were used. Results: Within our work, we could show that the morphology of B. cereus derived EVs is comparable to EVs secreted by other gram-positive bacteria. However, the EV secretion rate and FTIR spectral fingerprints varies significantly among different strains

This work is expected to pave the way to decipher EV contribution to B. cereus pathogenicity and aid the development of novel strategies to

Fungal extracellular particles mediate adaptation to environmental stress

EPs were isolated from conditioned media by sequential centrifugation followed by separation on sucrose density gradient or size exclusion chromatography. EPs morphology was characterized by TEM, concentration and size by Nanoparticle tracking analysis and molecular content by immunoblotting and spectrophotometry. The role of EPs was evaluated by in vitro functional assay in different conditions. (EV-TRACK ID: EV210293) Results: We successfully optimized isolation of EPs from non-stressed Hortaea cultures. TEM micrographs showed heterogeneous nature of EPs, which also included EVs with cupshaped morphology. Hortaea EPs had the average (±SE) mode diameter of 97(±3.7) nm and concentration of 1.7(±0.041) ×10ˆ9 particles per mL of media. They carried typical EVs marker proteins α-tubulin and GAPDH, as shown by immunoblotting, but additionally packed Hog1, the main kinase in osmotic stress response. Further separation of EPs (density or size) showed heterogeneous population that differed depending on the external stimulus: depleted media, high osmolarity and/or absence of melanin

Summary/Conclusion: Extremotolerant Hortaea releases EPs enriched in melanin and protein Hog1, which contribute to osmotic stress adaptation. Funding: The Slovenian Research Agency grants P1-0170

Lactobacillus-derived membrane vesicles modulate the production of pro-inflammatory cytokines in the Caco cell monolayer in context of IL-beta stimulation

Miriam Sandanusová 2

Eva Vojtková 1

Lukáš Kubala 2  Institute of Biophysics of the Czech Academy of Sciences

Methods: Lactobacillus-derived MVs were isolated by ultracentrifugation-based floatation within sucrose cushion followed by the characterization using DLS, TEM, and WB (LTA marker). The protein cargo was determined by BCA, SDS-PAGE, and proteomics. Evaluation of MV-mediated bacteria-host intestine interaction was carried out using Caco2 cells and IL-1beta as a stimulator followed by detection of pro-inflammatory cytokines using ELISA. Results: MVs were visualized by TEM and size range was established using DLS (100 nm being the most abundant). The purified MVs samples contain proteins in concentration 200-400 ug/ml per MVs pellet with similar protein profiles determined by SDS-PAGE. A total of 337 proteins were consistently identified in MVs prepared under the same standard conditions

IL-1beta-treated Caco2 cells displayed an increase in IL-6 and IL-8 release when compared to untreated cells, reflecting the pro-inflammatory effect. In the presence of MVs used as 24-hour pre-treatment of Caco2 cells, a decrease of IL-6 and IL-8 by IL-1beta

Summary/Conclusion: Lactobacillus rhamnosus CCM7091 produces MVs with similar protein profiles under standardized cultivation conditions. Functional analysis suggests that MVs could be used as a pre-treatment tool to alleviate the response of the intestinal epithelium to IL-1beta stimulation

María Trelis 1

Spain Introduction: The trematode Fasciola hepatica is the causative agent of liver fluke disease (fasciolosis) in mammalian species. Modulation of host immunity is partly due to the release of extracellular vesicles (EVs) that can be internalized by host cells. We have identified two proteins present in F. hepatica-derived EVs that have anti-inflammatory effects in macrophages and alter the proteomic profiles and EV

Methods: Enolase and Fh16.5 proteins were produced in Escherichia coli M15, and purified with a Ni-NTA kit (Qiagen)

All assays were performed on FBS-free culture media. Results: Enolase, but not Fh16.5 protein, showed anti-inflammatory properties in LPS-stimulated macrophages and neither protein produced cytotoxicity. 38 proteins were significantly up-regulated in LX-2 cells upon exposure to the Fh16.5 protein and 80 were down-regulated. Regarding the enolase treatment, 33 proteins were up-regulated and 44 down-regulated. After the treatment with each protein, EVs from LX-2 HSC cells were successfully isolated and characterized, showing a higher concentration after treatment with Fh16.5, but not enolase. The proteomic analysis of the EVs from HSC cells showed that a total of 11 proteins were significantly up-regulated and 14 proteins down-regulated after Fh16.5 treatment, while 2 proteins were up-regulated and 5 down-regulated after enolase treatment

is the recipient of a predoctoral fellowship (CPI-20-353) funded by Conselleria d'Educació, Cultura i Esports

PS: Technologies and methods: Characterization of EV-molecules Chair: Alice Gualerzi, Laboratory of Nanomedicine and Clinical Biophonics, IRCCS Fondazione Don Carlo Gnocchi

Roberta Verta 1

Adele Tanzi 1

Sharmila Fagoonee 1

Simona Scalabrin 1 ; Raffaele Nuzzi 1

Extracellular vesicles (EVs) are abundant in all biological fluids, however the EV characterization in the aqueous humor (AH) is still undetermined. The aim of this work was to characterize EVs from AH (AH-EVs) in term of surface markers of cellular origin and function. Methods: We obtained AHs from patients with cataract undergoing surgical phacoemulsification and insertion of intraocular lenses (n=10 patients), after approval by the ethical committee and patients' written consent. AH-EVs were isolated by ultracentrifugation and size and quantification performed using nanoparticle tracking analysis. The AH-EVs were subjected to TEM, MACSPlex and Super Resolution microscope analysis. Subsequently, we investigated their in vitro effects on proliferation and wound heling of human immortalized keratinocyte cells (HaCaT) compared to PBS

cup-shaped morphology and expression of tetraspanins. Super resolution microscopy confirmed co-expression of CD9, CD63 and CD81. Moreover, MACSPlex analysis showed expression of mesenchymal/stem (CD105, CD133) and epithelial (EPCAM) markers. The in vitro evaluation showed that HA-EVs but not serum-EVs induced a significantly higher proliferation rate of HaCaT cells and a faster wound repair

Summary/Conclusion: We here provided evidence of the presence of EVs in the AH and characterized them for exosomal, mesenchymal and epithelial cell marker expression, highlighting a possible origin from limbal stem cells and corneal epithelium

AH-EVs displayed in vitro proliferative and regenerative capacity, potential beneficial effects in eye pathophysiology

The anti-inflammatory and anti-oxidant properties of lemon-derived extracellular vesicles are achieved through the modulation of ERK/NF-κB and AhR/Nrf signaling pathways

Francesco Dieli 4

Alice Conigliaro 2

Alimentari e Forestali, Università degli Studi di Palermo, palermo, Italy Introduction: Plant-derived extracellular vesicles (PDEVs) are gaining increasing interest in the scientific community for their biological properties, in particular for their role in the cross-kingdom interaction. In this study, we have in-depth characterized the content of EVs from Citrus limon L. (LEVs) and we explored their possible protective effect against inflammatory and oxidative processes both in vitro and ex vivo models. Methods: We isolated PDEVs from the juice of Citrus limon L. through sequential centrifugation and ultracentrifugation. LEVs were characterized at dimensional (DLS, NTA), morphological (TEM), and biochemical (western blot) levels. We also identified flavonoids, limonoids, and lipid contents through liquid chromatography coupled to mass spectrometry. Finally, we carried out a Next-generation sequencing analysis to identify the RNA profile of LEVs. To investigate whether LEVs have a protective role on the inflammatory and oxidant processes, murine and primary human macrophages, as well as skin fibroblasts were pre-treated with LEVs and then stimulated with LPS and UV radiation. Results: LEVs possess a variety of flavonoids and limonoids that exert beneficial roles, such as anti-inflammatory, anti-oxidant, and anti-cancer. The identification of flavonoids in LEVs prompted us to investigate their role in inflammatory and oxidative stress models. We found that pre-treatment with LEVs decreased gene and protein expression of pro-inflammatory cytokines and reduced the nuclear translocation and phosphorylation of NF-κB in LPS-stimulated murine macrophages. The inhibition of NF

Summary/Conclusion: In conclusion, we demonstrated that LEVs exert anti-inflammatory and anti-oxidant effects by modulating the ERK1-2/NF-κB and the AhR/Nrf2 signaling pathways. Ongoing studies are aimed at correlating the observed biological effects with the Inc-RNAs and microRNAs identified in LEVs. Funding: Stefania Raimondo is supported by PON

Microalgal extracellular vesicles as nature designed delivery platforms for therapeutic and cosmetic applications

Samuele Raccosta 4

Introduction: Nanoalgosomes are microalgae-derived extracellular vesicles (EVs) that can be used as nanocarriers for precision medicine. Our previous works have thoroughly characterized these nanovesicles, eliciting their EV identity. Here, we exploit the natural properties of nanoalgosomes as effective carriers of bioactive ingredients for the development of novel pharmaceutical and cosmetic formulations. Specifically, we focused on their endogenous cargo bioactivities and then we evaluated nanoalgosomes loaded with different bioactive molecules to develop natural drug delivery systems. Methods: Nanoalgosomes are separated at pilot-scale from a suspension of Tetraselmis chuii cells using tangential flow filtration (TFF) and the physicochemical characterization was carried out according to our quality control check, based on MISEV2018 guidelines. Next, nanoalgosome bioactivity is evaluated using different approaches (e.g., antioxidant assay). different molecules (e.g., doxorubicin as chemotherapeutic drug model and RNAs) and protocols were used to efficiently perform nanoalgosomeloading. After the nanoalgosome engineering, the loaded nanoalgosome functionality is analysed in normal and tumour cells

Summary/Conclusion: These studies demonstrate how nanoalgosomes represent an efficient natural delivery system of highvalue microalgal substances and exogenous bioactive biomolecules and/or synthetic drugs, hence fostering the exploitation of such sustainable EVs in a scalable manner. Funding: VES4US and BOW projects funded by the EU-H2020 programmes under grant agreements

Broccoli-derived extracellular vesicles as nanocarriers of exogenous microRNAs for RNA-based therapy

López de las Hazas 1

Francisco Marín 2

Plant-derived EVs protect miRNAs against RNase digestion and increase miRNA stability. Characterization methods have shown that plant EVs have similar structures to mammalian EVs which may facilitate their uptake, tissue distribution and ability to produce biological effects in animal cells by a cross-kingdom regulation. These observations suggest the potential use of plantderived EVs from natural sources as delivery vehicles for oral miRNA-based therapy. Methods: In this study, we evaluated the use of plant EVs as potential vehicles for RNA drug delivery. EVs were isolated from broccoli combining ultracentrifugation and size exclusion chromatography. Isolated EVs were then loaded with exogenous ath-miR159a, a highly expressed miRNA in plant EVs. miRNA stability was assessed by RNase A protection assay and in-vitro digestion in GI simulated conditions. The suitability of EVs as delivery vehicles for extracellular RNAs was tested by evaluating the absorption of ath-miR159a by Caco-2 cells. The potential gene targets were predicted by bioinformatic tools. Results: Broccoli-derived EV isolation was confirmed by Transmission Electron microscopy and EV concentration was measured by Nanoparticle Tracking Analysis. In-vitro digestion revealed the protector effect of the EVs and the effective miRNA incorporation. In-vitro exposure analysis of exogenous ath-miR159a (loaded into broccoli EVs) revealed significant

Summary/Conclusion: Results support that plants are a cost-effective natural source of extracellular vesicles which can be used as nanocarriers of bioactive miRNAs with a potential use in RNA-based therapy

Nanoalgosomes from marine diatom Phaeodactylum tricornutum

Matej Hočevar 3

Matic Kisovec 4

Marjetka Podobnik 4

Ester Heath 7

Nanoalgosomes (NAs) have recently become a subject of growing interest due to their pharmaceutical and biotechnological potential. NAs have previously been studied in detail in the marine microalgae Tetraselmis Chuii. In this work, we harvested and characterized extracellular material of Phaeodactylum tricornutum. The production of NAs was studied in cultures grown at varying conditions and at different growth phases. Methods: P. tricornutum (CCAP 1052) was grown in artificial marine water enriched with f/2-Guillard's solution. Cultures grown at different conditions (varying culture medium, ventilation, illumination and temperature) were observed. The cell density was quantified by flow cytometry. NAs were isolated by differential centrifugation from selected cultures. The cells and NAs isolates were visualized by light (LM), scanning electron (SEM), and cryogenic transmission electron microscopy (cryo-TEM). Isolates of NAs were also characterized by dynamic light scattering (DLS), and SDS-PAGE electrophoresis. NAs were lysed in 0.2% RapiGest detergent using 5 freeze-thaw cycles in liquid nitrogen and sonication; digested with trypsin, and analysed by LC-ESI-MS/MSbased proteomic workflow. Results: Cells in different liquid and solid cultures exhibited different morphologies and organizations. The largest amount of colloidal nanoparticles was produced in cultures of fusiform cells in the exponential growth phase. Various roughly round particles ranging from 20 nm up to several micrometers were observed by SEM in the culture samples, while only the small ones (about 50-300 nm in size) were observed in the NAs isolates. Cryo-TEM of the NAs isolates revealed membrane-enclosed vesicles with a radially-oriented fibrous decoration

It is indicated that NAs formation is increased when the microalgae are in the exponential growth phase. Nanofilaments found in the samples affect the flotation and three-dimensional organization of the cells in the cultures, and may impact the isolation by differential centrifugation. Funding: This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 801338 and ARRS project

Cow and goat milk extracellular vesicle effects on two in vitro models of human intestinal inflammation

Among their known functions, Extracellular Vesicles (EVs) can modulate immunity and inflammation. An EV role in the pathogenesis of chronic inflammatory diseases such as inflammatory bowel disease (IBD) has been found. IBD, mainly composed of Crohn's Disease and Ulcerative Colitis, have a dramatically rising incidence in western countries and lacks a resolutive therapy. Several factors seem to contribute to its onset, but IBD are certainly characterized by immune dysregulation and barrier function disruption. Milk-derived EVs (mEVs) can have immunomodulatory and anti-inflammatory effects and milk is one of the most promising food sources of EVs. In this context, the aim of this study was to evaluate bovine and goat mEV anti-inflammatory and immunomodulating effects on two in vitro models of human bowel disease: a co-culture of Caco-2 and THP-1 cells, and IPEC-J2 cells, an accepted swine model. Methods: To this porpoise, mEVs were isolated and characterized following the methods used in our previous study

Fate of milk extracellular vesicles during infant gastro-intestinal digestion

Introduction: The aim of this study was to track the fate of bovine milk extracellular vesicles (EV) during human infant gastrointestinal digestion. Methods: Bovine milk was digested according to the INFOGEST digestion protocol modified for the infant gut

Food Chem). Samples were taken after gastric and intestinal phase and for indepth studies, bile or enzymes were excluded or diluted. For EV separation fat was removed from test samples by centrifugation (3000g, 15 min) and caseins were removed by acidification

contamination marker: β-lactoglobulin), nanoparticle tracking analysis and atomic force microscopy. Results: Milk EVs survived gastric phase conditions, but failure to detect markers TSG101, XDH and CD9 indicated that EVs were disrupted and digested during the intestinal phase. This was not due to bile concentration, but rather to intestinal enzymes. In addition, intact EVs were not detected after intestinal phase by atomic force microscopy. The particle concentration of milk EVs before digestion was 1.82x1010±1.34x1010 particles/mL of milk and it significantly

Summary/Conclusion: Intact milk EVs were present at the end of the gastric digestion, but intestinal digestive enzymes negatively impacted on EV structure and yield. Funding: Irish Department of Agriculture food and fisheries

Methods: HME isolation: The pH of human milk was adjusted to 4.6. Then a series of gradient ultracentrifuge and filtration were carried out. The exosome pellet was washed by PBS solution. Characterization of HME: a) Nanoparticle tracking analysis for size, b) Transmission electron microscopy for imaging, c) Exocheck exosome antibody arrays kit for protein determination Cell transport study for permeability of HME, the Caco-2 cell was incubated on a transwell. Cell uptake: The cell uptake ability was measured after cell transport studies. Cell proliferation study: 4-day Caco-2 cell proliferation study was conducted. The cell viability was measured by MTS every 24h after sampling. Stability of HME in intestinal digestion solution: Fasted State Simulated Intestinal Fluid (FaSSIF) and Fed State Simulated Intestinal Fluid (FeSSIF) were used to simulate intestinal fluid

Biocinchonic Acid (BCA) protein assay and NTA were used to measure the protein concentration and the size after digestion

or postural instability. No reliable biomarker is available either for ALS diagnosis, for the monitoring of disease progression or assessment of response to therapy. Literature showed that astrocytes derived-extracellular vesicles (EVs) carrying on the surface GLAST (GLAST+ EVs) were increased in ALS preclinical model but not data were available in the humans. Nowadays, EVs are recognized as biomarker of several human diseases, and they transfer their cargo to the recipient cells by affecting their functions. GLAST is the transporter of glutamate, the main neurotransmitter in the central nervous system. An excessive accumulation of glutamate is responsible for neuronal death. The aim of this pilot project was to search for GLAST+ EVs in ALS patients. Methods: We enrolled 25 ALS patients at the Hospital Maggiore della Carità and 17 healthy age-matched controls. GLAST-EVs were quantified in plasma by flow cytometry using a fast (1hr) method, recently validated by our laboratory. Results: We found that GLAST+ EV counts were significantly higher in ALS patients compared to HC. Importantly, by stratifying ALS patients in fast and slow progressors, we found that GLAST-EVs counts were even higher in fast patients compared with slow ones. Summary/Conclusion: We identified for the first time a neuronal tissue specific EV in ALS patients which allows, from the clinical point of view to discriminate fast and slow progressor patients. We applied a method which can be easily transferred to the hospitals, already equipped with a flow cytometer, thus entering the diagnostic process. Our results are preliminary and will be validated in a larger cohort. 

ABSTRACT Introduction: Environmental stimuli such as stress and diet alter the sperm epigenome, particularly the small RNAs carried by the sperm that are inherited to the next generation and mediates paternal epigenetic inheritance. Previous studies have shown that the extracellular vesicles secreted by the epididymis epithelia convey the small RNAs to sperms. However, although the sperm that is capable of fertilising an oocyte in assisted reproduction is produced in the testis, the effect of these stimuli on testicular EVs (tEVs) and subsequently the sperm epigenome remain unexplored. Methods: Mice were fed with a high-fat diet (HFD) or chow diet (SD) for 24-26 weeks. The tEVs were isolated from the testes using a recently developed method that involved a one-step tissue dissociation followed by an affinity column-based isolation of the membraneous vesicles. The physical properties of tEVs were compared by the transmission electron microscope. The small RNA cargoes of tEVs were investigated by small RNA sequencing. The uptake of tEVs by sperm was monitored by fluorescence microscopy and the small RNAs profile in sperm were validated by realtime-PCR. Results: Our results showed that tEVs obtained from HFD-and SD-fed mice displayed similar hallmark cup-shape morphologies. Interestingly, tEVs isolated from HFD-fed mice were significantly larger (HFD 320.5±99.8 nm vs SD 251.9±81.0nm, p< 0.001). The tEVs carried negligible levels of tRNA that mediates the paternal epigenetic inheritance of metabolic traits. The tEVs isolated from HFD-fed mice had lower levels of miRNAs and snoRNAs. We further identified eight miRNA cargoes that showed differential abundance in tEVs isolated from HFD-and SD-fed mice. Notably, while the ejaculated sperm took up the tEVs from HFD-and SD-fed mice with comparable efficiency, the increased miR34b and miR34c in HFD tEVs did not cause an increase of these two miRNAs in the ejaculated sperm. Summary/Conclusion: HFD alters the small RNA profiles of tEVs. However, the differential abundance of miRNA cargoes in tEVs did not correlate with the levels of miRNAs carried by the ejaculated sperm. 

Introduction: Ewing sarcoma (EwS) is a highly aggressive cancer and the second most common malignant bone tumor in children and young adults with high propensity for metastasis. Patients with metastasis have a poor long-term outcome. Novel targeted therapeutic strategies that are more efficacious and less toxic are therefore desperately needed. Intercellular communication within the tumor microenvironment (TME) is emerging as a crucial mechanism for cancer cells to establish immunosuppressive and cancer-permissive environment. Extracellular vesicles (EVs) offer a candidate mechanism as they are actively released by tumor cells and enriched with proteins and RNAs to communicate with other cells in the TME. Methods: For EV purification, Conditioned medium (CM) was subjected to sequential centrifugation at 2000 g for 10 min and 10,000 g for 20 min. CM was then concentrated using the Tangential Flow Filtration Easy columns, passed through 0.22 μm filter, diluted with equal volumes of PBS and subjected to ultracentrifugation (UC) at 100,000 g for 4hrs. EV pellets were then re-suspended in 3 ml of PBS and pelleted again by UC. Purified EVs were dissolved in 500 μl PBS. EVs were quantified using Nanoparticle Tracking Analysis. Purified EVs were tested for presence of exosome markers (CD63, CD9, and CD81) using western blot and using R-PLEX Human CD63/CD81/CD9 (EV) Antibody Set. Cytokine response from EwS ADAR1+/-cells and their EV treated fibroblasts was analyzed using the customized U-plex inflammatory panel (MSD). droplet-based digital PCR (ddPCR) and RNA-seq were used to analyze the expression of repeat elements in cells and their EVs. Results: In our recent study involving whole transcriptome RNA sequencing, it was found that EVs secreted by EwS cell lines as well as those detected in the plasma of EwS patients are selectively enriched with Adenosine to Inosine (A to I) edited RNAs. A high proportion of these A-to-I edited transcripts are derived from diverse long and short interspersed retrotransposon elements (LINEs and SINEs), human endogenous retroviral elements (HERVs) and pericentromeric genomic regions, where their abundance in plasma was associated with metastatic progression. A to I conversion is catalyzed by the ADAR1 enzyme. We therefore performed ADAR1 knock-down (KD) in EwS cells, which accumulated cellular levels of these repeat RNAs in donor cells and limited their packaging into EVs. Moreover, we observed secretion of cytokines characteristic of a type 1 interferon response in ADAR1 KD cells compared to wild-type cells. Notably, treatment of fibroblasts with EwS-derived EVs resulted in intracellular accumulation of HSAT2 RNAs, but accumulation was reduced when EVs were derived from ADAR1 KD compared to control EwS cells. Summary/Conclusion: These results suggest that tumor cells secrete EVs enriched in A to I edited RNAs derived from various repeat elements that can target non-tumor host cells, with functional consequences remaining to be elucidated.

Maria J. Introduction: Idiopathic inflammatory myopathies (IIM) are a heterogeneous group of autoimmune disorders with distinct phenotypes. Extracellular vesicles (EVs) act in autoimmune diseases pathogenesis and have been proposed as disease biomarkers. This study aims at characterizing and investigating EVs as potential biomarkers of IIM. Methods: EVs were isolated from platelet-free plasma of 45 IIM patients and 45 age/sex-matched healthy donors (HDs) through size exclusion chromatography and ultrafiltration steps. EVs were observed by transmission electron microscopy (TEM), quantified by nanoparticles tracking analysis (NanoSight NS300), and immuno-characterized by imaging flow cytometry (Amnis ImageStream MkII) for the tetraspanins CD63, CD81, CD9, and integrin CD11c. Informed consent and local ethics committee approval were obtained. Results: TEM images showed intact small roundish particles (Ø range: 30-150 nm). Mean EV concentration was slightly higher in IIM than in HDs (1.95x10ˆ10 ± 1.47x10ˆ10 SD [EVs/mL] vs. 1.45x10ˆ10 ± 7.82x10ˆ9, p=0.0478) with mode size of 150.9 ± 19.9 nm and 154.5 ± 14.8 nm, respectively. EV levels were increased in patients with interstitial lung disease (n=24) (2.20x10ˆ10 ± 1.64x10ˆ10, p=0.0416) and significantly higher in patients with cancer-associated myositis (CAM) (n=8) (3.28x10ˆ10 ± 2.84x10ˆ10, p< 0.0001) compared to HDs. Moreover, EVs concentration was higher in IIM patients with CAM than in those without (n=35) (1.59x10ˆ10 ± 6.12x10ˆ9, p=0.0004). EVs concentration in pharmacologically untreated patients (n=13) (2.77x10ˆ10 ± 2.36x10ˆ10) was increased compared to treated ones (n=31) (1.63x10ˆ10 ± 7.12x10ˆ9, p=0.0092) and HDs (p=0.0011). CD63+ EVs were those most frequently detected both in IIM (n=16) (3.04x10ˆ8 ± 9.67x10ˆ7) and HDs (n=16) (2.70x10ˆ8 ± 1.19x10ˆ8). Summary/Conclusion: EVs seem to be increased in IIM and may be associated with some disease phenotypes. Moreover, IIM therapy seems to normalize their concentration. These results suggest EVs as useful potential biomarkers of IIM. T-cell tolerance, Biomarkers and Therapies in Type  Diabetes Team, Université de Paris, Institut Co-chin, CNRS, INSERM" Paris, France;  Cardiometabolic and Renal Risk Research Group, Biomedical Research Institute Clinic Hospital -INCLIVA, Valencia, Spain Introduction: Non-coding RNA (ncRNA), released into circulation or packaged into exosomes, play important roles in many biological processes in the kidney. The purpose of the present study is to identify a common ncRNA signature from exosomes, urine and plasma associated with early renal damage and its related molecular pathways by constructing a RNA-based transcriptional network. Methods: This is an observational case-control study which included twenty-one patients with essential hypertension (n=21) and twenty-two without persistent elevated urinary albuminuria (UAE) (≥30 mg/g urinary creatinine). Three individual libraries (plasma and urinary exosomes and total plasma) were prepared from each hypertensive patient for ncRNA sequencing analysis. Next, a RNA-based transcriptional regulatory network was constructed. Results: The three RNA biotypes with the greatest number of differentially expressed transcripts were long-ncRNA (lncRNA), microRNA (miRNA) and piwi-interacting RNA (piRNA). We identified a common 24 ncRNA molecular signature related to hypertension-associated albuminuria, of which lncRNA was the most representative. In addition, the transcriptional regulatory network analysis showed five lncRNA (LINC02614, BAALC-AS1, FAM230B, LOC100505824 and LINC01484), and the miR-301a-3p to play a significant role in network organization and to target critical pathways regulating filtration barrier integrity, tubule reabsorption and systemic endothelial dysfunction. Summary/Conclusion: Our study found a combined ncRNA signature associated with albuminuria, independently of biofluid origin (urine or plasma, circulating or in exosomes) that identifies a handful of potential targets involved in filtration barrier, tubule reabsorption and endothelial function that may be utilized to treating hypertension-associated albuminuria and cardiovascular damage progression 

Alisa Petkevich 1 ; Aleksandr Abramov 1 ; Vadim Pospelov 2  Peoples' Friendship University of Russia (RUDN), Moscow, Russia;  RUDN University, Moscow, Russia Introduction: Extracellular vesicles (EVs) and its molecular cargo may serve as a valuable predictive and prognostic biomarker for cancer. Apparently, there are four main informative molecules containing in EVs -proteins, microRNA, mRNA and DNA. In some researches, it is assumed, that EV DNA may be a more sensitive and informative option in terms of cancer prognosis and prediction compare to cell free circulating DNA despite isolation of the latter may be more yielded than of EV DNA. The aim of this study was to compare the sequencing data and quantity of double-stranded DNA isolated from EVs of different human biological fluids -blood serum, plasma and urine. Methods: Biological fluids were obtained from healthy volunteers (n=17, male=9, female=8, mean age=45 (30 -61)). Blood was taken to EDTA K2 tubes (4 ml) and centrifugated immediately after collection (15 minutes, 2000 g, 4 • C) to get plasma, blood was taken to SiO2 tubes (4 ml) and centrifugated immediately after collection (15 minutes, 2000 g, 4 • C) to get serum, 20 ml of "mid-stream" urine was centrifugated immediately after collection (30 minutes, 2000 g, 4 • C); all samples were stored at +4 • C for around 24 hours before EVs isolations. Samples were filtered 0,45 μm and then EVs were isolated with differential ultracentrifugation protocol. Size of isolated EVs was measured with NTA, samples were treated with DNAse before DNA isolation, DNA was isolated with DNeasy (Qiagen, Germany), DNA was eluted in 50 mcl of AE buffer and stored at -20 • C. DNA quantity was determined with Nanodrop® 1000 (Thermo Fisher Scientific). Double-stranded DNA (ds DNA) was determined with Agilent 7500 DNA Kit (Agilent Technologies). Library preparation was performed with TruSight Tumor 170, sequencing was performed with HiSeq™ 4000, data processing was performed with Illumina software. Results: Size of isolated EVs ranged from 150 nm to 350 nm, DNA quantity after DNAse treatment was more than 10 ng in every sample. The least quantity of ds DNA was 8 ng and was identified in EVs from urine. Sequencing error rate varied from 0,2x10-5 to 6 x10-5, median coverage rate for allele variant of 0,6% frequency was at least 1200x in all samples types. Summary/Conclusion: There is a slight difference in ds DNA concentration and length from EVs of different biological fluids. Working with DNA from EVs of cancer patients it is may be reasonable to perform normalization depending on the source of EVs.Introduction: A growing body of evidence supports the hypothesis that extracellular vesicles (EVs) and their associated RNAs (EV-RNAs) represent informative circulating material for liquid biopsies in prostate cancer (PCa) patients. However, details on the EVs populations specifically released by PCa into the blood, as well as on the PCa-related EVs-associated transcripts have not been fully elucidated yet. Methods: We evaluated the detection of PCa biomarkers in EVs samples isolated through three methodological approaches. Specifically, EVs were isolated from cancer cell lines as well as from the plasma of healthy donors and PCa patients (from the PRIME consortium study) by using ultracentrifugation, size exclusion chromatography, and a charge-based isolation method. We profiled the EVs by nanoparticle tracking analysis technology, imaging flow cytometry, and western blotting analysis to characterize the heterogeneity of their size distribution and the expression of protein markers. Finally, we performed RNA-Seq to investigate whether the detection of PCa-specific EV-RNAs was dependent on the EV isolation method. Results: By imaging flow cytometry and western blotting analysis, we observed similar EV subpopulations and PCa biomarkers (i.e. AR and PSMA) on EVs isolated from both cell lines and plasma. Furthermore, for all sequenced EV-RNAs we identified fragments of coding transcripts (i.e. mRNA) as well as of non-coding transcripts (including miRNAs, lncRNA and a variety of miscellaneous small RNA) with comparable composition of RNA species. Furthermore, by ddPCR we were able to detect PCa-associated transcripts (such as MYC) on EV-RNAs derived from PRIME PCa patients. Summary/Conclusion: The evaluated EVs isolation methods represent valid approaches to investigate PCa biomarkers towards the exploitation of EV-RNA in liquid biopsies. Introduction: Liquid biopsies in patients with cancer have recently emerged as a tool for early detection and residual disease monitoring. The most studied source has been total circulating DNA where cancer-specific mutations or profiles have been analyzed. However, extracellular vesicles (EVs) containing tumor-derived materials could potentially be used as source of cancer-specific DNA. The aim of this study was to investigate and compare DNA from human melanoma-derived EVs and total circulating plasma DNA. Methods: EVs were isolated from human melanoma metastatic tissues (n=6) using enzymatic treatment followed by ultracentrifugation. Both large and small EVs were pooled and bottom loaded on a density cushion. Tissue-derived EVs were characterized by transmission electron microscopy (TEM) and EV markers analysis using ExoView™ and Western blot. Using the ultra-sensitive SiMSen-seq technique and a panel of 34 melanoma-related genes, DNA from the patients' tumors as well as DNA from corresponding tumor-derived EVs and patient plasma was isolated and investigated. Introduction: In our previous work, we have shown that vesicles released by infective forms of Trypanosoma cruzi, the agent of Chagas' disease, modulate the inflammatory response of macrophages, increase invasion of parasites, which could explain increased inflammation and the progression of disease in experimental models, and eventually in the progression of human Chagas' disease. We also found that this process varies among different parasite isolates that produce different patterns of infection. The EVs shedding from parasites are heterogeneous and contain glyconjugates that interact with host cells. To gain more information about the role of these different populations, we now characterized the populations released by trypomastigotes forms of (Y strain) and evaluate their interaction with THP-1 human monocytes into macrophages. Methods: he isolated populations of EVs were labeled with RNA (TO) and DNA marker (Hoechst dye) and incubated with host human monocytes cells in RPMI 10% FBS and from 5 to 60 minutes by using an Image steam apparatus. The cells were further maintained for 24 to 48 hours and observed by Scanning Electronic microscopy to observe changes in the morphology and by Confocal microscopy after immunofluorescence labeling with markers of cell differentiation. Results: Both populations of EVs released by T. cruzi could be labeled by RNA, but not by DNA markers. When incubated 10 minutes with cells, the RNA labeling was incorporated in 60% of the monocytes reaching maximal labeling withing 1 hour. The incorporation was higher when using EVs of larger sizes. The cells then start to undergo morphological changes, and after 24 hours became sprayed, with as typical macrophages. To confirm the transformation into macrophages, the cells were labeled with anti-actin and anti-vinculin, both expressed in differentiated macrophages. Summary/Conclusion: All EVs of T. cruzi contains RNA that are rapidly transferred to monocytes. In parallel, these EVs can induce monocyte in vitro differentiation of a monocyte cell line to macrophages. Experiments are in progress to identify the components and the role of RNA present in the EVs involved in the differentiation process. Introduction: Dicrocoelium dendriticum and Fasciola hepatica are two worldwide-distributed trematodes than can coexist in the same host, complicating their specific diagnosis. They can infect the liver and bile ducts of many mammalian species, causing liver damage associated with parasite migration, the activity of fluke secreted products, and inflammation. The manipulation of the host response is mostly due to the release of molecules at the host-parasite interface (excretory/secretory product (ESP)), which include extracellular vesicles (EVs). Although there are a few reports on ESP interactions with liver cells, the effects elicited by EVs have not been described. Methods: Parasites were obtained from local abbatoirs (Valencia, Spain) and cultured to obtain ESP. EVs were isolated by differential centrifugation and size-exclusion chromatography (SEC), and characterized by NTA, TEM and immunogold labeling for EV proteins (EVtrack ID EV220006). EVs were added to the LX-2 human hepatic stellate cell line (SCC064, Merck), cell pellets were obtained and analyzed through Label-free quantitative proteomics and Western Blotting. MTT assays were performed to assess EV cytotoxicity. All assays were performed using FBS-free culture media. Results: EVs from F. hepatica (FhEVs) and D. dendriticum (DdEVs) were successfully isolated and characterized. 63 proteins were significantly up-regulated in LX-2 HSC cells upon exposure to DdEVs, including proteins involved in the ROS response and ABSTRACT Introduction: Endothelial derived extracellular vesicles (EndoEVs) have been found to influence processes such as inflammation, coagulation, or the specific progression of atherosclerosis. Nevertheless, their small subfractions (< 200 nm) in general as well as possible differences between different vascular origins remain poorly understood. Hence, to enable the use of EndoEVs to their full potential in context of various pathologies regarding not only the blood but also the lymphatic vasculature further research is key. Methods: We used nanoparticle tracking analysis (NTA) and fluorescence triggered -flow cytometry (FT-FC) to study EndoEVs derived from different vascular beds including blood, human umbilical vein and lymphatic endothelial cells as well as plasmaderived EVs in coherence with the MISEV guidelines. Cell type specific markers such as CD31, CD41/61, CD34, Podoplanin and others alongside the EV marker CD81 were used to evaluate expression on EVs and their presence in both in vitro and in vivo samples. Furthermore, protein concentrations were approximated using spectrophotometry to assess influences on assays using lipid membrane specific dyes. Results: The analysis of samples with different protein concentrations resulted in significant decrease in detection rates of stained vesicles showing important indications for sample preparation and data evaluation. Both NTA and FT-FC resulted in the detection of both cell type specific CD31 as well as the EV marker CD81. NTA analysis of plasma EVs showed signals significantly higher than the background whereas CD31 did not exceed the isotype control. Summary/Conclusion: We conclude that our work provides a basis for further characterization and evaluation of EndoEVs using sophisticated fluorescence-based methods. To further add to the current understanding of physiological EndoEV levels additional markers will be included from here on to set necessary parameters for the assessment of pathological deviations. Funding: Conducted work was partially funded by Particle Metrix. Introduction: Matrix vesicles (MVs) are extracellular nanostructures, released by mineral competent cells, initiating the nucleation of apatite inside MVs and bind to collagen fibrils, where apatite is released and continues to grow sustained by Pi and Ca2+ in the extracellular media (ECM). Tissue-nonspecific alkaline phosphatase (TNAP), hydrolyses extracellular PPi, which inhibits apatite formation, forming extracellular Pi, while ectonucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) produces extracellular PPi from extracellular ATP. Na,K-ATPase (NKA), a plasma-membrane ATPase, can hydrolyse intracellular ATP. The three enzymes are present in MVs. However, the role of NKA during mineralization, and the orientation of NKA in the MVs' membrane remain unclear. Methods: Mineralization and phosphatase activity of purified NKA from rabbit kidneys and in proteoliposomes containing dipalmitoylphosphatidylcholine (PC) and dipalmitoylphosphatidylethanolamine (PE) 1:1 (w/w) was assessed with ATP and compared to isolated MVs from femurs of chicken embryos. The minerals composition was studied by ATR-FTIR. Lipidomics of MVs highlighted presence of phosphatidylethanolamine. Results: The mineralization ability of PC:PE-NKA induced by the addition of 3.0 mM ATP and 2.0 mM Ca2+ was higher compared to that of pure PC-NKA, suggesting the preference of the active site of NKA toward ECM of PC:PE-NKA. The addition of 3 mM ouabain, which inhibits ∼90% of NKA, in the presence of ATP and Ca2 did not hinder nucleation nor mineral propagation.

Introduction: Pancreatic Ductal Adeno carcinoma (PDAC) has a poor survival rate due to late stage diagnosis. Usually, upon discovery, this cancer type will mostly be resistant to chemotherapy and radiation, characterize with fibrotic tissue and highly metastatic. Also, 80-85% of advanced cases are found to be p53 mutated. As previously described, tumors expressing p53 gain of function mutations (GOF) are known to induce pro-invasive alterations to the extra cellular matrix (ECM) secreted by fibroblasts. Notably, over 70% of the tumor mass are cancer associated fibroblasts (CAFs). CAFs can be found as activated and naïve cells in the tumor tissue and presents heterogeneous phenotypes. In PDAC, fibroblasts activation leads to the secretion of tumorigenic factors, cytoskeleton rearrangements and remodeling of tumor-associated ECM. Hence, changes in the ECM were reported to modulate cancer cell phenotypes and found to be dependent on CAFs infiltration. Such rearrangements in the PDAC tissue might form dense ECM produced by CAFs also known as desmoplasia or fibrosis. Importantly, desmoplasia is detected in ∼85% of PDAC patients. ECM-secreting CAFs promote cancer progression fueled by a cross talk with PDAC. We hypothesized that such interaction mediated by extracellular vesicles (EVs). EVs are small nanoparticles (40-150 nm) which can be secreted from all cells. The molecular cargo shipped by EVs (typically nucleic acids and proteins) has the capacity to modulate cellular phenotypes at the receiving end. Cancer cells can secret EVs carrying oncogenes and regulatory factors to neighboring and distant cells. The uptake of this cancer EVs can potentially lead to changes in cells faith. In this study we examined whether EVs shed by PDAC harboring mutant p53 can facilitate CAFs activation in a 3D in vitro system. Methods: In this study, we based our method on a 3D system for ECM deposition. Additionally, EVs isolation was done according to MISEV 2018 guidelines. Results: Our analysis suggests that EVs taken up by Fibroblasts results in changes of the fibers, orientation, and ECM thickness. Summary/Conclusion: TP53 mutated PDAC EVs uptake leads to changes in ECM orientation. Further experiments will be dedicated to study the molecular mechanism through which TP53 mutations are leading to changes in ECM deposition. We believe that EVs play a crucial role in fibrosis creation. These findings might lead to better understanding of this aggressive cancer and open a door for a new treatment approach.

Daniel Alfandari 1 ; Hila Ben Ami Pilo 2 ; Paula Abou Karam 3 ; Osnat Dagan 2 ; Carine Joubran 2 ; Ron Rotkopf 3 ; Neta Regev-Rudzki 3 ; Ziv Porat 4  Faculty of Biochemistry, Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot , Israel, Rishon Lezion, Israel;  Faculty of Biochemistry, Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot , Israel, Rehovot, Israel;  Weizmann Institute of Science, Rehovot, Israel;  WIS, Rehovot, Israel Introduction: We have previously developed a method that allows us to label EVs without the need for antibodies but by labeling other cellular components. By doing so, we could monitor different EV populations according to their distinct cargo components, and the cargo distribution inside recipient cells post uptake. Here, we took this approach one step further, and monitored by live uptake assay the kinetics distribution of the signal of RNA cargo of Pf-derived EVs within two different recipient cells of the human host immune system, monocytes vs macrophages. By staining the RNA cargo of the vesicles, we were able to directly track the cargo's internalization over time and measure the dynamics of the RNA distribution inside human monocytes and macrophages. We examined three different parameters post uptake of the vesicles to the recipient cells over the course of 1 hour. Surprisingly, we observed significant differences in the dynamics of the RNA cargo distribution within monocytes vs macrophages, which might suggest a distinct roles for the RNA cargo post uptake to the target cells. These data demonstrate Introduction: Previously, we have shown that treatment of arthritic mice with BMEVs ameliorated cartilage damage but a direct effect on cartilage cells (chondrocytes) was not studied (1) . In this study, we evaluated the direct effect of BMEVs on cartilage proteoglycan depletion and chondrocyte gene expression. Furthermore, their effect to IL-1 induced catabolic effects in chondrocytes was studied. 1. Arntz O.J. Mol Nutr Food Res. 2015 Methods: Isolated BMEVs were characterized by NTA, microBCA, transmission electron microscopy, sucrose density gradient, and western blot (WB). Human cartilage explants, their enzymatically isolated chondrocytes, and immortalized chondrocytes were exposed ex vivo, to BMEVs. BMEVs were PKH-67 labelled and uptake in chondrocytes was determined by fluorescence microscopy. By RT-qPCR, mRNA expression levels of cartilage destructive enzymes (ADAMTS5, MMP1, MMP3, MMP13) and the enzyme inhibitor (TIMP-3) were measured in chondrocytes after BMEVs exposure. The effect on glycosaminoglycan (GAG) release from cartilage explants into the culture medium was studied by the 1,9-dimethylmethylene blue (DMMB) assay. The effects of BMEVs on IL-1-induced gene expression was determined by RT-qPCR. Results: Based on size, protein content, density and presence of exosome markers CD63, CD81, HSP-70, and Alix, we confirmed that BMEVs were pure and express markers of exosomes. Chondrocytes showed uptake of BMEVs. BMEVs exposure to chondrocytes resulted in >50% reduced expression of ADAMTS5, MMP1, MMP3 and MMP13, while TIMP-3 was enhanced (>300%). Cartilage explants exposed to BMEVs, showed reduced GAG release (>33%) and inhibition of MMP1 gene expression (>70%). IL-1 treated chondrocytes showed enhanced MMP1, 3 and 13 mRNA levels which were counteracted by BMEVs. Summary/Conclusion: This study showed a direct protective effects of BMEVs on human chondrocytes and cartilage, and suggest that the observed cartilage protection in murine arthritis models could be due to a direct effect of BMEVs on chondrocytes. Funding: This study was powered by Health-Holland, Top Sector Life Sciences & Health (grant 441 LSHM19108 SGF, REFIT project) allocated to Dr. Fons AJ van de Loo.

Introduction: RNA interference, which is mediated by small interfering RNA (siRNA), is a powerful therapeutic approach. However, oral administration of siRNA is currently not possible due to extremely poor delivery efficacy. Poor delivery efficacy in turn is a result of nucleic acid instability in the gastrointestinal (GI) tract and highly inefficient transfection due to poor transport across the biological barriers of the intestinal mucosa. Bovine milk exosomes have been demonstrated to be capable of crossing the intestinal mucosa and hence possess potential as delivery vehicles for facilitating intestinal (i.e. oral) delivery of siRNA. This work explored this potential. Methods: Exosomes were isolated from bovine milk by ultracentrifugation and purified by size exclusion chromatography. Exosomes were characterised for size, zeta-potential and expression of exosomal protein markers. GI stability of exosomes was measured by exposure exosomes to fasted-state and fed-state simulated small intestinal fluids (FaSSIF and FeSSIF, respectively). The ability of exosomes to transport across an in vitro intestinal model (Caco-2 monolayers) was tested before and after exposure of exosomes to simulated small intestinal fluids. siRNA was incorporated into exosomes via transfection and the effect of exosomemediated delivery on cell uptake (Caco-2 cells) of siRNA established. Results: Exosomes were characterized with the size around 130 nm and slightly negative charge. Isolated exosomes were identified with confirmed expression of typical protein markers. Both of FaSSIF and FeSSIF demonstrated no effect on exosomes size and DPH: HME was damaged in FaSSIF and FeSSIF and 1% SDS compared with that in PBS. The FaSSIF and FedSSIF groups have similar intensity. The size of HME in FedSSIF mostly decrease when Fastsif and PBS batches also have a decrease compared with stock exosome. There is no significant difference amount these digested exosomes. Summary/Conclusion: From the results so far, this isolation method can prepare exosomes from human milk. The characterization of HME was measured and determined. The cell permeability, cell uptake ability and the stability of HME show the potential use in oral drug delivery. The cell proliferation of HME shows the potential in the treatment of intestinal damage.

Laura Soriano-Romaní; Elías Hurtado; Begoña Ruiz AINIA, Parque Tecnológico de Valencia. C/ Benjamín Franklin -, E Paterna (Valencia). Spain, Valencia, Spain Introduction: EVs from fruit juices raise as new potential sources to obtain plant-derived functional ingredients for both food and topical use products. It is known that the isolation technology modifies the cargo of the EVs and, consequently, their functional effect. The objective was to characterize Citrus clementina derived EVs, isolated by scalable methodology, in terms of intestinal and dermal absorption and possible immunomodulatory role using different cell models Methods: To meet our goal, the scalable technology of ion exchange chromatography was used to isolate EVs from Citrus clementina juice. EVs were characterized by NTA and electronic microscopy. Then, EVs were fluorescent labeled with CFSE, and intestinal epithelial cells and keratinocytes were seeded in permeable filter supports for transport assays. In addition, the immunomodulatory role of EVs was evaluated in macrophages, analyzing changes in ARN expression of cytokines. High quality protein from EVs was isolated and characterized by LC-HRMS Results: EVs showed normal morphology and a mean size of 194±21 nm. The in vitro cell assays with labeled EVs showed a transport of 39.8% after 4h in contact with intestinal cells and 29.2% after 1h in contact with keratinocytes. EVs increased the expression of IL-8 and IL-10 but not IL-1b in macrophages, showing a clear immunomodulatory effect. Preliminary proteomic results showed several protein clusters, including different cysteine peptidases that may explain the biological effect in macrophages Summary/Conclusion: EVs from Citrus clementina isolated by scalable methodology for its industrial application, were transported by intestinal and dermal cells and showed potential as functional immunomodulatory ingredients. A more extensive omic study on its biocargo could identify and explain new potential effects and the mechanism of action of them Funding: Project financed by IVACE and the European Regional Development Fund, ERDF (Ref. IMDEEA/2020/97)