key: cord-0023916-qg8myvqa authors: Mammes, Aurelien; Pasquier, Jennifer; Mammes, Olivier; Conti, Marc; Douard, Richard; Loric, Sylvain title: Extracellular vesicles: General features and usefulness in diagnosis and therapeutic management of colorectal cancer date: 2021-11-15 journal: World J Gastrointest Oncol DOI: 10.4251/wjgo.v13.i11.1561 sha: 05e64a1cdfe9577c77b0df34b7760a721d508ae7 doc_id: 23916 cord_uid: qg8myvqa In the world, among all type of cancers, colorectal cancer (CRC) is the third most commonly diagnosed in males and the second in females. In most of cases, (RP1) patients’ prognosis limitation with malignant tumors can be attributed to delayed diagnosis of the disease. Identification of patients with early-stage disease leads to more effective therapeutic interventions. Therefore, new screening methods and further innovative treatment approaches are mandatory as they may lead to an increase in progression-free and overall survival rates. For the last decade, the interest in extracellular vesicles (EVs) research has exponentially increased as EVs generation appears to be a universal feature of every cell that is strongly involved in many mechanisms of cell-cell communication either in physiological or pathological situations. EVs can cargo biomolecules, such as lipids, proteins, nucleic acids and generate transmission signal through the intercellular transfer of their content. By this mechanism, tumor cells can recruit and modify the adjacent and systemic microenvironment to support further invasion and dissemination. This review intends to cover the most recent literature on the role of EVs production in colorectal normal and cancer tissues. Specific attention is paid to the use of EVs for early CRC diagnosis, follow-up, and prognosis as EVs have come into the spotlight of research as a high potential source of ‘liquid biopsies’. The use of EVs as new targets or nanovectors as drug delivery systems for CRC therapy is also summarized. Exosome and its cargo content. Small extravesicles (SEVs) are nano-sized membrane vesicles released by a variety of cell types and are thought to play important roles in intercellular communications. SEVs contain many kinds of proteins, either cytosolic or plasma membrane ones. Transporters, receptors, signaling proteins… but also enzymes can be evidenced. Metabolites are also present as well as nucleic acids. Genomic and mitochondrial DNAs, and multiple RNAs (mRNAs, miRNA, lncRNA, circRNA…) can be detected. Through horizontal transfer of these bioactive molecules, SEVS are emerging as local and systemic cell-tocell mediators of oncogenic information. MHC: Major histocompatibility complex; MVE: Multivesicular endosomes. Among highly representative proteins that can also be found in SEVs figure important regulators of EVs trafficking: (1) Members of the Rab family that play well-established roles in vesicle transfer between intracellular compartments such as MVEs driving to PM for SEVs secretion[54,55]; (2) SNARE membrane fusion machinery, through SNARE complexes recruitment, that is specifically required for MVEs docking then fusion with PM[30,35,56]; (3) ESCRT proteins and important ESCRT side molecules implicated in ESCRT assembly or nucleation like ALIX [57] ; and (4) Tetraspan transmembrane proteins (tetraspanins), highly enriched in SEVs, that are also involved in ESCRT-independent EVs release [58, 59] . Tetraspanins display high affinity for cholesterol and sphingolipids such as ceramides which may create PM microdomains as it occurs in membrane reconstitution experiments [60] . Their interaction with PM proteins, either by direct association or by entrapment in tetraspanin-enriched PM microdomains, facilitates their sorting into EVs[58,61-63]. Interestingly, EVs can also transport mitochondrial proteins that may be active. Two mitochondrial inner membrane proteins MT-CO2 (encoded by the mitochondrial genome) and COX6c (encoded by the nuclear genome) were highly prevalent in the plasma of melanoma patients, as well as in ovarian and breast cancer patients defining a new EVs subtype [64] . As not only mitochondrial membrane proteins but also mitochondrial enzymes are present in EVs, mt-EVs could affect the metabolic output of the recipient cells by either preventing inflammation [65] or promoting tumor growth [66] [67] [68] . SEVs specific endosomal-driven content allows their distinction from ectosomes that can directly bud and shed from PM at lipid-raft-like domains [69] . These vesicles, now generically referred to as MLEVs, are extremely heterogeneous in size, ranging from 200 nm to as large as 10 μm. They are generally enriched in cell surface or integral transmembrane proteins, reflecting their PM origin [70, 71] . For example, during reticulocyte maturation, autophagosomal exocytic event is coupled with plasma membrane blebbing that release glycophorin A, an integral plasma membrane protein, into budding vesicles [72] . Last, SEVs content is also distinct from apoptotic microparticles or apoptotic bodies (apoBD). ApoBDs are larger than SEVs and MLEVs as they have a diameter of 800-5000 nm [73] . ApoBDs encapsulate residual ingredients of dying cells. They are enriched with autoantigens and pro-inflammatory factors [74, 75] and bear key markers of cell disassembly such as ROCK1 and PANX1 and apoptotic markers such as CD31 or Annexin V. Aside proteomic studies that try to unravel the complex protein repertoire in EVs, metabolomic studies reveal that EVs contain different classes of lowmolecular-weight compounds. Organic acids, nucleotides, sugars and their derivatives, carnitines, vitamins and related metabolites, and amines are frequently evidenced in EVs [43] . Of course, most of these metabolites were generally derived from cytosolic cellular pathways, as large portions of cytosol are engulfed in ILVs then EVs [76] . Nevertheless, metabolites presence could also result from either specific metabolite sorting or ILVs/EVs in situ synthesis through residing metabolic enzymes as high metabolite concentrations over the cellular levels were reported in EVs [77] . Complete but more often partial metabolic routes can be evidenced in EVs explaining why EVs metabolite identification does not generally cover the whole parental cell metabolome but represents a miniature subset of it. Lipids are also frequently found in EVs. EVs lipidome analysis allows characterization of different classes of lipids, including glycerolipids, glycerophospholipids, sphingolipids, sterol lipids, and fatty acids confirming similarity between EVs lipid content and their parental cells membranes composition [78] . As it is important to preserve functional flexible lipid bilayer as well as right ion composition and pHhomeostasis[60], numerous ATP-driven transporters and ion-pumps are also found in EVs. To be fully functional, these elements need energy supply that may be given either by glycolytic enzymes [79] or even mitochondrial ATP synthase that is frequently found in EVs [64] . To optimize energy thresholds, such enzymes and substrates seems to be organized in metabolons that have been found to be fully functional in EVs [80] . Every cell may send out a range of messages to distinct still unknown targets, and both messages and targets may vary depending on the metabolic state of the producing cell. In EVs metabolic composition is of importance as it may represent a specific environment ("climate") the parental cell is going to transfer to the recipient one. By providing substrates for biosynthesis, EVs-transported aminoacids (glutamine, leucine…) have been shown to strongly affect the tricarboxylic acid (TCA) cycle of the recipient cancer cells thus improving nutrient status of fast growing and proliferating cells [81] . By providing both enzymes and substrates, adipocytes EVs stimulate melanoma fatty acid oxidation (FAO) that increase mitochondrial activity redistributes mitochondria to membrane protrusions of migrating cells, which is necessary to increase cell migration [82] . Interestingly, using various cell culture protocols, several reports have shown that EVs production in quantity and composition is largely influenced by external factors[83], the most striking variation being in the EVs metabolomes [84] . As slight metabolic variations could drive cancer cell reprogramming[85], the role of EVs seems central in that process. Valadi and Skog both demonstrated that EVs transported mRNAs that can be translated into protein, providing the first evidence of virus-independent genetic material horizontal transfer between cells [86] . Since these pioneering studies, the presence of RNAs, within EVs have been reliably shown with either microarrays or real-time quantitative polymerase chain reaction techniques in numerous reports [47] . This presence can easily be explained as cytosolic proteins engulfment, resulting from a microautophagy process[87], involve proteins located close to the MVE outer membrane during its inward budding and can comprise RNAs molecules [86] . Those RNA species include not only mRNAs but also rRNA, tRNA, snRNA, snoRNA, piRNA, Y-RNA, scRNA, SRP-RNA, 7SK-RNA and lncRNAs. All these RNAs can be transferred to the recipient cells [88, 89] . In addition, two major components of the RNA-Induced Silencing Complex, namely DICER and Argonaute, aimed at producing miRNAs have been shown to associate with MVE and to be sorted into exosomes [48, 90] . This suggests that miRNAs are likely to be packaged into EVs along with proteins required for their processing or function) [91] . As largely protected from RNAses when packaged in EVs, miRNAs driven-gene regulation will be able to generate a multifaceted signaling response in the target cell. As EVs mRNAs are also functional and can be translated in the target cell[86], both mechanisms provide a direct modulation of recipient cell protein production. This new signaling pathway play specific roles in intercellular communication during various physiological [14, 92] Considering the many cell types that interact at the mucosal interface, the intestinal lumen could be a rich source for EVs in large bowel tissues as well as an interesting source of disease-specific EVs in pathological conditions. Normal colonic cells as a primary source of EVs: As most of our tissues, colonic tissue may be an important source of EVs. Intestinal epithelial cells (IEC) are located at the strategic interface between external environment and the body most extensive lymphoid compartment. Aside their essential role in nutrients absorption, IEC have been shown to play a key role in immune response by promoting and regulating luminal antigens presentation to mucosal immune cells [109] through EVs release at both apical and basolateral sides as IEC display all the elements needed for either antigen processing or EVs production [110] . These EVs contain molecules that are implicated in adhesion and antigen presentation, such as major histocompatibility complex (MHC) class I molecules, MHC class II molecules, CD63… [111] . As these EVs may also contain CD133, whose presence in lipid rafts play a pivotal role in the maintenance of stem cell features [112] , it has been suggested that CD133-containing EVs release may contribute to cell differentiation by reducing and/or modifying stem cell characteristic membrane microdomains composition within IEC apical plasma membrane [113] . Numerous studies have demonstrated that circulating EVs increased in patients with intestinal pathologies while EVs fractions are different in cancers, compared to patients with inflammatory intestinal diseases such as Crohn's or inflammatory bowel diseases (CD or IBD) [128] . EVs release in colorectal cancer: Acidity and hypoxia are key features in cancer that could affect exosome release. Tumor pH may range from 6.0 to 6.8, and the level of acidity is directly associated to the tumor level of malignancy as it selects among cancer cells those that will resist [136] . One consequence of acidity-driven cancer cell selection pressure is an increased EVs release by human cancer cells [137, 138] . Hypoxia is also a common characteristic of solid tumors and is associated with cancer progression and poor outcomes. It is generally associated with hypoxic environment that has also been shown to be an important cause of EVs release [139] . Hypoxic CRC cells can transfer Wnt4 mRNA to normal CRC cells by exosome, which can activate β-catenin signal and potentiate the invasive ability of normal CRC cells [140] . In hypoxic microenvironment, CRC cells-secrete miR-410-3p in EVs that promotes progression and metastatic potential of normoxic CRC cells via PTEN/ PI3K/Akt pathway[141]. Epithelial cancers may be driven by a relatively rare sub-population of self-renewing, multipotent cells, named cancer stem cells or cancer-initiating cells (CSCs). Increasing data show that CSCs play a crucial role not only in primary colorectal tumor formation but also in metastasis [142] . In addition, CSCs play a critical role in CRC relapse [143] . They display unique properties of self-renewal, infinite division and multi-directional differentiation potential [144] . Asymmetrical growth and slow-cycling cellular turnover renders them resistant to therapies that target rapidly replicating cells [145] . Not all CSCs in primary lesions are metastatic, allowing distinction between stationary cancer stem cells (SCSCs) and migrating cancer stem cells (MCSCs) [146] . SCSCs exist in colonic epithelial tissues and are active even in benign precursor lesions, contributing to tumor mass proliferation in situ[147]. On the contrary, MCSCs, which have undergone EMT, possess motility characteristics and are able to spread in other tissue to form metastatic tumor mass [148, 149] . Untreated colorectal tumors contain a population of quiescent/slow cycling cells resembling CSCs and overexpressing EMT markers such as Zeb2 [150] . As for ISC, maintenance of these scarce CSCs generally resides in very specialized niches [151] , allowing them to stay dormant for various to long periods of time [152, 153] . These niches represent a positive specific microenvironment which is able to maintain stemness and pluripotency [154] . The release of EVs by mesenchymal stromal niche surrounding cells drive hematopoietic stem cell clonogenic potential maintenance and survival, by preventing apoptosis through EV gene expression regulation [155] . This continuous crosstalk between CSC and their surrounding microenvironment is critical as a tiny variation in its modulation could induce important deregulation and subsequent tumor progression [156] . For example, miR-196b-5p, which is highly enriched in CRC patients serum EVs [157] has been shown to promote either CRC cells stemness or chemoresistance to 5-fluorouracil (5-FU) via targeting negative regulators of the STAT3 signaling pathway. Understanding the importance of EVs transfer in that context is a key feature for future CRC therapy [158] . Tumor microenvironment (TME) is a complex and dynamic network including both cancer and stromal cells. Stress conditions such as hypoxia, starvation, and acidosis increase tumor cells EVs release leading to TME changes and expansion. Such specific behavior is the consequence of a complex combinatory of bioactive molecules present in EVs [159] . Not only different form of RNAs but also proteins or lipids could account for these important changes. The release of CD133+ EVs by poorly differentiated CRC cells was found to increase Src and ERK phosphorylation in surrounding cells, with subsequent MAPK intracellular signaling activation and promotion of tumor growth [113] . Among TME, fibroblasts such as cancer associated fibroblasts (CAFs), endothelial cells and infiltrating immune cells are likely to be the major cell types that interacts with tumor cells through EVs signaling [162, 163] . Both nature and composition of TME-derived EVs is of importance as cellular origin of the EVs cargo will determine specific changes within the recipient cell [164] . Analyzing their effect on CRC tumor cells, TME-originating EVs have been evidenced to play a central role in cell proliferation[165], acquisition of invasive properties and increased migration [166, 167] , resistance to chemotherapy[168], angiogenesis development [169] , and escape from the immune system. On the other side, several tumorigenic signals are derived from CRC cells and conveyed to stromal cells through EVs. From the very beginning of CRC progression, CRC cells secrete EVs that can deeply modify TME cells [170] . CAFs are prompted by CRC cells EVs to harbor a highly pro-proliferative and pro-angiogenic phenotype [171] . These important stromal changes are driven by CRC cells EVs composition that is itself largely modulated by different factors such as differentiation or hypoxia [113] . All along the natural history of cancer, malignant cells should exhibit high metabolic plasticity to adapt themselves to tumor and surrounding environment continual changes [180] . Tumor cell proliferation continuously demand the highest nutrient capacity to fulfill enhanced biosynthetic and bioenergetics requests. In normal cells, metabolism of glucose is mainly performed through cytosolic glycolysis then mitochondrial TCA and OXPHOS that produce ATP. As mitochondrial PDH is inhibited and pyruvate cannot be transformed into acetyl-coA, cancer cells enhance glycolysis to produce sufficient ATP and generate high lactate content even in aerobic conditions (the "Warburg effect"), both being hallmarks of cancer [181] . High lactate production and release induces TME acidification promoting immune surveillance escape and metastasis [182] . As lipids, amino-acids, and nucleotides are strongly required for cancer cell multiplication, either fatty acids synthesis and FAO[183], or glutamine and serine metabolisms are all increased in tumor cells. Glutamine appears as a major energy substrate in cancer cells. Glutamine could produce TCA cycle intermediates to provide an additional energy source for cancer cells [184] . It has been recently shown that TME metabolism can largely modulate cancer cells progression. CAFs can provide metabolites that will facilitate tumor cells ATP production. Lactate, exported through CAFs MCT4 lactate shuttle then up-taken through cancer cells MCT1 Lactate transporter, could be used to fuel surrounding cancer cells, a process called "reverse Warburg effect" [185] [186] [187] . TME can also induce cancer cells FAO through cancer-associated adipocytes free fatty acid (FFA) release then cancer cells FFA CD36 uptake, hereby promoting cancer progression [188] . TME associated endothelial cells that mediated tumor angiogenesis are highly glycolytic[189] while tumor-associated macrophages (TAMs) polarization to immunostimulatory M1 or immunosuppressive M2 phenotype is largely driven by metabolism, M1 cells being highly glycolytic whereas M2 cells mostly relying on FAO and OXPHOS [190] . All these TME cells can shed EVs that will modulate cancer cells metabolism and play a role in their proliferation. EVs can contain metabolites but also metabolism enzymes that can modulate cancer cells metabolism. Uptake of EVs enriched in metabolic enzymes ALDOA and ALDH3A1 accelerated glycolysis thus promoting unirradiated lung cancer cells proliferation [191] . EVs lncRNA SNHG3 sponging miR-330-5p in recipient cells positively regulated pyruvate kinase M expression inhibiting OXPHOS, increasing glycolysis, and promoting breast cancer cells proliferation [192] . As EVs can be produced bi-directionally (Figure 3) , cancer cells can also modulate TME cells fate through metabolism reprogramming. Human melanoma-associated EVs miR-210 and miR-155 can reprogram CAFs metabolism to enhance glycolytic phenotype leading to extracellular acidification that favors pre-metastatic niche formation [193] . Prostate cancer cells EVs transfer of PKM2 protein to stromal cells leads to pre-metastatic niche formation [194] . Breast cancer cells EVs were found to contain miR-122 which could remodel metabolism to exacerbate metastasis [195] . VEGF-containing EVs can enhance EC glycolytic phenotype, inducing vascular permeability and cancer cells transendothelial migration [196] or promoting chemoresistance [197] . By increasing glycolysis and reprograming myeloid cells to an immunosuppressive phenotype, pancreatic ductal adenocarcinoma EVs could create an immunosuppressive background favoring tumor progression [198] . EVs can be involved in directional cell movement through tissues [199] . Distant spread can arise in two steps. The first one concerns local tumor cell dissemination where epithelial cell migrate through TME at the front of the tumor through generation of membrane protusions (invadopodia) and basal lamina break-in [200] . The second involves vascular disruption to allow tumor cells hematogenous spread. Once in the circulation, tumor cells migrate and must found a premetastatic niche where they can settle then proliferate. To initiate both process, CRC cells will recruit then educate stromal cells to induce CAFs, tumor-associated macrophages with the immune-suppressive M2 phenotype, and endothelial cells that promote tumor angiogenesis [147] . CXCR4, present in HT29 EVs may also contribute to stromal cells recruitment [201] . CRC cells can induce CAF generation by EVs transfer of TGF-β[202] promoting also two CAFs distinct phenotypes, i.e., proliferative or invasive, by reprogramming their proteome [171] . Concerning macrophages, mutant p53 CRC cells are able to reprogram them into M2 phenotype through EVs miR-1246 transfer [203] . In both steps, loss of epithelial characteristics in favor of mesenchymal-like phenotype through epithelial to mesenchymal transition (EMT) process is involved [140, 204] . During the local movement phase, stromal cells support EMT induction in tumor cells through stromal EVs. CAFs EVs can induce EMT in CRC cells by transfer of miR-92a-3p that promotes beta-catenin ubiquitination then degradation [205] . Similarly, EVs mediated transfer of miR-21 from CAFs to CRC cells increases their metastatic potential [166] . Aside CAFs, M2 macrophages can induce CRC cell migration through EVs cotransfer of miR21-5p and miR-155-5p [206] . M2 cells can also secrete Wnt-containing EVs to induce CRC stem cell activity that is involved in metastasis development [120] . This EMT transition is largely influenced by EVs matrixins transfer. Cotransfer of claudin 7 and MMP14 induces MMP2 and MMP9 recruitment that enhance invasiveness [207] . By EVs release, tumor cells can themselves induce up-or down-regulation of EMTrelated genes in neighboring tumor cells, leading to distant invasion and/or migration [208] . EVs EMT inducers such as caveolin-1, HIF1α, beta-catenin, TNFa, TGF-β transfer can result in directional tumor cell migration [199, 209] by either regulating ECM composition [210] or driving fibroblast differentiation into myofibroblast [211] . An important characteristic of tumor cells relies on their capacity to colonize preferentially specific organs (organotropic metastasis) that is often determined by anatomic aspects. Indeed, an important subset of CRCs will develop through distant metastasis, mostly to the liver. CRC capacenenity to colonize liver is primarily due to the hepatic portal system that drains the colon and by the facilitating defenestrated architecture of liver sinusoid endothelium [212] . Nevertheless, a crosstalk between CRC circulating cells and hepatocytes through bidirectional EVs transfer is also mandatory. It is now well accepted that primary tumor educates metastatic microenvironment, commonly defined as the "premetastatic niche," allowing circulating tumor cells (CTC) to find a suitable environment in which they can settle then proliferate. Such niche generation is characterized by local tissue inflammation, immune suppression, stromal cell activation, and ECM remodeling [213] . EVs proteins or miRNAs have been shown to be involved in establishing this niche [167] . EVs can modify ECM to support circulating CRC cells adhesion by increasing fibronectin deposits within the liver [214] . Such ECM modifications increase CRC cell adhesion, promoting mesenchymal-to-epithelial transition (MET), and enabling liver metastasis colonization. EVs miR-25-3p promotes pre-metastatic niche formation by inducing vascular permeability and angiogenesis [169] while EVs miR-21 through toll like receptor (TLR) 7/IL-6 axis in macrophages pathway as well as EVs miR-203 seem to induce an inflammatory niche that can potentiate liver metastasis [215, 216] . EVs derived from CRC cell lines are involved in the modulation of the innate immune response, which is considered as a central step in the formation of the metastatic niche. Circulating EVs miRNAs after internalization by target cells can also act as ligands of TLRs [217] . Like in primary tumors, cancer cell EVs can reprogram resident cells to promote metastatic niche achievement and attract newly released CTCs. For example, in the niche, gastric cancer cells drive epidermal growth factor receptor (EGFR) EVs transfer to liver stromal cells that upregulate HGF expression through miR-26a/b downregulation inducing CTC attraction and further metastatic proliferation [218] . Angiogenesis is important for tumor proliferation and distant metastasis. Endothelial cells (ECs) can uptake via the endocytic pathway EVs from various origins[219]. Uptake of tumor-derived exosomes by normal endothelial cells activates angiogenic signaling pathways in endothelial cells and stimulates new vessel formation[67,68, 220]. Once internalized, EVs are immediately directed to the perinuclear zone and actin filaments enriched area. When tubules are formed, EVs move to cell periphery and enter advanced pseudopods [221] . After complete remodeling, adjacent ECs probably transport EVs to neighboring ECs and to other cells in the TME [222] . In hypoxic conditions, tumor cells can secrete angiogenic factors, such as VEGF-A, inducing ECs migration and tumor angiogenesis. Higher levels of circulating proangiogenic basic bFGF originating from CRC cells have been detected in the serum of CRC patients [223] . EVs are also released by hypoxic CRC cells. Wnt4 enriched EVs increased β-catenin nuclear translocation in ECs enhancing angiogenesis and tumor growth [224] . It holds the same for Wnt5a[225] and Wnt5b whose increased expression in CRC cells correlates with aggressiveness. Caco-2 cells, one of the mostly used human CRC cell lines, secrete Wnt5b containing EVs that stimulates cell migration and proliferation of A549 cells [210] . Mutations in adenomatous polyposis coli (APC) gene are common in CRC patients and are associated with the deregulation in Wnt signaling. Restoration of APC expression in CRC SW480 cells induces DKK4 release through EVs, a mechanism restoring Wnt signaling pathway that may be lost during CRC progression [226] . In CRC ascites, EVs released by CRC tumor cells have been shown to carry proangiogenic proteins like Plexin B2 and tetraspanin [227] . Interestingly, CRC cell lines (HCT116 and DLD-1) secrete EVS that carry high levels of tissue factor, which is involved in blood coagulation, but is also a known modulator of angiogenesis and metastasis processes [228] . Aside proteins, EVs miRs have also been involved in angiogenesis induction[229], miR-183-5p was first found to be highly expressed in CRC cell-derived EVs, which triggers a marked increase in the proliferation, migration and tube formation abilities of HMEC-1 cells by targeting FOXO1 [230] . CRC-derived miR-1229 containing EVs, by inhibiting HPIK2 expression, promote through VEGF pathway activation HUVECs tubulogenesis, transfection with exomiR-1229 inhibitor anta-miR-1229 significantly suppressing tube formation[231]. EVs from 5-FU-resistant CRC cells promoted angiogenesis through dipeptidyl peptidase IV, a potent inducer of this angiogenesis [232] . TAMs were also proven to be beneficial for angiogenesis. M2 macrophages were positively correlated with microvessel density of pancreatic ductal adenocarcinoma tissues. M2 macrophage-derived EVs could promote mouse aortic ECs angiogenesis in vitro and subcutaneous tumors growth in vivo, increasing vascular density in mice [233]. While tumor cell dissemination seems to be an early event of tumorigenesis, metastasis development ability is strongly associated with immune evasion. It seems that in CRC, the immune system influences tumor heterogeneity and sculpts clonal evolution. Tumor clones development is linked to the intra-metastatic immune microenvironment via an immune editing process [234] . CRC EVs induce recruitment to the pre-metastatic niche of suppressive immune cells, such as TAMs, tumor-associated neutrophils, Tregs leading to a strong inhibition of the antitumor response and facilitating CRC growth [ (Figure 4) . While it is well admitted that EVs from metastatic tumor cells display protumorigenic functions, it seems that, in poorly metastatic cancer, tumor cells EVs induce expansion of patrolling monocytes in bone marrow, promoting metastasis eradication via NK cells and macrophages recruitment [241] . Such discrepancies highlight the fact that cancer cell EVs may play heterogeneous functions in tumor immunity that remain to be elucidated. Despite improvement and diversification of therapeutics for CRC patients (surgery, targeted therapy, radiotherapy and chemotherapy) and the emergence of new drugs during the last years, resistance to treatment still exists and remains one of the deadlocks for patients with an advanced CRC for whom medicines no longer work [242] . Today, administration of FOLFOX, a combination of folinic acid, 5-FU and oxaliplatin (OXA), is one of the most widely used chemotherapeutic regimens for treating CRC but these treatments generate serious systemic side effects and have an impact on the patients quality of life. More recently, the use of targeted drugs (for example bevacizumab, cetuximab, regorafenib ...) allow improvement of metastatic CRC survival times but malignant tumors drug resistance still persist [243] . Resistance to conventional chemotherapy: Aside classical mechanisms of resistance to 5-FU and OXA such as impaired drug inflow or efflux, drug inactivation, or single nucleotide polymorphisms of fluoropyrimidine or platinum targets, EVs generated by CRC cells have been reported to play a critical role in resistance to treatments [244] . Cancer stemness acquisition could be a possible feature that induces chemoresistance in CRC [245] . Wnt activity may reflect stem cell features. EVs-mediated Wnt secretion by CAFs is able to induce CRC reprogramming into CSCs then potentiate CRC resistance to chemotherapy [246] . In addition, CAFs release of H19 EVs also potentiated cancer stem cell resistance to OXA. LncRNA H19 was highly expressed in CAFs and upregulated in EVs. H19 activated the Wnt/β-catenin signaling pathway and potentiated drug resistance of CSCs [247] . The role of CAFs in exporting EVs that will confer chemoresistance to CRC cells is significant as it was reported that CAFs Evs can activate CRC cells ERK/AKT pathway inducing a protective effect to OXA[162]. CAFs can export urothelial carcinoma-associated 1 (UCA1), a lncRNA with three exons that has been found to display oncogenic functions in various types of cancer [248] . In CRC, UCA1 was found to be associated with resistance to cetuximab and 5-FU [249, 250] . UCA1 suppresses miRNA-204-5p expression [251] that induces drug resistance. miR-196b-5p promotes CRC cells chemoresistance to 5-FU by targeting SOCS1 and SOCS3 negative regulators of STAT3 signaling pathway, resulting in global activation of STAT3 signaling [157] . Interestingly, UCA1 and miR-196b-5p are highly expressed in CRC patients EVs as compared to healthy control subjects and may represent interesting CRC biomarkers ( Figure 5 ). Resistance to targeted therapies: Cetuximab or panitumumab, that target the extracellular domain of EGFR preventing downstream activation of the MAPK or mTOR pathways, increases survival times in CRC patients [252] . Nevertheless, a subset of mutations involving either BRAF or PIK3 and amplifications of MET or HER2 induce resistance to these monoclonal antibodies (Mab) therapy [253] . Cetuximab CRCresistant EVs have been shown to restrict the PI3K negative regulator PTEN in CRC cells [254] through UCA1 overexpression [250] . Aside EVs nucleic acids or proteins inhibition of EGFR-driven cellular process in the recipient cell, EGFR positive EVs could bind anti-EGFR mAbs reducing mAb bioavailability. Such mechanism has been described for anti VEGFA mAb bevacizumab in metastatic and lung cancers. VEGFA positive EVs neutralize bevacizumab inducing cancer cell chemotherapeutic escape [255] . Being able to quantify and use EVs as relevant biological markers may improve CRC screening in the future. Nowadays, CRC is currently detected by different methods. Colonoscopy is widely used in clinical practice, which is regarded as the gold standard for detecting CRC. However, it has several limitations such as invasive nature, high cost and bothering bowel preparation [256] . Aside this invasive procedure, noninvasive screening tests such as iterative fecal occult blood testing (FOBT) [257] or November 15, 2021 Volume 13 Issue 11 plasma carcinoembryonic antigen (CEA) quantification have also been used. Unfortunately, both are of limited value mainly because poor sensitivity and specificity [258, 259] urging the need to find new methods aimed to quickly, easily and robustly diagnose and monitor CRC. This is where EVs can certainly play an important role. EVs can be detected in many biological fluids of patients, such as blood, urine, CSF and saliva [13] and can now be easily isolated [260] even though a universal standardized and widely accepted method for isolating then analyzing EVs is still mandatory [244] . Thanks to their lipid bilayers, EVs are stable in circulation and protected from degradation of serum ribonucleases and DNases [261] . As several miRNAs, lncRNAs and proteins are differently expressed in EVs originating from tumor and normal cells, they are potential sources of biomarkers and become a promising field in CRC diagnosis ( Figure 6 ). EVs miRs have been regularly involved in CRC development holding promise that their quantification in plasma or serum could serve as relevant CRC biomarkers. Some of them, that have been associated to specific events in CRC natural history, have been found in blood of CRC patients [262] . Among them, miR-25-3p[169] and miR-21 [216] , both promoting premetastatic niche formation by respectively inducing vascular permeability and macrophages differentiation towards a pro-inflammatory phenotype, and miR-203 that induces TAM activation [215] , have been reported to be highly expressed in plasma CRC patients EVs and related to a poor prognosis. Recently, miR-410-3p was found highly enriched in hypoxic CRC-derived EVs in a HIF1α or HIF2α-dependent manner. miR-410-3p decreases PTEN in recipient cancer cells thus activating PI3/Akt axis and leading to tumor progression. miR-410-3p levels were positively associated with poor prognosis of CRC[141]. Nevertheless, while several specific miRNAs panels November 15, 2021 Volume 13 Issue 11 have been found in EVs from CRC patients, only a few have yet been clinically validated [263] . A panel of 7 miRNAs (let-7a, miR-1229, miR-1246, miR-150, miR-21, miR-223, and miR-23a) was first validated by qRT-PCR, indicating that it may be a suitable biomarker to detect CRC [264] . Among this, miR-23a, miR-1246 and miR-21 are highly interesting as all three display high specificity and sensibility [262] . If both miR-23a and miR-1246 are positive and both CA19-9 and CEA negative, one can say that it is probably an early stage CRC [265] . In addition, miR-125a-3p and miR-320c were found to be significantly increased in EVs of early-stage CRC patients, combination of miR-125a-3P and CEA improving drastically the screening power for early-stage CRCs [266] . Another interesting work showed that miR-6803-5p was significantly increased in serum samples from CRC patients and correlated to a poor prognosis as compared to healthy subjects [267] . While associated increased levels of both miR-17-5p and miR-92a-3p levels may serve as an early indicator of liver metastases [268] , EVs overexpression of miR-486-5p, miR-19a, miR-17-92a correlate with CRC recurrence [269, 270] . Last, increased expression of EVs miRs that can be released by CAFs can be also an early indicator of chemotherapy resistance. High expression of miR-92a-3p activates Wnt/β-catenin pathway and inhibits mitochondrial apoptosis by directly inhibiting FBXW7 and MOAP1, contributing to cell stemness, EMT, metastasis and 5-FU resistance in CRC [205] . On the opposite, aside plasma EVs miRs increased levels, down-regulation of some miRNAs could be predictive factors of CRC. Five EVs miRNAs (miR-638, miR-5787, miR-8075, miR-6869-5p and miR-548c-5p) were decreased among CRC patients. These miRNAs may be involved in the development and progression of CRC by regulating glucose metabolism. Besides, in this study, 2 miRNAs (miR-486-5p and miR-3180-5p) have been shown to be significantly increased [271] , results that were further confirmed [269] . Low levels of tumor suppressor miR-6869-5p that targets TLR4/NF-κB signaling pathway inhibiting proliferation and promoting CRC cells apoptosis have been reported in CRC patients serum EVs [272] . More recently, decreased expression of miR-1505p [273] and miR-548c-5p [274] were both associated to CRC poor prognosis. November 15, 2021 Volume 13 Issue 11 vesicles, only a subset (proteins, miRNAs, lncRNAs) have been shown to be of potential clinical value on colorectal cancer detection, diagnosis, prognosis and treatment response evaluation. All referenced markers were found to be differentially expressed in cancer patients and in healthy people. The yellow ones were useful for diagnosis, the green ones for progression, the blue ones for prognosis and the pink ones were associated with chemoresistance. TAM: Tumor associated macrophages. LncRNAs, non-coding RNAs greater than 200 nucleotides, were once considered as junk DNA and transcriptional noise but emerging evidences demonstrate that they are evolutionarily conserved and that their strongly regulated expression plays critical roles in regulating gene expression [275] . As they can be differentially expressed in blood EVs of CRC patients, they could be new interesting biomarkers [276] . LncRNAs have been involved in CRC initiation and progression. Colorectal cancer-associated lncRNA (CCAL) seems to be a key regulator of CRC progression [277] and it was reported that CCAL promotes OXA resistance of CRC cells [278] . It has been also demonstrated that both down-regulation of lncRNA UCA1 and up-regulation of circRNA homeodomain interacting protein kinase 3 is found in CRC patients EVs. UCA1 LncRNAs, upregulated in CRC biopsies and downregulated in serum EVs, serves as a miR143 sponge that modulate MYO6 expression [279] . Six lncRNAs (LNCV6_116109, LNCV6_98390, LNCV6_38772, LNCV_108266, LNCV6_84003, and LNCV6_98602) are significantly up-regulated in patients with CRC as compared to healthy individuals [280] . High serum EVs expression of lncRNA 91H have been associated to CRC poor prognosis [281] and an increase of growth arrest-specific 5 and colon cancer-associated transcript 2 (CCAT2) lncRNAs in CRC patients have also been reported [282] . Interestingly, CCAT2 lncRNA levels were significantly decreased after surgery and removal of the tumor[283]. Finally, several lncRNAs have been associated to treatment resistance [284] . HOTAIR [285], XIST [286] and LINC00473[287] lncRNAs have been found to confer 5-FU resistance through respective miR-218 and miR-203a-3p, miR15a and miR-152 regulations [288, 289] . LncRNA CRNDE induces CRC OXA resistance via miR-181a-5pmediated regulation of Wnt/beta-catenin signaling and miR 136 sponging [290, 291] . EVs proteins as a source of cancer biomarkers: Finally, aside nucleic acids, EVs proteins could also be measured to diagnose CRC as they may differ between healthy and CRC individuals. A primary study has shown that 36 proteins were upregulated and 22 proteins downregulated in CRC patients EVs compared to normal volunteers EVs. Moreover, upregulation of these proteins was associated with a pretumorigenic microenvironment for metastasis and on the opposite, downregulation was associated with tumor growth and cell survival [292] . Several studies have identified a number of proteins that can be considered as potential biomarkers. For example, among them, glypican-1[293,294] was suggested to be a specific diagnosis marker because it is highly expressed in CRC patient EVs and normalized after surgery. Identically, EVs lower expression of Copine III, a protein highly expressed in CRC tumors, was associated to better survival [295] . Additionally, S100 calcium-binding protein A9 (S100A9) levels were noticeably higher in plasma EVs of CRC relapse patients than those in tumor resection patients [296] . S100A9 has been related to CRC worsening as its overexpression could enhance TME CRC cells stemness. High levels of cytokeratin 19, CA125, and tumor-associated glycoprotein 72 (TAG72) have been quantified in CRC patients plasma EVs [297] . Interestingly, TAG72 protein overexpression was found to contribute to CRC patients chemoresistance to 5-FU. The emergence of quantitative measurements that will be simple, inexpensive, easily performed and non-invasive for the patient is strongly mandatory. Analysis of EVs content (miRNAs, lncRNAs and proteins) may allow early diagnosing CRC and even predicting its relapse, metastasis and potential chemotherapy resistance. EVs have been shown to be a source of patient's resistance to chemotherapy. It is mandatory to explore new therapeutic possibilities aimed to both suppress tumor progression and reduce EVs-related drug resistance. The first possibility to treat cancer would be to target EVs by inhibiting EVs uptake [298] . Indeed, EVs endocytosis is an active process but a rather complex one leading its inhibition a new therapeutic perspective but a very difficult one to achieve. Many studies have found molecules that could inhibit EVs internalization. Heparin can inhibit in a dose-dependent manner EVs absorption through direct action on heparan sulfate proteoglycans which themselves play a role EVs endocytosis [299] . Cytochalasin D that inhibits phagocytosis and other endocytosis mechanisms through an inhibitory effect of actin polymerization has been shown to inhibit EVs uptake[300]. Inhibition of EVs internalization by Methyl-βcyclodextrin (MβCD) in glioblastoma cells has been reported [301] . MβCD depletes cholesterol from natural membranes and decreases EVs uptake by interfering with lipid rafts stability. Another molecule, dynamin, already described as an inhibitor of endocytosis, has been shown to interfere with EVs uptake in cancer [302] . Nevertheless, the large repertoire of mechanisms involved in EVs uptake in cancer impairs the overall efficiency of these molecules. A recent study showed that antibodies targeting CD9 and CD63 tetraspanins stimulate EVs macrophages phagocytose inhibiting cancer EVs-mediated communication [303] . However, such antibodies do not only target cancer EVs but also "physiological" CD9 and CD63 EVs. The role of these specific EVs being not yet known, additional studies must be carried out to know the viability of such method. One other possibility of EVs targeting would be to inhibit EVs biogenesis. Inhibiting EVs biogenesis also involves complex issues, primarily due to the large number of proteins that are concerned in this cellular process. However many pharmacological agents have been found and seem promising. Fluidity of cell plasma membrane is fundamental during membrane lipid bilayer re-organization and thus EVs formation. During EVs biogenesis, ceramide regulate EVs production [24] . Ceramide synthesis required an ubiquitous enzyme, neutral sphingomyelinase 2 (nSMase2) that can be specifically targeted by GW4869 inhibiting cancer cells EVs release in a dosedependent manner [304] and consequently limiting miRNAs hematogenous release [305] . On the opposite, nSMase2 overexpression increases miRNAs quantity in blood [306] . The link between nSmase2 and EVs has been shown in breast cancer aggressiveness [307] . GW4869 therapeutic effects have been observed on murine melanoma. GW4869-induced B16BL6-derived EVs secretion inhibition decreased B16BL6 cells proliferation and increased apoptosis-related proteins. Treatment of GW4869-treated cells with B16BL6-derived EVs restore their proliferation [308] . As GW4869 seems to be promising, imipramine which is a tricyclic anti-depressant is also a source of interest because of its inhibitory activity on acid sphingomyelinase (aSMase) that catalyzes sphingomyelin hydrolysis to ceramide [309] . Thus, imipramine is reported to prevent the translocation of aSMase, inhibiting EVs secretion. So, both GW4869 and imipramine can stop the production of ceramide November 15, 2021 Volume 13 Issue 11 TSG101 is a protein involved on endosomes trafficking and exosomes biogenesis [310] . In CRC cells that express Wnt5b, knockdown of TSG-101 generates Wnt5b EVs downregulation decreasing Wnt5b-driven cell proliferation suggesting TSG101 as a potential therapeutic target in cancer [311] . A third possibility to target EVs is to limit or inhibit their release by secreting cells. A drug that inhibits EVs release is manumycin A, an antibiotic which is a selective and strong inhibitor of Ras farnesyltransferases. Farnesyltransferase inhibitors inhibit Ras activity and therefore EVs release [312] . Aside Ras proteins figure Rab proteins that are also modulators of EVs biogenesis [7] . Rab2b, Rab5a, Rab9a, Rab27a and Rab27b impacts in EVs release have been studied, the two latter playing also a role in EVs docking and exocytosis [29] . Knockdown of Rab27a decreased EVs-release amount [313] and Rab27a inhibition reduced tumor growth and lowered metastatic cells dissemination [314, 315] . Gold nanoparticles conjugated with anti-sense RAB27a oligonucleotides to mute Rab27a generate 80% inhibition of EVs release in breast cancer [316] . Plectin enables EVs secretion in pancreatic cancer. Downregulation of plectin in pancreatic cancer cells reduced EVs release in the same way Rab27a and Rab27b knockdowns do suggesting that combining both mechanisms could be a therapeutic combination that enables greater results [317] . As plasma membrane fluidity is important for EVs shedding, drugs aimed at targeting either lipid rafts formation or cholesterol synthesis will interfere with EVs release. Lipid depletion results in EVs release reduction [318] . Pantethine, a pantothenic acid (vitamin B5) derivative is used as an intermediate in the production of co-enzyme A and it plays a role in the metabolism of lipids and reduction of total cholesterol levels. Panthetine inhibits by 80% cholesterol synthesis as well as fatty acid synthesis [319] . Panthetine has been shown to limit EVs release in systemic sclerosis [320] . Its use on chemoresistant breast cancer cells significantly reduced EVs release [321] . Actin and actin-regulating proteins are also strongly involved in EVs secretion. Invadopodia are cellular structures used by cancer cells to degrade extracellular matrix and invade. Because of high levels of actin, such structures are key sites for EVs release. Indeed, invadopodia inhibition limits EVs release [322] . Furthermore, knockdown of cortactin, that acts as an actin dynamics regulatory protein, decreased whereas its overexpression led to an increase of EVs release [323] . Rho-associated protein kinases (ROCK) are a family of serine-threonine kinases belonging to the PKA-G-C family and involved in cells shape and movement regulation, by acting on the cytoskeleton. Cytoskeleton organization as well as cellular contractility through activity on actin filaments is important features for EVs shedding. Y27632 is a commonly used ROCK competitive inhibitor which is able to compete with ATP at ROCK catalytic sites [324] . Y27632 causes a reduction in the release of EVs as well as a change in cell surface morphology[325] by sustaining activation of proteolytic enzymes, such as stathmin and calpain, that destabilized cell plasma membrane. Thus, Y27632 can be used alone or in combination with Calpeptin, the most studied calpain inhibitor [326] . Calpains, once activated through calcium binding, can activate different cellular processes including cell migration, cell invasion and EVs formation and release. Calpeptin has also been used alone to inhibit EVs release [327] . PEG-SMRwt-Clu, a drug derived from the secretion region of HIV-1 Nef protein, regulates exosomal pathway trafficking and seems promising. PEG-SMRwt-Clu was able to inhibit cell growth in breast cancer cell lines and more interesting to partially increase chemosensitivity. The use of PEG-SMRwt-Clu was also associated with a decrease in the number of released EVs [328] . Despite the current efforts and the number of EVs endocytosis, biogenesis and release inhibitors that are already available, inhibition of EVs is still a very complex issue because of the multifactorial nature of the different pathways involved in these processes. Nevertheless, EVs uptake, biogenesis or release inhibition remains a potential and interesting therapeutic cancer target in the near future. EVs are major players in tumor progression via the transfer of cargo within them. One other possible way to cure CRC would be an EVs-based therapy that uses EVs as therapeutic vectors. In very recent years, studies have mainly focused on the idea that EVs could be natural delivery vehicles to transport therapeutic drugs, antibodies or RNA to modify gene expression [329] . In the cancer field, it would be indeed a specific and effective therapy delivery method to specifically treat cancer cells. EVs are biocompatible and biodegradable and therefore, less toxic and immunogenic than other nanoparticular drug delivery systems such as liposomes or polymeric nanoparticles [330] . EVs have innate limited immunogenicity and cytotoxicity [331, 332] . Moreover, drug stability is largely enhanced as EVs avoid drugs degradation by extracellular enzymes [333] . Thus EVs capacity to target tumor cells is 10 times higher than liposomes of a similar size. Such property is certainly linked to particular ligand-receptor interactions and to efficient endocytosis mechanisms linked to the EVs membrane lipid composition that contributes significantly to cellular adherence and internalization [334] . Last, EVs can penetrate through anatomical barriers [335, 336] and their lipid composition protects them from reticuloendothelial system phagocytosis [244] . Several reports have demonstrated the potential of using EVs therapy and clinical trials are currently underway to find treatments that extend patient survival. Many kinds of EVs-based therapies have been shown to improve chemotherapy effectiveness. EVs have been used to deliver many kinds of drugs such as curcumin [337] , paclitaxel[338] and doxorubicin [339] . While loading doxorubicin in EVs reduces cardiotoxicity[340], its packaging into EVs increases its efficacy when compared to free doxorubicin in cancer-bearing mice treatment. Inside EVs, doxorubicin has a better stability and will be even more collected within the tumor, significantly suppressing mice CRC growth and extending survival time [341] . EVs loaded with paclitaxel were tested in the treatment of multiple drug resistance cancers. Loaded exosomes can overcome drug efflux transporter adverse effect, decreasing metastasis growth when compared to controls [342] . EVs are also natural carriers of nucleic acids molecules and can be genetically engineered to deliver specific nucleic acid molecules such as miRNA [343] , and more recently gene editing system CRISPR/Cas9 [344] . EVs-based nucleic acid delivery in cancer treatment have shown promising therapeutic effects [38] . EGFR expressing cells can be targeted with GE11-positive exosomes loaded with microRNA let-7a, a tumor suppressor microRNA. The results showed an efficient delivery of exosomes cargo and consequent tumor growth inhibition [345] . EVs can also be used as a new type of tumor vaccine. Phase I clinical trials have shown that ascites EVs combination with granulocyte-macrophage colony stimulating factor induces a safe and effective response from specific anti-tumor cytotoxic T-cell in the treatment of advanced CRC [346] . EVs have also been explored as modulators of the immune response against tumor cells. Dendritic cells are antigen-presenting cells inducing immune responses. Dendritic cells have been shown to secrete antigenpresenting EVs that coexpress molecules of the major histocompatibility complex. Such exosomes activate specific cytotoxic T lymphocytes in vivo that can reduce or even suppress tumor growth [347] . EVs loading of anti-tumor peptides has also been used. A specific mutated form of survivin-T34A induces caspase activation leading to apoptosis. In vitro treatment of cancer cell lines with survivin-T34A EVs increased cell death [348] . Different cell-derived EVs may be home to specific cell types [7] . EVs derived from hypoxic tumor cells tend to be taken up by hypoxic tumor cells [349] . Different cells under different conditions determine EVs heterogeneity, generating huge and complex combinatorial possibilities. Thus, to better use EVs in cancer, engineering EVs with ligands that can specifically bind to targeted cancer cells is mandatory. Either EVs surface expression of receptor/ligand, antibody/ligand or microenvironment specific molecules can be used to specifically modify EVs. Recently, bioengineered EVs have been shown to be able to specifically bind to HER2/Neu by expressing designed ankyrin repeat proteins on their membrane surface [350] . Engineering both CD3 and EGFR expression on EVs membranes allows cross-linking of T cells with EGFR positive cancer cells enhancing antitumor immunity [351] . As hyaluronan has been evidenced in EVs [352] , hyaluronidase engineered EVs have been shown to degrade tumor extracellular matrix and enhance the permeability of T cells and drugs within the tumor [353] . Using EVs as therapeutic vectors in cancer seems very promising and clinical trials are nowadays being carried out [354] . Unfortunately, no major breakthrough still occurs certainly because of the complexity to handle such new therapeutic methods in vivo. To accelerate their use in cancer patient treatment, there is also an urgent need to better understand both EVs biology and nature [298] . EVs exert a wide variety of biological functions, mainly via delivering signaling molecules that regulate a vast repertoire of cellular processes. Their role in cancer development is central as they participate through bidirectional signaling between cancer cells and TME cells to every step of CRC carcinogenesis up to metastatic dissemination. Their detection in a large variety of biological fluids represents the future of cancer detection, an easy and reproducible mean to identify specific biomarkers of diagnostic and prognostic relevance. Moreover, they also represent new targets for treatment as their inhibition could limit or stop cancer development. Additionally, as extracellular signaling molecules, they could be used as very specific nanovectors to transport conventional or innovative therapies to cancer cells of interest. However, although pre-clinical data appear very promising, validation from large clinical trials are needed to support EVs use as either tumor biomarkers for monitoring cancer progression and driving treatment decisions or new vectors for specifically targeted treatments. Such data are mandatory to better understand EVs function in cancer progression and translate EVs use in clinical practice. Biological properties of extracellular vesicles and their physiological functions Oncosomes -large and small: what are they, where they came from? Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation Linking stemness with colorectal cancer initiation, progression, and therapy Colorectal cancer stem cells: from the crypt to the clinic Cancer stem cells in progression of colorectal cancer Cancer stem cells in colorectal cancer: a review Opinion: migrating cancer stem cells -an integrated concept of malignant tumour progression Metastatic stem cells: sources, niches, and vital pathways Isolation and phenotypic characterization of colorectal cancer stem cells with organ-specific metastatic potential Characterization of Cancer Stem Cells in Colon Adenocarcinoma Metastasis to the Liver A pre-existing population of ZEB2 + quiescent cells with stemness and mesenchymal features dictate chemoresistance in colorectal cancer Investigations into the cancer stem cell niche using in-vitro 3-D tumor models and microfluidics The cancer stem cell niche: how essential is the niche in regulating stemness of tumor cells? Drug Resistance Driven by Cancer Stem Cells and Their Niche Embryonic stem cellderived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery Extracellular vesicles of stromal origin target and support hematopoietic stem and progenitor cells The Crosstalk between Cancer Stem Cells and Microenvironment Is Critical for Solid Tumor Progression: The Significant Contribution of Extracellular Vesicles Maintenance of cancer stemness by miR-196b-5p contributes to chemoresistance of colorectal cancer cells via activating STAT3 signaling pathway Mutant p53 cancers reprogram macrophages to tumor supporting macrophages via exosomal miR-1246 Hypoxic Microenvironment Induces EMT and Upgrades Stem-Like Properties of Gastric Cancer Cells CAFs secreted exosomes promote metastasis and chemotherapy resistance by enhancing cell stemness and epithelial-mesenchymal transition in colorectal cancer M2 Macrophage-Derived Exosomes Promote Cell Migration and Invasion in Colon Cancer Claudin-7 promotes the epithelialmesenchymal transition in human colorectal cancer Emerging roles of exosomes during epithelial-mesenchymal transition and cancer progression Exosomes: composition, biogenesis, and mechanisms in cancer metastasis and drug resistance Focal Adhesion Kinase Promotes the Progression of Aortic Aneurysm by Modulating Macrophage Behavior Cancer exosomes trigger fibroblast to myofibroblast differentiation Metastasis Organotropism: Redefining the Congenial Soil Pre-metastatic niches: organ-specific homes for metastases Pancreatic cancer exosomes initiate premetastatic niche formation in the liver Circulating exosomal microRNA-203 is associated with metastasis possibly via inducing tumor-associated macrophages in colorectal cancer Colorectal cancer-derived small extracellular vesicles establish an inflammatory premetastatic niche in liver metastasis MicroRNAs bind to Toll-like receptors to induce prometastatic inflammatory response Exosome-delivered EGFR regulates liver microenvironment to promote gastric cancer liver metastasis Exosomal transfer of vasorin expressed in hepatocellular carcinoma cells promotes migration of human umbilical vein endothelial cells ] by K562 chronic myeloid leukemia cells promote angiogenesis in a Src-dependent fashion Internalization of Exosomes through Receptor-Mediated Endocytosis Clinical significance of fibroblast growth factor (FGF) expression in colorectal cancer Exosomes Derived From Hypoxic Colorectal Cancer Cells Promote Angiogenesis Through Wnt4-Induced β-Catenin Signaling in Endothelial Cells Pre-metastatic cancer exosomes induce immune surveillance by patrolling monocytes at the metastatic niche Drug resistance and new therapies in colorectal cancer Molecular Targeted Drugs and Treatment of Colorectal Cancer: Recent Progress and Future Perspectives Revisiting the Role of Exosomes in Colorectal Cancer: Where Are We Now? Colorectal cancer stem cells are enriched in xenogeneic tumors following chemotherapy Exosomal Wnt-induced dedifferentiation of colorectal cancer cells contributes to chemotherapy resistance Carcinoma-associated fibroblasts promote the stemness and chemoresistance of colorectal cancer by transferring exosomal lncRNA H19 Urothelial cancer associated 1: a long noncoding RNA with a crucial role in cancer LncRNA-UCA1 enhances cell proliferation and 5-fluorouracil resistance in colorectal cancer by inhibiting miR-204-5p Predictive role of UCA1-containing exosomes in cetuximab-resistant colorectal cancer MicroRNA-204 modulates colorectal cancer cell sensitivity in response to 5-fluorouracil-based treatment by targeting high mobility group protein A2 Prognostic and predictive value of primary tumour side in patients with RAS wild-type metastatic colorectal cancer treated with chemotherapy and EGFR directed antibodies in six randomized trials Mechanisms of resistance to anti-EGFR therapy in colorectal cancer Exosomes promote cetuximab resistance via the PTEN/Akt pathway in colon cancer cells Shedding of bevacizumab in tumour cells-derived extracellular vesicles as a new therapeutic escape mechanism in glioblastoma Colorectal Cancer Screening With Colonoscopy Circulating and stool nucleic acid analysis for colorectal cancer diagnosis The sensitivity, specificity, predictive values, and likelihood ratios of fecal occult blood test for the detection of colorectal cancer in hospital settings Colorectal cancer: evolution of screening strategies New Technologies for Analysis of Extracellular Vesicles Characterization of human plasma-derived exosomal RNAs by deep sequencing Exosomes as potential sources of biomarkers in colorectal cancer MicroRNA signatures: novel biomarker for colorectal cancer? Circulating exosomal microRNAs as biomarkers of colon cancer Serum overexpression of miR-301a and miR-23a in patients with colorectal cancer Circulating exosomal miR-125a-3p as a novel biomarker for early-stage colon cancer Exosomal miR-6803-5p as potential diagnostic and prognostic marker in colorectal cancer Circulating Exosomal miR-17-5p and miR-92a-3p Predict Pathologic Stage and Grade of Colorectal Cancer DNAmethylation-mediated silencing of miR-486-5p promotes colorectal cancer proliferation and migration through activation of PLAGL2/IGF2/β-catenin signal pathways Exosomal microRNA in serum is a novel biomarker of recurrence in human colorectal cancer Exosome-encapsulated microRNAs as circulating biomarkers for colorectal cancer MicroRNA-6869-5p acts as a tumor suppressor via targeting TLR4/NF-κB signaling pathway in colorectal cancer Downregulation of serum exosomal miR-150-5p is associated with poor prognosis in patients with colorectal cancer Downregulation of exosome-encapsulated miR-548c-5p is associated with poor prognosis in colorectal cancer Non-coding RNAs in human disease Exosomal long noncoding RNA CRNDE-h as a novel serum-based biomarker for diagnosis and prognosis of colorectal cancer Long non-coding RNA CCAL regulates colorectal cancer progression by activating Wnt/βcatenin signalling pathway via suppression of activator protein 2α Long noncoding RNA CCAL transferred from fibroblasts by exosomes promotes chemoresistance of colorectal cancer cells Upregulated in CRC Biopsies and Downregulated in Serum Exosomes, Controls mRNA Expression by RNA-RNA Interactions Plasma Exosomal Long Non-Coding RNAs Serve as Biomarkers for Early Detection of Colorectal Cancer Exosomal lncRNA 91H is associated with poor development in colorectal cancer by modifying HNRNPK expression Prognostic and predictive value of long non-coding RNA GAS5 and mircoRNA-221 in colorectal cancer and their effects on colorectal cancer cell proliferation, migration and invasion Circulating long non-coding RNA colon cancerassociated transcript 2 protected by exosome as a potential biomarker for colorectal cancer The emerging role of noncoding RNAs in colorectal cancer chemoresistance lncRNA HOTAIR Contributes to 5FU Resistance through Suppressing miR-218 and Activating NF-κB/TS Signaling in Colorectal Cancer Long noncoding RNA XIST is a prognostic factor in colorectal cancer and inhibits 5-fluorouracil-induced cell cytotoxicity through promoting thymidylate synthase expression LINC00473 promotes the Taxol resistance via miR-15a in colorectal cancer LncRNA HOTAIR is a Prognostic Biomarker for the Proliferation and Chemoresistance of Colorectal Cancer via MiR-203a-3p-Mediated Wnt/ß-Catenin Signaling Pathway Knockdown of long non-coding RNA XIST exerts tumor-suppressive functions in human glioblastoma stem cells by up-regulating miR-152 The lncRNA CRNDE promotes colorectal cancer cell proliferation and chemoresistance via miR-181a-5p-mediated regulation of Wnt/β-catenin signaling Long noncoding RNA CRNDE functions as a competing endogenous RNA to promote metastasis and oxaliplatin resistance by sponging miR-136 in colorectal cancer Protein content and functional characteristics of serum-purified exosomes from patients with colorectal cancer revealed by quantitative proteomics GPC1 exosome and its regulatory miRNAs are specific markers for the detection and target therapy of colorectal cancer Heat shock protein 60 levels in tissue and circulating exosomes in human large bowel cancer before and after ablative surgery Circulating exosomal CPNE3 as a diagnostic and prognostic biomarker for colorectal cancer Granulocytic Myeloid-Derived Suppressor Cells Promote the Stemness of Colorectal Cancer Cells through Exosomal S100A9 The potential of exosomes derived from colorectal cancer as a biomarker Exosomes in cancer: Use them or target them? Cancer cell exosomes depend on cell-surface heparan sulfate proteoglycans for their internalization and functional activity Routes and mechanisms of extracellular vesicle uptake Exosome uptake depends on ERK1/2-heat shock protein 27 signaling and lipid Raft-mediated endocytosis negatively regulated by caveolin-1 Tumor-derived microvesicles induce proangiogenic phenotype in endothelial cells via endocytosis Circulating Extracellular Vesicles as a Novel Therapeutic Strategy against Cancer Metastasis Exosomes Promote Ovarian Cancer Cell Invasion through Transfer of CD44 to Peritoneal Mesothelial Cells Neutral sphingomyelinases control extracellular vesicles budding from the plasma membrane Secretory mechanisms and intercellular transfer of microRNAs in living cells Neutral sphingomyelinase 2 (nSMase2)-dependent exosomal transfer of angiogenic microRNAs regulate cancer cell metastasis Accelerated growth of B16BL6 tumor in mice through efficient uptake of their own exosomes by B16BL6 cells Small molecule inhibitors of acid sphingomyelinase Syndecan-syntenin-ALIX regulates the biogenesis of exosomes Wnt5b-associated exosomes promote cancer cell migration and proliferation Inhibiting extracellular vesicles formation and release: a review of EV inhibitors Rab27A regulates exosome secretion from lung adenocarcinoma cells A549: involvement of EPI64 Rab27a supports exosome-dependent and -independent mechanisms that modify the tumor microenvironment and can promote tumor progression Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET Smuggling gold nanoparticles across cell types -A new role for exosomes in gene silencing Unexpected gain of function for the scaffolding protein plectin due to mislocalization in pancreatic cancer Lipids in exosomes: Current knowledge and the way forward Effect of pantethine on the biosynthesis of cholesterol in human skin fibroblasts Pantethine Prevents Murine Systemic Sclerosis Through the Inhibition of Microparticle Shedding Targeting microparticle biogenesis: a novel approach to the circumvention of cancer multidrug resistance Exosome secretion is enhanced by invadopodia and drives invasive behavior Cortactin promotes exosome secretion by controlling branched actin dynamics Extracellular vesicles: Novel promising delivery systems for therapy of brain diseases Exosome as a Novel Shuttle for Delivery of Therapeutics across Biological Barriers Treatment of brain inflammatory diseases by delivering exosome encapsulated anti-inflammatory drugs from the nasal region to the brain Milk-derived exosomes for oral delivery of paclitaxel Genetically Engineered Liposome-like Nanovesicles as Active Targeted Transport Platform A33 antibodyfunctionalized exosomes for targeted delivery of doxorubicin against colorectal cancer Exosomes increase the therapeutic index of doxorubicin in breast and ovarian cancer mouse models Development of exosome-encapsulated paclitaxel to overcome MDR in cancer cells γδTDEs: An Efficient Delivery System for miR-138 with Anti-tumoral and Immunostimulatory Roles on Oral Squamous Cell Carcinoma Exosome-Liposome Hybrid Nanoparticles Deliver CRISPR/Cas9 System in MSCs Systemically injected exosomes targeted to EGFR deliver antitumor microRNA to breast cancer cells Phase I clinical trial of autologous ascitesderived exosomes combined with GM-CSF for colorectal cancer Eradication of established murine tumors using a novel cell-free vaccine: dendritic cell-derived exosomes Enhancement of Gemcitabine sensitivity in pancreatic adenocarcinoma by novel exosome-mediated delivery of the Survivin-T34A mutant Development and MPI tracking of novel hypoxiatargeted theranostic exosomes Engineered Exosomes for Targeted Transfer of siRNA to HER2 Positive Breast Cancer Cells Reprogramming Exosomes as Nanoscale Controllers of Cellular Immunity Identification of Biomarker Hyaluronan on Colon Cancer Extracellular Vesicles Using Correlative AFM and Spectroscopy Degradation of tumour stromal hyaluronan by small extracellular vesicle-PH20 stimulates CD103 + dendritic cells and in combination with PD-L1 blockade boosts anti-tumour immunity Extracellular vesicles for personalized medicine: The input of physically triggered production, loading and theranostic properties We are indebted to Mrs Pasquet for her fine work in the correction of the English text of our manuscript.