key: cord-0795236-tylequgg authors: Sounart, Hailey; Lázár, Enikő; Masarapu, Yuvarani; Wu, Jian; Várkonyi, Tibor; Glasz, Tibor; Kiss, András; Borgström, Erik; Hill, Andrew; Jurek, Aleksandra; Niesnerová, Anezka; Druid, Henrik; Bergmann, Olaf; Giacomello, Stefania title: Dual spatially resolved transcriptomics for SARS-CoV-2 host-pathogen colocalization studies in humans date: 2022-03-21 journal: bioRxiv DOI: 10.1101/2022.03.14.484288 sha: c163f12978ae054f732f728fd58425b2e3ed7000 doc_id: 795236 cord_uid: tylequgg To advance our understanding of cellular host-pathogen interactions, technologies that facilitate the co-capture of both host and pathogen spatial transcriptome information are needed. Here, we present an approach to simultaneously capture host and pathogen spatial gene expression information from the same formalin-fixed paraffin embedded (FFPE) tissue section using the spatial transcriptomics technology. We applied the method to COVID-19 patient lung samples and enabled the dual detection of human and SARS-CoV-2 transcriptomes at 55 μm resolution. We validated our spatial detection of SARS-CoV-2 and identified an average specificity of 94.92% in comparison to RNAScope and 82.20% in comparison to in situ sequencing (ISS). COVID-19 tissues showed an upregulation of host immune response, such as increased expression of inflammatory cytokines, lymphocyte and fibroblast markers. Our colocalization analysis revealed that SARS-CoV-2+ spots presented shifts in host RNA metabolism, autophagy, NFκB, and interferon response pathways. Future applications of our approach will enable new insights into host response to pathogen infection through the simultaneous, unbiased detection of two transcriptomes. Much is still unknown about how hosts react to pathogens and how pathogen infection underlies various biological 52 processes and disease states. Although single-cell transcriptomics methods have improved the elucidation of cell-type 53 specific effects caused by pathogens and how these relate to disease outcomes 1,2 , such approaches remove pathogens To independently validate the viral detection by our set of SARS-CoV-2 probes, we compared the ST viral detection 94 to the viral detection offered by the orthogonal imaging-based RNAScope technology 11 using consecutive sections. Specifically, we compared the S gene signal detected by ST and RNAScope across all COVID-19 and control samples. In COVID-19 sections, 9.5% of spots (i.e., 1,132 spots in total) presented SARS-CoV-2 transcriptional signal with 116 highly reproducible capture of SARS-CoV-2 gene expression between consecutive sections (r=0.98, p-value < 1.5e-117 6, Figure 1f ). Overall, we captured up to 9 different SARS-CoV-2 genes (Supplementary Table 3 ) with an average per 118 spot of ∼1.7 unique molecules and ∼1.5 unique genes, respectively. These relative low levels of viral load are likely 119 associated with the longer disease duration (13-17 days) of these patients (Supplementary Although the SARS-CoV-2 transcripts differed in their abundances across genes, we observed a fairly even spatial 132 distribution of each gene across samples 1C and 3C, while for 2C the transcripts showed a more localized spatial We did not find viral entry factors ACE2, TMPRSS2, PCK5, or PCSK7 to be differentially expressed between 175 COVID-19 and control lung sections, in line with a recent study 3 . However, we found three other known SARS-CoV-176 2 entry factors, CTSL 26,27 , CTSB 26 , and NRP1 28,29 to be upregulated in the COVID-19 lungs (Supplementary Table 177 6). Previous studies 30,31 observed that CTSL and CTSB have increased expression in COVID-19 lungs, with SARS- Table 183 7). Besides many consensus fibrosis markers (COL1A1, COL3A1, COL5A1, SPP1, FN1, POSTN), we found the 184 CTHRC1 gene, recently described as a marker of pathological, pulmonary fibrosis-associated fibroblasts 32 , to be 185 highly expressed in the fibroblast-rich cluster 6 of COVID-19 lungs (Supplementary Table 7 ). At the same time, we 186 identified markers of alveolar fibroblasts, thought to be a cellular source of the CTHRC1 + fibroblast subpopulation 32 , 187 as either mildly downregulated (TCF21, PDGFRA) or upregulated (NPNT1) in the same cluster (Supplementary Table 188 7). To investigate the biological changes occurring in the epithelial cell compartments, we performed further sub- In conclusion, the proposed method enables insights into highly localized host response to pathogen infection within 269 the spatial context of the tissue microenvironment at the whole-transcriptome level in an unbiased and high-throughput The filtered count matrices (filtered_feature_bc_matrix.h5), and tissue images from spaceranger output were analyzed 429 in R using the Load10X_Spatial function available in Seurat (version 4.0.4) 58 . The filtered count matrices were 430 separated into human count data, and SARS-CoV-2 count data matrices. Spot level filtering was performed on the 431 human count matrices to keep spots with at least 400 genes, 500 UMIs, and a novelty score of 0.87. Gene level filtering 432 was applied to omit genes that did not appear in at least 1 spot. These count matrices were also filtered for Hemoglobin 433 gene counts (Supplementary Table 1 The computational validation was performed as follows: the RNAScope signal was detected with an ad hoc Matlab 467 (version R2021b) algorithm, which is specified in the next section "Automatic detection of RNAScope signal"; then Host-Microbiome Interactions in the Era of Single A deadly dance: the choreography of host-pathogen 507 interactions, as revealed by single-cell technologies COVID-19 tissue atlases reveal SARS-CoV-2 pathology and cellular targets Temporal and spatial heterogeneity of host response to SARS-CoV-2 pulmonary infection Systemic Tissue and Cellular Disruption from SARS-CoV-2 Infection Shotgun transcriptome, spatial omics, and isothermal profiling of SARS-CoV-2 infection reveals 515 unique host responses, viral diversification, and drug interactions The spatial landscape of lung pathology during COVID-19 progression Spatial mapping of SARS-CoV-2 and H1N1 lung injury identifies differential transcriptional 519 signatures Profiling of lung SARS-CoV-2 and influenza virus infection dissects virus-specific host 521 responses and gene signatures RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues Quantification of HER2 and estrogen receptor heterogeneity in breast cancer by single-526 molecule RNA fluorescence in situ hybridization MicroRNA quantification from tissue using nanoliter well arrays Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ Highly multiplexed subcellular RNA sequencing in situ Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and 533 tissues Genome-wide spatial expression profiling in formalin-fixed tissues Spatial transcriptome sequencing of FFPE tissues at cellular level Multiplex digital spatial profiling of proteins and RNA in fixed tissue Visium Spatial Gene Expression Reagent Kits for FFPE User Guide In situ sequencing for RNA analysis in preserved tissue and cells Probabilistic cell typing enables fine mapping of closely related cell types in situ Two distinct immunopathological profiles in autopsy lungs of COVID-19 The Architecture of SARS-CoV-2 Transcriptome SARS-CoV-2 Subgenomic RNAs: Characterization, Utility, and Perspectives SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically 550 Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and 552 is a promising target for new drug development Neuropilin-1 is a host factor for SARS-CoV-2 infection Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity Transcriptional and proteomic insights into the host response in fatal COVID-19 cases Multi-organ proteomic landscape of COVID-19 autopsies Collagen-producing lung cell atlas identifies multiple subsets with distinct localization and 560 relevance to fibrosis COVID-19 pulmonary pathology: a multi-institutional autopsy cohort from Italy and New Lung epithelial and endothelial damage, loss of tissue repair, inhibition of fibrinolysis, and 564 cellular senescence in fatal COVID-19 SON and SRRM2 are essential for nuclear speckle formation SARS-CoV-2 nucleocapsid protein phase-separates with RNA and with human hnRNPs Attenuating innate immunity and facilitating β-coronavirus infection by NSP1 of SARS-CoV-2 569 through specific redistributing hnRNP A2/B1 cellular localization Manipulation of autophagy by SARS-572 CoV-2 proteins SARS-CoV-2-mediated dysregulation of metabolism and autophagy uncovers host-targeting 574 antivirals SARS-CoV-2 Proteins Exploit Host's Genetic and Epigenetic 576 Mediators for the Annexation of Key Host Signaling Pathways The role of membrane-trafficking small GTPases in the 578 regulation of autophagy COVID-19: immunopathogenesis and Immunotherapeutics Chemokine Regulation During Epidemic Coronavirus Infection NF-κB Pathway as a Potential Target for Treatment of Critical Stage COVID-19 Patients Immunomodulation in COVID-19 Activation of NF-κB and induction of proinflammatory cytokine expressions 587 mediated by ORF7a protein of SARS-CoV-2 Serum CCL17 level becomes a predictive marker to distinguish between mild/moderate and 589 severe/critical disease in patients with COVID-19 An immune-based biomarker signature is associated with mortality in COVID-19 patients A molecular single-cell lung atlas of lethal COVID-19 Type I and III interferon responses in SARS-CoV-2 infection Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity Interferon gamma induces synthesis of complement alternative pathway proteins by human 598 endothelial cells in culture The complement system in COVID-19: friend and foe? Infection prevention and control for the safe management of a dead body in the 601 context of COVID-19: interim guidance Visium Spatial Gene Expression for FFPE-Tissue Preparation Guide, Document Number 604 CG000408 Visium Spatial Gene Expression for FFPE: RNA-templated Ligation Probe Design Visium Spatial Gene Expression for FFPE-Deparaffinization Integrated analysis of multimodal single-cell data Fast, sensitive and accurate integration of single-cell data with Harmony Proteomics. Tissue-based map of the human proteome PanglaoDB: a web server for exploration of mouse and human 614 single-cell RNA sequencing data SARS-CoV-2 Infects Endothelial Cells In Vivo and In Vitro