SEARCH · bioRxiv Science

Results for “single cell RNA sequencing”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 307 records · Page 17Linked to original sources

bioRxiv2024

Single-cell RNA sequencing of iPSC-derived brain organoids reveals Treponema pallidum infection inhibiting neurodevelopment

Congenital syphilis is a vertically transmitted bacterial infection caused by Treponema pallidum, often causing multidomain neurodevelopmental disabilities. However, little is known about the pathogenesis of this disease. Brain organoids platform derived from the induced pluripotent stem cell (iPSC) is exposed to T. pallidum infection for modelling congenital neurodevelopmental impairment. Single-cell RNA sequencing is used for identifying the subpopulations of differentially expressed genes and cellular heterogeneity and reconstructing differentiation trajectories following T. pallidum infection. The results reveal that T. pallidum infection influences the formation of neural rosette structures, reduces the cell number of the neural progenitor cell subcluster 1B (subNPC1B) and hindbrain neurons, and affects the neurodevelopment of the brain organoid. Moreover, it is speculated that T. pallidum inhibits the hindbrain neuron cell number through the suppression of subNPC1B subgroup in the organoids and inhibits transcription factor 3 activity in the subNPC1B-hindbrain neuronal axis. This is the first report on the inhibited effects of T. pallidum on the neurodevelopment of the iPSC-derived brain organoid model. T. pallidum could inhibit the differentiation of subNPC1B in brain organoids, thereby reducing the differentiation from subNPC1B to hindbrain neurons, and ultimately affecting the development and maturation of hindbrain neurons.

neuroscience↗

bioRxiv2022

Single-cell RNA sequencing reveals molecular features of postnatal maturation in the murine retinal pigment epithelium

Transcriptomic analysis of the mammalian retinal pigment epithelium (RPE) aims to identify cellular networks that influence ocular development, maintenance, function, and disease. However, available evidence points to RPE cell heterogeneity in the native tissue, which adds complexity to transcriptomic analysis. Here, to assess cell heterogeneity, we performed single-cell RNA sequencing of RPE cells from two young adult male C57BL/6J mice. Following quality control to ensure robust transcript identification limited to cell singlets, we detected 13,858 transcripts among 2,667 and 2,846 RPE cells, respectively. Dimensional reduction by principal component analysis and uniform manifold approximation and projection revealed six distinct cell popu-lations. All clusters expressed transcripts typical of RPE cells; the smallest (C1, containing 1-2% of total cells) exhibited hallmarks of stem and/or progenitor cells. Placing C1-6 along a pseudotime axis suggested a relative decrease in melanogenesis and stem/progenitor gene expression, and a corresponding increase in visual cycle gene expression upon RPE maturation. K-means testing of all detected transcripts identified additional expression patterns that may advance understanding of RPE stem/pro-genitor cell maintenance and the evolution of cellular metabolic networks during development. This work provides new insights into the transcriptome of the mouse RPE and a baseline for identifying experimentally induced transcriptional changes in future studies of this tissue.

systems biology↗

bioRxiv2021

Droplet-based single cell RNA sequencing of bacteria identifies known and previously unseen cellular states

Clonal bacterial populations rely on transcriptional variation to differentiate into specialized cell states that increase the communitys fitness. Such heterogeneous gene expression is implicated in many fundamental microbial processes including sporulation, cell communication, detoxification, substrate utilization, competence, biofilm formation, motility, pathogenicity, and antibiotic resistance1. To identify these specialized cell states and determine the processes by which they develop, we need to study isogenic bacterial populations at the single cell level2,3. Here, we develop a method that uses DNA probes and leverages an existing commercial microfluidic platform (10X Chromium) to conduct bacterial single cell RNA sequencing. We sequenced the transcriptome of over 15,000 individual bacterial cells, detecting on average 365 transcripts mapping to 265 genes per cell in B. subtilis and 329 transcripts mapping to 149 genes per cell in E. coli. Our findings correctly identify known cell states and uncover previously unreported cell states. Interestingly, we find that some metabolic pathways segregate into distinct subpopulations across different bacteria and growth conditions, suggesting that some cellular processes may be more prone to differentiation than others. Our high throughput, highly resolved single cell transcriptomic platform can be broadly used for understanding heterogeneity in microbial populations.

microbiology↗

bioRxiv2021

Single cell RNA Sequencing Identifies G-protein Coupled Receptor 87 as a Novel Basal Cell Marker of Distal Honeycomb Cysts in Idiopathic Pulmonary Fibrosis

Idiopathic Pulmonary Fibrosis (IPF) is a progressive and fatal lung disease with limited therapeutic options. Epithelial reprogramming and honeycomb cysts are key pathological features of IPF, however, the IPF distal bronchiole cell subtypes and their potential contribution to IPF development and progression still remain poorly characterized. Here, we utilized single-cell RNA sequencing on enriched EpCAM+ cells of the distal IPF and Donor lung. Using the 10x Genomics platform, we generated a dataset of 47,881 cells and found distinct cell clusters, including rare cell types, such as suprabasal cells recently reported in the healthy lung. We identified G-protein coupled receptor (GPR) 87 as a novel surface marker of distal Keratin (KRT)5+ basal cells. GPR87 expression was localized to distal bronchioles and honeycomb cysts in IPF in situ by RNA Scope and immunolabeling. Modulation of GPR87 in primary human bronchial epithelial cells cultures resulted in impaired airway differentiation and ciliogenesis. Thus, GPR87 is a novel marker and potentially druggable target of KRT5+ basal progenitor cells likely contributing to bronchiole remodeling and honeycomb cyst development in IPF.

cell biology↗

bioRxiv2022

Single Cell RNA Sequencing Driven Characterization of Pediatric Mixed Phenotype Acute Leukemia

BackgroundMixed phenotype acute leukemia (MPAL) is a rare subgroup of leukemia characterized by blast cells that display both myeloid and lymphoid lineage features, making this cancer difficult to diagnose and treat. A deeper characterization of MPAL at the molecular level is essential to better understand similarities/differences to the more common and better-studied leukemias, acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). Therefore, we performed single-cell RNA sequencing (scRNAseq) on MPAL bone marrow (BM) samples in an attempt to develop a more granular map of the MPAL microenvironment landscape. MethodsWe analyzed [~]16,000 cells from five pediatric MPAL BM samples collected at diagnosis to generate a single-cell transcriptomic landscape of B/Myeloid (B/My) and T/Myeloid (T/My) MPAL blasts and associated microenvironment cells. Cell clusters were identified using principal component analysis and uniform manifold approximation and projection (UMAP). Unsupervised analysis was performed to determine the overall relationship among B/My MPAL, T/My MPAL, and other acute leukemias - B-ALL, T-ALL, and AML. Supervised differentially expressed gene (DEG) analysis was performed to identify B/My and T/My MPAL blast-specific signatures. MPAL sample transcriptome profiles were compared with normal BM stem and immune cells to identify MPAL-specific dysregulation. Gene set enrichment analysis (GSEA) was performed, and significantly enriched pathways were compared in MPAL subtypes. Comparative analysis was performed on diagnostic samples based on their future minimal residual disease (MRD) and relapse status. ResultsB/My MPAL and T/My MPAL blasts displayed distinct subtype-specific blast signatures. UMAP analysis revealed that B/My MPAL samples had greater overlap with B-ALL samples, while T/My MPAL samples clustered separately from other acute leukemia subtypes. Genes overexpressed in both MPAL subtypes blasts compared to other leukemias and healthy controls included PLIN2, CD81, and UBE2S. B/My MPAL blast-specific genes included IRS2, SMIM3, and HBEGF, whereas T/My MPAL blast-overexpressed genes included IER5, BOD1L1, and HPGD. Sirtuin signaling, p38 MPAK signaling, and PI3K signaling pathways were upregulated in B/My MPAL blasts while oxidative phosphorylation and Rho family GTPases signaling pathways were upregulated in T/My MPAL blasts. Transcriptomic, pathways, and cell communication level differences were observed in the MPAL samples based on future MRD and clinical outcome status. ConclusionsWe have for the first time described the single-cell landscape of pediatric MPAL and demonstrate that B/My and T/My MPAL have unique scRNAseq profiles distinct from each other as well as from ALL and AML.

genomics↗

bioRxiv2026

Single-Cell RNA Sequencing Reveals Commensal Microbes Amplify Sex-Specific Immune Programming in the Murine Lung

Sex-based differences in respiratory disease outcomes are well recognized. However, the underlying immunological mechanisms driving this dimorphism remain incompletely understood. While sex hormones influence immune cell development and function, the role of commensal microbes in shaping sex-specific lung immunity has not been explored. Here, we used single-cell RNA sequencing (scRNAseq) and flow cytometry to profile lung immune cells in male and female mice housed under specific pathogen-free (SPF) or germ-free (GF) conditions. Under SPF conditions, males exhibited a striking myeloid bias, with increased monocytes and macrophages, along with broad upregulation of inflammatory mediators, including S100a8, S100a9, and Il1b, across multiple cell types, and enrichment of TNF and interferon (IFN) signaling pathways. In contrast, females displayed lymphocyte-skewed profiles, with higher frequencies of T cells and natural killer (NK) cells. Interestingly, these sex-based differences in immune composition and inflammatory programs were largely absent in GF mice, indicating that microbial exposure amplifies baseline immunological dimorphism between males and females. Notably, select sex-associated features, including female-biased NK cell enrichment, persisted irrespective of microbial status, suggesting intrinsic, microbiota-independent programming. Together, these findings indicate that commensal microbes modulate sex-specific lung immunity by amplifying pre-existing intrinsic differences, highlighting the intersection of extrinsic (microbial) and intrinsic (sex-linked) factors in shaping baseline mucosal immunity.

immunology↗

bioRxiv2024

Bacterial single-cell RNA sequencing captures biofilm transcriptional heterogeneity and differential responses to immune pressure

Biofilm formation is an important mechanism of survival and persistence for many bacterial pathogens. These multicellular communities contain subpopulations of cells that display vast metabolic and transcriptional diversity along with high recalcitrance to antibiotics and host immune defenses. Investigating the complex heterogeneity within biofilm has been hindered by the lack of a sensitive and high-throughput method to assess stochastic transcriptional activity and regulation between bacterial subpopulations, which requires single-cell resolution. We have developed an optimized bacterial single-cell RNA sequencing method, BaSSSh-seq, to study Staphylococcus aureus diversity during biofilm growth and transcriptional adaptations following immune cell exposure. We validated the ability of BaSSSh-seq to capture extensive transcriptional heterogeneity during biofilm compared to planktonic growth. Application of new computational tools revealed transcriptional regulatory networks across the heterogeneous biofilm subpopulations and identification of gene sets that were associated with a trajectory from planktonic to biofilm growth. BaSSSh-seq also detected alterations in biofilm metabolism, stress response, and virulence that were tailored to distinct immune cell populations. This work provides an innovative platform to explore biofilm dynamics at single-cell resolution, unlocking the potential for identifying biofilm adaptations to environmental signals and immune pressure.

microbiology↗

bioRxiv2025

Single Cell RNA Sequencing and Spatial Profiling Identify Mechanisms of Neonatal Brain Hemorrhage Development and Resolution

Precise control of cell-cell communication networks within brain neurovascular units (NVUs) promotes normal tissue physiology, and dysregulation of these networks can lead to pathologies including intracerebral hemorrhage (ICH). The cellular and molecular mechanisms underlying ICH development and subsequent tissue repair processes remain poorly understood. Here we employed quantitative single cell RNA sequencing coupled with spatial in situ gene expression profiling to characterize NVU signaling pathways associated with ICH in neonatal mouse brain tissue. The initial stages of ICH pathogenesis are characterized by downregulation of extracellular matrix (ECM)-associated signaling factors (Adamtsl2, Htra3, and Lama4) that functionally connect to canonical TGF{beta} activation and signaling in vascular endothelial cells. Conversely, the progressive resolution of ICH involves upregulation of neuroinflammatory signaling networks (Gas6 and Axl) alongside activation of iron metabolism pathway components (Hmox1, Cp, and Slc40a1) in astrocytes and microglial cells. Integrated computational modeling identifies additional ligand-receptor signaling networks between perivascular glial cells and endothelial cells during both ICH pathogenesis and resolution. Collectively, these findings illuminate the molecular signaling networks that promote NVU maturation and provide novel mechanistic insights into the pathways controlling ICH pathogenesis and repair.

developmental biology↗

bioRxiv2025

Single-cell RNA sequencing identifies accumulation of Fcgr2b+ virtual memory like CD8 T cells with cytotoxic and inflammatory potential in aged mouse white adipose tissue

Aging and obesity are associated with pro-inflammatory changes in adipose tissue. Overlapping mechanisms, such as the infiltration of inflammatory macrophages and T cells into visceral adipose tissue, have been implicated in contributing inflammation. However, a comparative analysis from both states is needed to identify distinct regulatory targets. Here, we performed single-cell RNA sequencing of stromal vascular fractions (SVF) isolated from gonadal white adipose tissue (gWAT) of young mice fed either a normal or a high-fat diet, and aged mice fed a normal diet. Our analysis revealed that physiological aging, compared to high-fat diet induced obesity, was associated with accumulation of phenotypically distinct CD8 T cells resembling virtual memory (VM) CD8 T cells. These cells expressed high levels of Cd44, Sell, Il7r, Il2rb, lacked Itga4, and exhibited elevated Fcgr2b expression which was associated with pseudotime differentiation trajectories. Flow cytometry confirmed an age-associated increase in Fcgr2b+CD49d- VM like CD8 T cells in gWAT. Notably, these Fcgr2b-expressing cells exhibited a cytotoxic profile, and expressed granzyme M. Functional analysis using recombinant granzyme M revealed its potential in inducing inflammation in mouse fibroblasts and macrophages. Together, our study has identified Fcgr2b+CD49d- VM-like CD8 T cells in the adipose tissue of aged mice with regulatory, cytotoxic and inflammatory potential.

immunology↗

bioRxiv2020

Revealing cellular heterogeneity and in vitro differentiation trajectory of cultured human endometrial mesenchymal-like stem cells using single-cell RNA sequencing

Endometrial mesenchymal-like stem cells (eMSCs) are adult stem cells contributing to endometrial regeneration. One set of perivascular markers (CD140b+CD146+) have been widely used to enrich eMSCs. Although eMSCs are easily accessible for regenerative medicine and have long been studied, their cellular heterogeneity and molecular program controlling their expansion and differentiation in vitro remains largely unclear. In this study, we applied 10X genomics single-cell RNA sequencing to eMSCs cultured in vitro after microbeading from 7 donors to investigate cellular heterogeneity in an unbiased manner. Corresponding clonogenic progenies of eMSCs after culture for 14 days were also sequenced to construct the in vitro differentiation trajectory of eMSCs. Transcriptomic expression based clustering revealed several subpopulations in eMSCs. Each subpopulation manifested distinct functional characteristics associated with immunomodulation, proliferation, extracellular matrix organization and cell differentiation. Pseudotime trajectory analysis on eMSCs and their differentiated progenies identified in vitro differentiation hierarchy of eMSCs. Further ligand-receptor pair analysis found that WNT signaling, NOTCH signaling, TGF-beta signaling and FGF signaling were important regulatory pathways for eMSC self-renewal and differentiation. By comparing eMSCs to Whartons Jelly MSCs and adipose-derived MSCs, we found these 3 kinds of MSCs expressed largely overlapping differentiation (CD) genes and highly variable genes. In summary, we reveal for the first time high molecular and cellular heterogeneity in cultured eMSCs, and identify the key signaling pathways that may be important for eMSC differentiation.

cell biology↗

bioRxiv2019

Single-cell RNA sequencing reveals functional heterogeneity and sex differences of glioma-associated brain macrophages

Microglia are resident myeloid cells in the central nervous system (CNS) that control homeostasis and protect CNS from damage and infections. Microglia and peripheral myeloid cells accumulate and adapt tumor supporting roles in human glioblastomas that show prevalence in men. Cell heterogeneity and functional phenotypes of myeloid subpopulations in gliomas remain elusive. Single-cell RNA sequencing (scRNA-seq) of CD11b+ myeloid cells in naive and GL261 glioma-bearing mice revealed distinct profiles of microglia, infiltrating monocytes/macrophages and CNS border-associated macrophages. We demonstrated an unforeseen molecular heterogeneity among myeloid cells in naive and glioma-bearing brains, validated selected marker proteins and showed distinct spatial distribution of identified subsets in experimental gliomas. We found higher expression of MHCII encoding genes in glioma-activated male microglia, which was corroborated in bulk and scRNA-seq data from human diffuse gliomas. Sex-specific gene expression in glioma-activated microglia may be relevant to sex differences in incidence and outcomes of glioma patients.

cancer biology↗

bioRxiv2023

Reanalysis of single-cell RNA sequencing data does not support herpes simplex virus 1 latency in non-neuronal ganglionic cells in mice

Most individuals are latently infected with herpes simplex virus type 1 (HSV-1) and it is well-established that HSV-1 establishes latency in sensory neurons of peripheral ganglia. However, it was recently proposed that latent virus is also present in immune cells recovered from ganglia in a mouse model used for studying latency. Here, we reanalyzed the single-cell RNA sequencing (scRNA-Seq) data that formed the basis for this conclusion. Unexpectedly, off-target priming in 3 scRNA-Seq experiments enabled the detection of non-polyadenylated HSV-1 latency-associated transcript (LAT) intronic RNAs. However, LAT reads were near-exclusively detected in a mixed population of cells undergoing cell death. Specific loss of HSV-1 LAT and neuronal transcripts during quality control filtering indicated widespread destruction of neurons, supporting the presence of contaminating cell-free RNA in other cells following tissue processing. In conclusion, the reported detection of latent HSV-1 in non-neuronal cells is best explained by inaccuracies in the data analyses.

microbiology↗

bioRxiv2026

Insights into tick-pathogen interactions - a single cell RNA sequencing approach of transcriptional changes during ehrlichial infection

Tick-borne diseases represent a significant threat to human and animal health worldwide. In the United States, the blacklegged tick, Ixodes scapularis (I. scapularis), serves as a competent vector for several bacterial pathogens, including Ehrlichia muris eauclairensis (EME). The I. scapularis embryonic cell line (ISE6) is a valuable tool for propagating tick-borne pathogens and studying tick-pathogen interactions. In this study, we examined the cellular complexity of ISE6 cells and their response to EME infection. Single-cell RNA sequencing revealed 15 distinct cell clusters present. Although ISE6 cells are heterogeneous, they do not display transcriptional similarity to any known tick tissues. Notably, this lack of similarity did not influence their susceptibility to EME infection. Our results demonstrated that EME infection induces time-dependent transcriptional changes in ISE6 cells: early infection is characterized by upregulation of genes associated with stress adaptation, mitochondrial function, and metabolic pathways, whereas late infection leads to broad downregulation of genes involved in the cell cycle, DNA replication, and cytoskeletal organization. These findings enhance our understanding of ehrlichial interactions with ISE6 cells and reinforce the utility of this cell line as a resource for isolating and propagating arthropod endosymbionts and tick-borne pathogens. IMPORTANCEThis study provides a single-cell resolution framework for interpreting tick cell line biology during infection with a medically relevant ehrlichial pathogen. Using scRNA-seq, we show that the I. scapularis embryonic-derived ISE6 cell line comprises multiple transcriptionally distinct cell states, yet these states do not map cleanly onto canonical tick tissue signatures, even when compared against a curated reference tissue atlas. Despite this heterogeneity, EME broadly infects ISE6 cell population, indicating that susceptibility is not restricted to a specific cell type. We further define a time-dependent arthropod vector response in which early infection is marked by activation of stress and metabolic adaptation response, followed by late-stage inhibition of key signaling, transcriptional, and proliferative pathways as bacterial burden increases. Together, these findings strengthen the biological interpretation of ISE6 as an in vitro model for tick-pathogen interactions and provide a resource for future mechanistic studies of ehrlichial persistence, replication, and vector competence.

molecular biology↗

bioRxiv2021

Chicken peripheral blood lymphocyte response to ALV-J infection assessed by single-cell RNA sequencing

Chicken peripheral blood lymphocytes (PBLs) exhibit wide-ranging cell types, but current understanding of their subclasses, immune cell classification, and function is limited and incomplete. Previously, we found that viremia caused by avian leukosis virus subgroup J (ALV-J) was eliminated by 21 days post infection (DPI), accompanied by increased CD8+ T cell ratio in PBLs and low antibody levels. Here we performed single-cell RNA sequencing (scRNA-seq) of PBLs in ALV-J infected and control chickens at 21 DPI to determine chicken PBL subsets and their specific molecular and cellular characteristics, before and after viral infection. Eight cell clusters and their potential marker genes were identified in chicken PBLs. T cell populations (clusters 6 and 7) had the strongest response to ALV-J infection at 21 DPI, based on detection of the largest number of differentially expressed genes (DEGs). T cell populations of clusters 6 and 7 could be further divided into four subsets: activated CD4+ T cells (cluster A0), Th1-like cells (cluster A2), Th2-like cells (cluster A1), and cytotoxic CD8+ T cells. Hallmark genes for each T cell subset response to viral infection were initially identified. Furthermore, pseudotime analysis results suggested that chicken CD4+ T cells could potentially differentiate into Th1-like and Th2-like cells. Moreover, ALV-J infection probably induced CD4+ T cell differentiation into Th1-like cells in which the most immune related DEGs were detected. With respect to the control group, ALV-J infection also had an obvious impact on PBL cell composition. B cells showed inconspicuous response and their numbers decreased in PBLs of the ALV-J infected chickens at 21 DPI. Percentages of cytotoxic Th1-like cells and CD8+ T cells were increased in the T cell population of PBLs from ALV-J infected chicken, which were potentially key mitigating factors against ALV-J infection. More importantly, our results provided a rich resource of gene expression profiles of chicken PBL subsets for a systems-level understanding of their function in homeostatic condition as well as in response to viral infection.

immunology↗

bioRxiv2026

Transcriptomic Characterization of Terminal Complement Complex-bound Cells in Human Choroid Using Single-Cell RNA Sequencing

Age-related macular degeneration (AMD) is among the leading causes of blindness worldwide. Early AMD is characterized by dysfunction in the choroid, including early dropout of endothelial cells and increased deposition of the complement cascade's membrane attack complex (MAC) in the choriocapillaris. In this study, we used a single-cell RNA sequencing-based approach with barcoded antibodies to measure abundance of the MAC and other surface proteins at single cell resolution on RPE/choroid samples from four aged human donor eyes. We included antibodies to detect the MAC, CD34, CD45, and complement regulators CD55 and CD59, in addition to control antibodies. Our analysis of these data revealed cell clusters with expected gene expression profiles and antibody-based detection of CD34 and CD45 congruent with transcriptome-based cell identity. We also detected surface complement regulators CD55 and CD59 across a wide variety of cell types. Across endothelial cells, surface CD55 and CD59 appeared more abundant on venous clusters, and abundance of each was correlated with expression of a third complement regulator, clusterin (CLU). The MAC was detected on a variety of cell types, but was most abundant on the surface of cells in the macrophage family, smooth muscle cells, and pericytes. We confirmed these findings by identifying MAC deposition on choriocapillaris pericytes using immunohistochemistry for MAC, endothelial, and pericyte markers. Ultimately, these data showcase a valuable new approach to analyze gene expression and surface complement in human donor eyes, and provide novel insight into patterns of MAC deposition and complement protection in the aging human choroid.

cell biology↗

bioRxiv2025

Novel Insights into T-Cell Exhaustion and Cancer Biomarkers in PDAC Using Single Cell RNA Sequencing

One of the aggressive and lethal cancers, Pancreatic ductal adenocarcinoma (PDAC) is characterised by poor prognosis and resistance to conventional treatments. Moreover, tumor immune microenvironment (TIME) plays a crucial role in the progression and therapeutic resistance of PDAC. It is associated with T-cells exhaustion, leading to the progressive loss of T-cell functions with impaired ability to kill tumor cells. Therefore, this study employed single cell RNA sequencing (scRNA-seq) analysis identifying upregulated genes of T-cells; and of cancer cells in two groups ("cancer cells_vs_all-PDAC" and "cancer-PDAC_vs_all-normal"). Common and unique markers of cancer cells from both groups were identified. The Reactome pathways of cancer and T-cells were selected; while the genes implicated in those pathways were used to perform PPI analysis, revealing the hub-genes of cancer and T-cells. The gene expression validation of cancer and T-cells hub-genes was performed using GEPIA2 and TISCH2, while overall survival analysis of cancer cells hub-genes was performed using GEPIA2. Conclusively, this study unravelled 16 novel markers of cancer and T-cells providing the groundwork for future research into the immune landscape of PDAC, particularly T-cell exhaustion. However, further clinical studies are needed to validate these novel markers as potential therapeutic targets in PDAC patients. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/632574v2_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@24703borg.highwire.dtl.DTLVardef@443ddforg.highwire.dtl.DTLVardef@39fb9corg.highwire.dtl.DTLVardef@b5bee1_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2022

Inflammatory Signaling and Fibroblast-Cancer Cell Interactions Transfer from a Harmonized Human Single-cell RNA Sequencing Atlas of Pancreatic Ductal Adenocarcinoma to Organoid Co-Culture

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by a heterogeneous tumor microenvironment (TME) that is enriched with cancer associated fibroblasts (CAFs)1. Cell-cell interactions involving these CAFs promote an immunosuppressive phenotype with altered inflammatory gene expression. While single-cell transcriptomics provides a tool to dissect the complex intercellular pathways that regulate cancer-associated inflammation in human tumors, complementary experimental systems for mechanistic validation remain limited. This study integrated single-cell data from human tumors and novel organoid co-cultures to study the PDAC TME. We derived a comprehensive atlas of PDAC gene expression from six published human single-cell RNA sequencing (scRNA-seq) datasets2-7 to characterize intercellular signaling pathways between epithelial tumor cells and CAFs that regulate the inflammatory TME. Analysis of the epithelial cell compartment identified global gene expression pathways that modulate inflammatory signaling and are correlated with CAF composition. We then generated patient-derived organoid-CAF co-cultures to serve as a biological model of the cellular interactions learned from human tissue in the atlas. Transfer learning analysis to additional scRNA-seq data of this co-culture system and mechanistic experiments confirmed the epithelial response to fibroblast signaling. This bidirectional approach of complementary computational and in vitro applications provides a framework for future studies identifying important mechanisms of intercellular interactions in PDAC.

cancer biology↗

bioRxiv2020

Single-cell RNA sequencing of Tocilizumab-treated peripheral blood mononuclear cells as an in vitro model of inflammation

COVID-19 has posed a significant threat to global health. Early data has revealed that IL-6, a key regulatory cytokine, plays an important role in the cytokine storm of COVID-19. Multiple trials are therefore looking at the effects of Tocilizumab, an IL-6 receptor antibody that inhibits IL-6 activity, on treatment of COVID-19, with promising findings. As part of a clinical trial looking at the effects of Tocilizumab treatment on kidney transplant recipients with subclinical rejection, we performed single-cell RNA sequencing of comparing stimulated PBMCs before and after Tocilizumab treatment. We leveraged this data to create an in vitro cytokine storm model, to better understand the effects of Tocilizumab in the presence of inflammation. Tocilizumab-treated cells had reduced expression of inflammatory-mediated genes and biologic pathways, particularly amongst monocytes. These results support the hypothesis that Tocilizumab may hinder the cytokine storm of COVID-19, through a demonstration of biologic impact at the single-cell level.

genomics↗