Single-cell mRNA-seq Research Articles

Osteomacs promote maintenance of murine hematopoiesis through megakaryocyte-induced upregulation of Embigin and CD166

Maintenance of hematopoietic stem cell (HSC) function in the niche is an orchestrated event. Osteomacs (OM) are key cellular components of the niche. Previously, we documented that osteoblasts, OM, and megakaryocytes interact to promote hematopoiesis. Here, we further characterize OM and identify megakaryocyte-induced mediators that augment the role of OM in the niche. Single-cell mRNA-seq, mass spectrometry, and CyTOF examination of megakaryocyte-stimulated OM suggested that upregulation of CD166 and Embigin on OM augment their hematopoiesis maintenance function. CD166 knockout OM or shRNA-Embigin knockdown OM confirmed that the loss of these molecules significantly reduced the ability of OM to augment the osteoblast-mediated hematopoietic-enhancing activity. Recombinant CD166 and Embigin partially substituted for OM function, characterizing both proteins as critical mediators of OM hematopoietic function. Our data identify Embigin and CD166 as OM-regulated critical components of HSC function in the niche and potential participants in various invitro manipulations of stem cells.

Bile exosomal miR-182/183-5p increases cholangiocarcinoma stemness and progression by targeting HPGD and increasing PGE2 generation.

Cholangiocarcinoma (CCA) is a highly lethal malignancy originating from the biliary ducts. Current CCA diagnostic and prognostic assessments cannot satisfy the clinical requirement. Bile detection is rarely performed, and herein, we aim to estimate the clinical significance of bile liquid biopsy by assessing bile exosomal concentrations and components. Exosomes in bile and sera from CCA, pancreatic cancer, and common bile duct stone were identified and quantified by transmission electronmicroscopy, nanoparticle tracking analysis, and nanoFCM. Exosomal components were assessed by liquid chromatography with tandem mass spectrometry and microRNA sequencing (miRNA-seq). Bile exosomal concentration in different diseases had no significant difference, but miR-182-5p and miR-183-5p were ectopically upregulated in CCA bile exosomes. High miR-182/183-5p in both CCA tissues and bile indicates a poor prognosis. Bile exosomal miR-182/183-5p is secreted by CCA cells and can be absorbed by biliary epithelium or CCA cells. With xenografts in humanized mice, we showed that bile exosomal miR-182/183-5p promotes CCA proliferation, invasion, and epithelial-mesenchymal transition (EMT) by targeting hydroxyprostaglandin dehydrogenase in CCA cells and mast cells (MCs), and increasing prostaglandin E2 generation, which stimulates PTGER1 and increases CCA stemness. In single-cell mRNA-seq, hydroxyprostaglandin dehydrogenase is predominantly expressed in MCs. miR-182/183-5p prompts MC to release VEGF-A release from MC by increasing VEGF-A expression, which facilitates angiogenesis. CCA cells secret exosomal miR-182/183-5p into bile, which targets hydroxyprostaglandin dehydrogenase in CCA cells and MCs and increases prostaglandin E2 and VEGF-A release. Prostaglandin E2 promotes stemness by activating PTGER1. Our results reveal a type of CCA self-driven progression dependent on bile exosomal miR-182/183-5p and MCs, which is a new interplay pattern of CCA and bile.

Abstract B041: Bone microenvironment cues shift Ewing sarcoma cells towards pro-metastatic, ECM-secreting cell states

Abstract Solid tumors are complex ecosystems comprised of subpopulations of heterogeneous cancer cells that are altered by interactions with diverse cellular and acellular factors present in tumor microenvironments (TMEs). Accruing evidence suggests that heterogeneity of tumor cells and the TME, and bidirectional crosstalk between these spatially and temporally dynamic components, contributes to the generation of metastatic and therapy-resistant cell subpopulations. Ewing sarcomas are highly metastatic bone and soft tissue tumors driven by pathognomonic FET:ETS transcription factor fusions, predominantly EWS::FLI1. Despite sharing this single oncogenic driver, we and others have shown that Ewing sarcoma tumor cells are highly plastic cells that exist in transcriptionally and phenotypically distinct states. Notably, the extent of EWS::FLI1 transcriptional activity, ranging from low to high, has been shown to in part define the Ewing sarcoma cell state. Cells with high levels of fusion protein activity are generally proliferative and non-motile, while cells with lower levels of EWS::FLI1 expression or activity display enhanced metastatic potential. In this study we have leveraged single cell mRNA-seq and CITE-seq of Ewing sarcoma cell lines, gene expression and immunohistochemical profiling of primary tumor samples, and 3D culture invasion assays to define the complex Ewing sarcoma ecosystem and to investigate the role of tumor cell subpopulations and tumor: TME cross talk on tumor biology. Our results show cells with low levels of EWS::FLI1 activity activate an epithelial-mesenchymal transition (EMT)-like shift in gene expression. This activation of a partial EMT program is notable for an increase in expression and secretion of extracellular matrix (ECM) proteins by subpopulations of tumor cells and by increased matrix invasion. These ECM-related genes serve as transcriptional markers of the EMT-like cell state in both Ewing sarcoma models and in primary patient tumors. Moreover, the local TME surrounding these subpopulations is rich in tumor cell-derived ECM proteins including Tenascin-C, biglycan, and SPARC, proteins implicated in promotion of metastasis. We propose that subpopulations of ECM-secreting Ewing sarcoma tumor cells remodel the TME in a manner reminiscent of cancer associated fibroblasts (CAFs) in carcinomas. Further, we propose that this CAF-like matrix deposition contributes to local tumor progression and metastatic dissemination. Significantly, this ECM-secreting tumor cell state can be induced by canonical Wnt and TGF-beta ligands, suggesting that factors present in the bone TME can serve as inducers of a metastatic cell state. Together these findings demonstrate that Ewing sarcoma metastasis may be promoted by specific cell subpopulations which respond to and remodel the bone tumor microenvironment. Citation Format: Emma Wrenn, April Apfelbaum, Trisha Lipson, Erin Rudzinski, Xuemei Deng, Katherine Braun, Nicolas Garcia, Shruti Bhise, Olivia Waltner, Scott Furlan, Beth Lawlor. Bone microenvironment cues shift Ewing sarcoma cells towards pro-metastatic, ECM-secreting cell states [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B041.

Abstract 15540: Single Cell Transcriptional-Epigenetic Profiling and Sub-Cellular Proteomics Demonstrate That HIV-Nef Extracellular Vesicles Modulate Human Macrophage Heterogeneity to Promote Atherosclerosis

Background: In the era of modern Anti-Retroviral Therapy, people living with HIV continue to remain at a higher risk than the general population for cardiovascular diseases including atherosclerosis. We hypothesized that circulating extracellular vesicles (EV) containing HIV-Nef protein promote inflammation by switching the pattern of macrophage heterogeneity towards a pro-inflammatory state that underlies cardiovascular disease. Methods & Results: Human primary macrophages were stimulated with EV engineered to contain HIV-Nef. Macrophage heterogeneity was characterized by simultaneous single cell mRNA-seq and single cell ATAC-seq integrated with WNN mapping in 50,931 cells. Among 16 clusters (Fig. A), pseudotime (Slingshot), gene regulatory (DoRothEA and SCENIC) and transcription factor motif accessibility (ChromVar) analyses demonstrated that HIV-Nef regulates 3 inflammatory clusters as gauged by differentially active and accessible transcription factors, e.g., JUN, YY1 and STAT1. Sub-cellular proteomics identified differentially abundant proteins associated with DNA (662 proteins), nucleus (261), cytosol (461), cell surface (72) and EV (132). Pathway enrichment analysis of these proteins revealed changes in biological processes in immune responses, cytoskeletal regulation, and metabolism. Functional phenotyping showed HIV-Nef shifts macrophages towards a pro-inflammatory phenotype by suppressing atheroprotective efferocytosis, promoting chemotaxis and altering metabolic state. Furthermore, short-term injection of HIV-Nef EV into WT mice induced inflammatory chemokine secretion, while long-term injection into Ldlr -/- mice enhanced atherogenesis (Fig.B). Conclusions: Persistent Nef in EV of HIV survivors may modulate macrophage heterogeneity to promote chronic inflammation. These findings may help identify patients at risk for cardiovascular complications and develop novel atheroprotective therapies.

Antigen-specific immune reactions by expanded CD8+ T cell clones from HLA-B*27-positive patients with spondyloarthritis

Spondyloarthritis (SpA) is a chronic inflammatory disease that is tightly linked to HLA-B*27 but the pathophysiological basis of this link is still unknown. It is discussed whether either the instability of HLA-B*27 molecules triggers predominantly innate immune reactions or yet unknown antigenic peptides presented by HLA-B*27 induce adaptive autoimmune reactions by CD8+ T cells. To analyze the pathogenesis of SpA, we here investigated the T cell receptor (TCR) usage and whole transcriptomes of CD8+ single cells from synovial fluid of HLA-B*27-positive SpA patients and HLA-B*27-negative controls. In HLA-B*27-positive patients, we confirmed preferential expression of several TCR β-chain families, found even more restricted usage of particular TCR α-chains, assigned matching TCR αβ-chain pairs with homologous CDR3-sequences, and detected identical TCR-chains in different patients. Gene expression analyses by single cell mRNAseq revealed that genes specific for the tissue resident memory phenotype, exhaustion, and apoptosis were particularly highly expressed in expanded clonotypes from HLA-B*27-positive SpA patients. Together, several independent lines of evidence argue in favor of an (auto)antigenic peptide related pathogenesis.

Minimal gene set discovery in single-cell mRNA-seq datasets with ActiveSVM

Sequencing costs currently prohibit the application of single-cell mRNA-seq to many biological and clinical analyses. Targeted single-cell mRNA-sequencing reduces sequencing costs by profiling reduced gene sets that capture biological information with a minimal number of genes. Here we introduce an active learning method that identifies minimal but highly informative gene sets that enable the identification of cell types, physiological states and genetic perturbations in single-cell data using a small number of genes. Our active feature selection procedure generates minimal gene sets from single-cell data by employing an active support vector machine (ActiveSVM) classifier. We demonstrate that ActiveSVM feature selection identifies gene sets that enable ~90% cell-type classification accuracy across, for example, cell atlas and disease-characterization datasets. The discovery of small but highly informative gene sets should enable reductions in the number of measurements necessary for application of single-cell mRNA-seq to clinical tests, therapeutic discovery and genetic screens.

A 2-Gene Signature Related to Interferon-Gamma Predicts Prognosis and Responsiveness to Immune Checkpoint Blockade of Glioma.

BackgroundGliomas represent the most common and aggressive brain malignancy. Interferon-gamma (IFNG) is a potent inducer of immune response, developing IFNG-related gene signature may promote the diagnosis and treatment of this disease.MethodsBulk tumor and single-cell mRNA-seq datasets of glioma ranging from WHO grade II to IV with corresponding demographics were included. Multiple bioinformatics and machine learning algorithms were performed to develop an IFNG-related prognostic signature and evaluate immune checkpoint blockade (ICB) therapy response.ResultsIFNGR1 and IFNGR2 were used as concise IFNG-related gene signature based on which the IFNGR score well-characterized the IFNG response in the glioma microenvironment. Increased IFNGR score was associated with clinicopathological parameters relating to tumor malignancy and prevailing molecular pathological markers. Notably, K-M and Cox regression analysis found that the IFNGR score was an effective prognostic biomarker, and was associated with tumor relapse for a subset of patients. Notably, IFNGR1 and IFNGR2 were preferentially expressed by the Mono/Macro cells in the glioma microenvironment and were significantly correlated with M2 macrophage. Thus, the IFNGR score-high group had increased expression of immune checkpoints and had the potential to predict ICB responsiveness.ConclusionIn conclusion, we have developed a concise IFNG-related gene signature of clinical significance, which may improve the current diagnosis and treatment of glioma.

Identification of Frameshift Variants in POLH Gene Causing Xeroderma Pigmentosum in Two Consanguineous Pakistani Families.

Xeroderma pigmentosum (XP) is a rare autosomal recessive genetic disorder characterized by severe sensitivity of skin to sunlight and an increased risk of skin cancer. XP variant (XPV), a milder subtype, is caused by variants in the POLH gene. POLH encodes an error-prone DNA-polymerase eta (pol eta) which performs translesion synthesis past ultraviolet photoproducts. The current study documents the clinical and genetic investigations of two large consanguineous Pakistani families affected with XPV. In family 1, whole exome sequencing (WES) revealed a novel frameshift variant, c.1723dupG (p.(Val575Glyfs*4)), of POLH, which is predicted to cause frameshift and premature truncation of the encoded enzyme. Indeed, our ex vivo studies in HEK293T cells confirmed the truncation of the encoded protein due to the c.1723dupG variant. In family 2, Sanger sequencing of POLH exons, revealed a recurrent nonsense variant, c.437dupA (p.Tyr146*). POLH forms a hetero-tetrameric POLZ complex with REV3L, REV7, POLD2 and POLD3. Next, we performed in silico analysis of POLH and other POLZ complex genes expression in publicly available single cell mRNAseq datasets from adult human healthy and aging skin. We found overlapping expression of POLH, REV3L and POLD2 in multiple cell types including differentiated and undifferentiated keratinocytes, pericytes and melanocytes in healthy skin. However, in aging human skin, POLH expression is reduced in compare to its POLZ complex partners. Insights from our study will facilitate counseling regarding the molecular and phenotypic landscape of POLH-related XPV.

Tbx5 navigates through the labyrinth of adult cardiac regeneration

Abstract Background Heart failure is the major cause of death and morbidity in industrialized countries with an estimated 23 million people affected per year, representing 30% of all global deaths. Injury to the adult mammalian cardiac muscle, often leads to a heart attack due to irreversible loss of a large number of cardiomyocytes (CM) and other cardiac interstitial cells, creating an unmet need for identifying a cardiac progenitor cell (CPC) population for cardiac replenishment. In contrast, amphibians and neonatal rodents possess the ability to regenerate their heart upon injury. It has been suggested recently that idle cardiac regenerative mechanisms may be present in adult mammals, inhibited by exogenous cues, or lack of. Murine and human CPCs can be isolated through the expression of Pdgfra, Kdr, and our novel surface marker, Gfra2. In addition, the expression of the embryonic transcription factor TBX5, is paramount for differentiation towards a cardiomyocyte fate. Therefore, Tbx5-expressing CPCs could be an effective target for proof-of-concept studies in the heart repair field, inclined to pharmacological modulation in patients with ischemic heart disease. Purpose To characterise an adult Tbx5-expressing CPC population in the injured heart. Using a developmental approach to two adult heart injury murine models, we show that Tbx5-expressing CPC exist in the injured adult mammalian heart, with a molecular signature that strongly correlates with that of embryonic and neonatal CPCs. Methods A well-defined tamoxifen-induced Tbx5-Cre; Rosa26R-eYFP/eYFP transgenic mouse model was employed, where myocardial infarction (MI) was induced through reperfusion/ischemia or chemical injury. Cardiac cells expressing YFP+ cells were collected from the adult injured hearts five to seven days post-injury. Flow-cytometric and single-cell mRNA-seq analysis was performed in order to collect and compare those YFP+ cells to control adult uninjured cardiac interstitial cells, and early neonatal-derived CPCs. Results Immunohistochemical analysis indicated that YFP+ interstitial cells were mostly present in the injury site, but also in distal cardiac areas. Flow cytometric analysis of live cells pinpoint these YFP+ cells are part of a CPC-like population. Single-cell mRNA transcriptomic analysis revealed that YFP+ cells resemble early postnatal CPC. Yet, YFP+ cells never expressed CM markers in vivo, but they did differentiate into CM-like cells, in vitro. Conclusions Upon MI, the adult heart possess an interstitial cell population that transiently re-activates the pioneer cardiac embryonic transcription factor Tbx5. The Tbx5-expressing cell population transcriptomically resembles that of CPC, which could promote CM regeneration upon neonatal injury. We show that Tbx5 lies in the centre of a novel adult CPC population. The adult heart microenvironment may hinder mammalian CM regeneration through regulation of the Tbx5-induced cardiac gene program. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Hellenic Foundation for Research & Innovation

CaMKIIα Expressing Neurons to Report Activity-Related Endogenous Hypoxia upon Motor-Cognitive Challenge.

We previously introduced the brain erythropoietin (EPO) circle as a model to explain the adaptive ‘brain hardware upgrade’ and enhanced performance. In this fundamental circle, brain cells, challenged by motor-cognitive tasks, experience functional hypoxia, triggering the expression of EPO among other genes. We attested hypoxic cells by a transgenic reporter approach under the ubiquitous CAG promoter, with Hif-1α oxygen-dependent degradation-domain (ODD) fused to CreERT2-recombinase. To specifically focus on the functional hypoxia of excitatory pyramidal neurons, here, we generated CaMKIIα-CreERT2-ODD::R26R-tdTomato mice. Behavioral challenges, light-sheet microscopy, immunohistochemistry, single-cell mRNA-seq, and neuronal cultures under normoxia or hypoxia served to portray these mice. Upon complex running wheel performance as the motor-cognitive task, a distinct increase in functional hypoxic neurons was assessed immunohistochemically and confirmed three-dimensionally. In contrast, fear conditioning as hippocampal stimulus was likely too short-lived to provoke neuronal hypoxia. Transcriptome data of hippocampus under normoxia versus inspiratory hypoxia revealed increases in CA1 CaMKIIα-neurons with an immature signature, characterized by the expression of Dcx, Tbr1, CaMKIIα, Tle4, and Zbtb20, and consistent with accelerated differentiation. The hypoxia reporter response was reproduced in vitro upon neuronal maturation. To conclude, task-associated activity triggers neuronal functional hypoxia as a local and brain-wide reaction mediating adaptive neuroplasticity. Hypoxia-induced genes such as EPO drive neuronal differentiation, brain maturation, and improved performance.

Animal regeneration in the era of transcriptomics.

Animal regeneration, the ability to restore a lost body part, is a process that has fascinated scientists for centuries. In this review, we first present what regeneration is and how it relates to development, as well as the widespread and diverse nature of regeneration in animals. Despite this diversity, animal regeneration includes three common mechanistic steps: initiation, induction and activation of progenitors, and morphogenesis. In this review article, we summarize and discuss, from an evolutionary perspective, the recent data obtained for a variety of regeneration models which have allowed to identify key shared mechanisms that control these main steps of animal regeneration. This review also synthesizes the wealth of high-throughput mRNA sequencing data (bulk mRNA-seq) concerning regeneration which have been obtained in recent years, highlighting the major advances in the regeneration field that these studies have revealed. We stress out that, through a comparative approach, these data provide opportunities to further shed light on the evolution of regeneration in animals. Finally, we point out how the use of single-cell mRNA-seq technology and integration with epigenomic approaches may further help researchers to decipher mechanisms controlling regeneration and their evolution in animals.

Androglobin gene expression patterns and FOXJ1-dependent regulation indicate its functional association with ciliogenesis

Androglobin (ADGB) represents the latest addition to the globin superfamily in metazoans. The chimeric protein comprises a calpain domain and a unique circularly permutated globin domain. ADGB expression levels are most abundant in mammalian testis, but its cell-type-specific expression, regulation, and function have remained unexplored. Analyzing bulk and single-cell mRNA-Seq data from mammalian tissues, we found that—in addition to the testes—ADGB is prominently expressed in the female reproductive tract, lungs, and brain, specifically being associated with cell types forming motile cilia. Correlation analysis suggested coregulation of ADGB with FOXJ1, a crucial transcription factor of ciliogenesis. Investigating the transcriptional regulation of the ADGB gene, we characterized its promoter using epigenomic datasets, exogenous promoter-dependent luciferase assays, and CRISPR/dCas9-VPR-mediated activation approaches. Reporter gene assays revealed that FOXJ1 indeed substantially enhanced luciferase activity driven by the ADGB promoter. ChIP assays confirmed binding of FOXJ1 to the endogenous ADGB promoter region. We dissected the minimal sequence required for FOXJ1-dependent regulation and fine mapped the FOXJ1 binding site to two evolutionarily conserved regions within the ADGB promoter. FOXJ1 overexpression significantly increased endogenous ADGB mRNA levels in HEK293 and MCF-7 cells. Similar results were observed upon RFX2 overexpression, another key transcription factor in ciliogenesis. The complex transcriptional regulation of the ADGB locus was illustrated by identifying a distal enhancer, responsible for synergistic regulation by RFX2 and FOXJ1. Finally, cell culture studies indicated an ADGB-dependent increase in the number of ciliated cells upon overexpression of the full-length protein, confirming a ciliogenesis-associated role of ADGB in mammals.

Abstract 968: Distinct pathways of DC-induced CD8+T cell differentiation revealed by single-cell mRNA sequencing analysis

Abstract Purpose of Study: Dendritic Cells (DC) maturing in the conditions of acute/early versus late/chronic inflammation (such as alpha-type-1-polarized DCs [αDC1s] and “standard” nonpolarized DCs; sDCs) deliver differential “signal 3” to resting T cells, resulting in their different induction of effector functions in tumor-specific CD8+ T cells. Here, using single-cell mRNAseq (scRNAseq), a unique technology allowing for unbiased analysis of gene expression (GEX), we evaluated the GEX patterns and cellular heterogeneity of CD8+ T cells expanded by cancer-cell loaded αDC1s or sDCs. Procedure: Freshly isolated autologous (compared to DCs) CD8+ T cells were in vitro sensitized for 1 week, using either αDC1s loaded with UV/gamma-radiation-killed ovarian cancer (OvCa) cell lines OVCAR or SKOV3, or sDCs loaded with the same antigens. αDC1s and sDCs not loaded with exogenous antigen were used as controls. The expanded CD8+ T cells were harvested, captured at the single-cell level and immobilized in a vertical flow array chip (VFAC), which contain 100-microchambers packed with 105 beads immobilizing 1010 oligo(dT) probes with unique cell-ID, UMI, and PCR-tags. Each cell was lysed and its mRNA was individually tagged with unique cell ID sequences prior to library preparation and targeted scRNAseq using a custom 94-gene inflammation RNA-seq panel was performed. Post-clustering (Scanpy), unsupervised cell-type identification (louvain), and downstream analyses were performed on GEX data to determine impact of αDC1s versus sDCs (loaded with OVCAR or SKOV3 versus not loaded with antigens) on the effector and memory pathways of differentiation of the expanded T cells and their unique functional status. Results: PCA and unsupervised clustering revealed sample groups with distinct phenotypic signatures associated with loaded versus unloaded aDC1s, with differential GEX subclusters identified for the αDC1- activated T cells (independently on DC loading with either OVCAR or SKOV3 cells) to include at least two populations with differential expression of genes typical of central memory, effector memory and effector cells (killer molecules and chemokine receptors typical of both lymphoid and peripheral homing potential). In sharp contrast, sDC1 activated T cell showed a more uniform gene distribution patterns lacking the effector T cell component. These differences correlated with the differential ability of αDC1- and sDC-activated T cells to respond to migrate to different chemokines and to recognize and kill cancer cells. Conclusion: scRNAseq was able to identify distinct single-cell T-cell phenotypes caused by induction of αDC1s or sDCs (loaded with OVCAR or SKOV3), consistent with the differential ability of these two DC types to induce effector, effector memory and central memory CD8+ T cells in response to different tumor targets and with their differential ability to modulate peripheral tissue homing patterns. Citation Format: Bowen Dong, Sarabjot Pabla, Vincent Giamo, Weijian Jiang, Kiyomi Taniguchi, Sean T. Glenn, Masataka Shirai, Pawel Kalinski. Distinct pathways of DC-induced CD8+T cell differentiation revealed by single-cell mRNA sequencing analysis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 968.

Abstract 970: Single-cell mRNA sequencing analysis of the synergistic impact of double-stranded RNA (dsRNA) and IFNα on human monocyte-derived macrophages

Abstract Purpose of Study: Single-cell mRNAseq (scRNAseq) is a promising technology allowing for unbiased analysis of gene expression at the cellular level. In this study, scRNAseq was used to identify cellular sub-types and differential gene expression functional units in monocyte-derived macrophages in response to double-stranded RNA (dsRNA, viral mimic, TLR3 ligand), IFNα or their combination. Procedure: Human monocyte-derived macrophages were cultured overnight in the absence (U) or presence of dsRNA (rintatolimod/Ampligen-A), IFNα (I) or their combination (IA), which we previously identified as synergistic in induction of chemokines attracting CTLs, Th1 and NK cells. The cells were harvested after 1 day, captured at the single-cell level and immobilized in a vertical flow array chip (VFAC), which contains 100-microchambers packed with 105 beads immobilizing 1010 oligo(dT) probes with unique cell-ID, UMI, and PCR-tags. Each single cell was captured and lysed on the small hole (3-micro meter in diameter) above the microchamber on the VFAC and cDNA libraries were synthesized and tagged with unique cell-IDs post mRNAs hybridization. ScRNAseq using a custom 94- gene inflammation RNA-seq panel was then performed. Post-clustering (Scanpy), unsupervised cell-type identification (louvain), and downstream analyses were performed on the gene expression data to determine impact of A, I and IA treatment on the cellular composition of the induced macrophages. Results: PCA and unsupervised clustering revealed sample groups with distinct phenotypic signatures. The analyses revealed the expected synergy between the dsRNA and IFNα in the induction of CXCR3- and CCR5-binding chemokines, but antagonism in the induction of Treg attractants. Unexpectedly, the single cell analyses revealed striking heterogeneity of the responses of the individual myeloid cells within the morphologically uniform macrophage population activated by the individual stimuli and their IA combination. Conclusion: scRNAseq was able to identify distinct single-cell macrophage phenotypes caused by induction of dsRNA and IFNα. Of the 5 subclusters identified, inducement by IFNα alone or in combination with TLR3 identified subsets of cell populations representing lineage and activation markers. These unexpected observations facilitate the identification of intracellular signaling pathways underlying the heterogenous response of individual myeloid cells to exogenous and endogenous activators, helping to develop improved vaccine adjuvants and cancer immunotherapies. Citation Format: Vincent Giamo, Melissa Grimm, Sarabjot Pabla, Ellen Karasik, Jesse Luce, Sean T. Glenn, Jeffrey Conroy, Jeffrey Conroy, Barbara Foster, Kiyomi Taniguchi, Pawel Kalinski, Masataka Shirai. Single-cell mRNA sequencing analysis of the synergistic impact of double-stranded RNA (dsRNA) and IFNα on human monocyte-derived macrophages [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 970.

THE ROLE OF OSTEOMACS IN REGULATING STEM CELL FUNCTION AND THE HEMATOPOIETIC NICHE

Networking between hematopoietic stem cells (HSC) and cells of the hematopoietic niche is not only critical for the maintenance of stem cell function, but also for the competence of the hematopoietic niche. Among the key cellular components of the niche are a group of specialized bone-resident macrophages known as osteomacs (OM). We previously documented that close interactions between OM, osteoblasts (OB) and megakaryocytes (MK) are critical for sustaining in vitro and in vivo stem and progenitor cell function. To understand the importance of OM-OB-MK interactions within the niche, we performed 3D tissue cytometry on fixed and stained unperturbed bone marrow sections. This approach identified the spatial relationships between OM, OB, MK and HSC within the niche and defined parameters, under which these cell types coexist in undamaged bone marrow. Next, we performed single cell mRNAseq and CyTOF to assess genetic and proteomic expression changes in OM following their interaction with MK. These studies revealed the upregulation of CD166 and embigin on OM via MK interactions. We then examined how these molecules impact hematopoietic function in clonogenic assays. When these assays were initiated with CD166 KO OM or treated with anti-embigin blocking antibodies, we established that loss of these surface molecules on OM caused a decline in the normal OM-mediated hematopoietic enhancing activity. Conversely, recombinant CD166 and embigin partially substituted for OM activity thus identifying potential mediators through which OM maintain hematopoietic function. Our data, for the first time, reveal intimate spatial interactions between OM, OB, MK and HSC in the hematopoietic niche. They also illustrate the importance of crosstalk between OM, OB and MK and reveal novel mediators such as CD166 and embigin that cooperate with other elements of the niche to support HSC function.

Abstract PD5-03: Characterization of high TIL breast cancers reveals a prognostic and functionally distinct tissue-resident memory subpopulation

Abstract Background: Tumor infiltrating lymphocytes (TILs) assessed via light microscopy are prognostic and predictive in the early stage and advanced triple negative and HER2-amplified breast cancer (BC). Higher TILs can also identify patients more likely to benefit from anti-PD-1 therapy. In this study we interrogated T cell subsets that comprise high TILs to determine if distinct subpopulations are key mediators of anti-tumor immunity. Methods: We characterised TILs with a focus on CD3+ T cells in 129 primary and metastatic BC samples using flow cytometry, bulk RNASeq on flow sorted T cell populations, multiplex immunohistochemistry and microdroplet based single cell 3' mRNA sequencing on the 10X Genomics Chromium platform. Cell type specific gene expression signatures were determined from differential expression between putative T cell subpopulations. These signatures were investigated in clinical cohorts, including trial cohorts treated with pembrolizumab. Results: High TIL Infiltrates consisted primarily of CD3+ T cells, with both CD8 and CD4 populations. Unsupervised clustering of single cell sequencing identified 9 CD8 and CD4 subpopulations with distinct gene expression profiles. In addition to Tregs and CD8 effector memory (TEM) T cells, we found a CD8+ tissue resident memory (TRM) population expressing greater levels of T-cell checkpoints and cytotoxic markers compared to effector memory cells. In 2 primary tumours and 1 liver metastasis, bulk RNASeq of flow sorted TEM and TRM corroborated the single cell mRNASeq results. T cell receptor profiling (TCR) in the 3 samples found non-overlapping repertoires in the 2 primary tumours, but overlap in one metastatic lesion, suggesting divergent developmental origins in the breast, but the potential for nascent TRM differentiation in a metastatic niche. Clustering of these TCRs suggested differing antigen specificities between TRM and non-TRM CD8 T cells. Using Metabric data, the CD8 TRM gene expression signature was prognostic for disease free survival (DFS) in primary TNBCs (n=329, log-rank p=0.003), and was able to further stratify cases with high and low CD8A expression for DFS (log-rank p = 0.03). The CD8 TRM signature was enriched in baseline tumour samples of responders (n = 9) compared with non-responders (n=36) in 45 patients with metastatic melanoma treated with T cell checkpoint blockade (p < 0.0001). Additional single cell sequencing data with TCR sequencing will be combined with these initial results, and an independent data set of single cell mRNASeq and TCR Seq on CD3+ BC TILs will be used to confirm our findings. Cell type specific signatures will be explored in additional clinical cohorts including KEYNOTE-086, and presented at the meeting. Conclusion: Using single cell profiling of the immune microenvironment in BC we demonstrate that high TIL BCs contain multiple T cell subpopulations with different functional and prognostic significance. Our approach identified a CD8 TRM population with a distinct gene expression profile and strong expression of key immune checkpoints likely representing the presence of true tumor specific immunity. This population may be a key target of immune checkpoint blockade. Citation Format: Savas P, Virassamy B, Ye C, Salim A, Mintoff CP, Caramia F, Salgado R, Teo ZL, Dushyanthen S, Byrne A, Luen SJ, Fox SB, Speed TP, Mackay LK, Neeson PJ, Loi S. Characterization of high TIL breast cancers reveals a prognostic and functionally distinct tissue-resident memory subpopulation [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD5-03.

Abstract IA20: Targeting the immune microenvironment in breast cancer

Abstract Immunotherapy has revolutionized cancer treatment, but the benefits are not ubiquitous or absolute. In breast cancer, higher levels of lymphocytic infiltrate confer significant prognostic advantage, predict response to chemotherapy in early stage disease and are associated with benefit from PD-1 inhibition. To enhance the modest benefits of immunotherapy seen in breast cancer thus far, it is expected that a greater understanding of the immune subpopulations in the tumor microenvironment will be necessary, emphasizing that the quality and quantity of tumor infiltrating lymphocytes are important. We have profiled the T cell infiltrate in primary and metastatic breast cancers, using flow cytometry, single cell mRNASeq, multiplex immunohistochemistry and bulk RNASeq on flow sorted CD8 populations. In this work, we found that CD8 T cells display tissue resident specialization. In particular, we confirmed the presence of a CD8 T cell population with tissue resident memory features. This population was notable for high expression of T cell checkpoint receptors and cytotoxic markers, active proliferation, and a distinct T cell receptor repertoire in contrast to CD8 effector memory T cells. A gene signature derived from this population was found to be prognostic in early stage breast cancer and associated with benefit from PD-1 checkpoint inhibition in melanoma. We find that single cell profiling in human samples is a powerful technique which unveils tissue resident aspects of the anti-cancer immune response. This approach may lead to better immunotherapy biomarkers and optimization of immunotherapeutic strategies. Citation Format: Sherene Loi, Peter Savas. Targeting the immune microenvironment in breast cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr IA20.

Single-Cell Dosing and mRNA Sequencing of Suspension and Adherent Cells Using the PolarisTM System.

Single-cell functional analysis provides a natural next step in the now widely adopted single-cell mRNA sequencing studies. Functional studies can be designed to study cellular context by using single-cell culture, perturbation, manipulation, or treatment. Here we present a method for a functional study of 48 single cells by single-cell isolation, dosing, and mRNA sequencing with an integrated fluidic circuit (IFC) on the Fluidigm® Polaris™ system. The major procedures required to execute this protocol are (1) cell preparation and staining; (2) priming, single-cell selection, cell dosing, cell staining, and cDNA generation on the Polaris IFC; and (3) preparation and sequencing of single-cell mRNA-seq libraries. The cell preparation and staining steps employ the use of a universal tracking dye to trace all cells that enter the IFC, while additional fluorescence dyes chosen by the user can be used to differentiate cell types in the overall mix. The steps on the Polaris IFC follow standard protocols, which are also described in the Fluidigm user documentation. The library preparation step adds Illumina® Nextera® XT indexes to the cDNA generated on the Polaris IFC. The resulting sequencing libraries can be sequenced on any Illumina sequencing platform.

A Bioinformatic Toolkit for Single-Cell mRNA Analysis.

The recent technological developments in the field of single-cell RNA-Seq enable us to assay the transcriptome of up to a million single cells in parallel. However, the analyses of such big datasets present a major challenge. During the last decade, a wide variety of strategies have been proposed covering different steps of the analysis. Here, we introduce a selection of computational tools to provide an overview of a generic analysis pipeline.The first step of every scRNA-Seq experiment is proper study design, which does not require sophisticated experimental or informatics skills but is nonetheless presumably the most important step. The quality of the resulting data strictly depends on the proper planning of the experiment, including the selection of the most suitable technology for the biological question of interest as well as an elaborated study design to minimize the influence of confounding factors. Once the experiment has been conducted, the raw sequencing data needs to be processed to extract the gene expression information for each cell. This task comprises quality assessment of the sequenced reads, alignment against a reference genome, demultiplexing of the cell barcodes, and quantification of the reads/transcripts per gene. As any other transcriptomics technology, single-cell mRNA-Seq requires data normalization to assure sample-to-sample, here cell-to-cell, comparability and the consideration of confounding factors.Once gene expression values have been extracted from the reads and normalized, the researcher has the agony of choosing between a plethora of analysis approaches to investigate diverse aspects of the single-cell transcriptomes, such as dimensionality reduction and clustering to explore cellular heterogeneity or trajectory analysis to model differentiation processes.In this chapter, we present a wrap-up of the abovementioned steps to conduct single-cell RNA-Seq analyses and present a selection of existing tools.

Analysis of Clonal Evolution/Heterogeneity of MDS By Simultaneous Detection of Both Mutation and Gene Expression By Single Cell Sequencing

Recent advances in single-cell sequencing (sc-Seq) technologies have enabled high-throughput transcriptome analysis in thousands of cells to understand the heterogeneity among cancer populations in terms of genome-wide gene expression. However, its application to the analysis of clonal evolution of cancer populations is largely limited by the lack of an efficient sc-Seq platform that allows for accurate detection of gene mutations at the same time with transcriptome analysis. The major challenge here is a frequent allele dropout of just two copies per single cell, which results in an inaccurate genotype assignment for many cells, preventing identification of relevant genotype-phenotype correlations. To overcome this, we developed a novel sc-Seq platform (scMutSeq) that allows for precise determination of both genotype and genome-wide gene expression simultaneously with negligible allele dropouts, on the basis of the Fluidigm C1 Single-Cell mRNA Seq HT system and applied it to the analysis of clonal evolution and intratumor heterogeneity of myelodysplastic syndromes (MDS) characterized by frequent clonal evolution to acute amyloid leukemia (AML).We first evaluated the performance of our plat form using an AML-derived cell line with heterozygous SF3B1K700E mutation, HNT-34, for which efficiency of the detection of both wild-type and mutant allele, together with global gene expression, was evaluated. Among 400 cells subjected to scMutSeq analysis, a total of 125 passed QC, in which cell viability was assessed in terms of expression of mitochondrial genes. Global gene expression and heterozygous SF3B1mutation were successfully detected in all the QC-confirmed cells with none of the cells showing the wild-type allele or homozygous SF3B1mutation, where evaluable transcript reads (unique molecular identifier >=1) were obtained for a median of 2,753 genes, designated as nGene. The performance was also tested for flow-sorted hematopoietic stem/progenitor cells (HSPCs) (Lin−CD34+) from an MDS patient positive for the SF3B1K700E mutation. Gene expression was successfully analyzed all the QC-confirmed cells (n=81) with a median nGene of 1,953. No substantial allele dropouts were suspected, because none of the cells genotyped had homozygous SF3B1mutation.We then applied scMutSeq to the analysis of TP53-mutated AML/MDS with complex karyotype, including del(5q) and del(7q), for which longitudinal samples were obtained for the assessment of clonal evolution. scMutSeq successfully analyzed the mutation status of TP53and global gene expression profiles at a single-cell level, where copy number abnormalities were also evaluated on the basis of gene expression. We identified two discrete clones in the HSPC fraction, carrying both del(5q) and del(7q) and del(5q) alone, respectively, even though the analysis of bulk DNA had failed to detect the latter clone, indicating that a minor clone having a distinct genotype came under detection with scMutSeq. Moreover, the HSPCs with both del(5q) and del(7q) showed aberrant expression of erythroid and megakaryocytic genes, increased expression of inflammatory signals and decreased expression of cell cycle-related genes, exhibiting a clear genotype phenotype correlation. Subsequent analysis of samples at later time points further disclosed evolution of clones having discrete del(5q) deletions and expression, revealing a complexity of clonal evolution in MDS.Next, to investigate the early process of MDS development, we analyzed clonal hematopoiesis found in a minor fraction (1.2-12%) of bone marrow samples from three elder individuals having hip replacement surgery, in which DNMT3A(n=1) (R882H) and TET2(n=2) (D905fs and Q1540fs) mutations had been detected by ddPCR or targeted deep sequencing, respectively. scMutSeq analysis of the HSPCs from these individuals revealed that mutant HSPCs showed distinct gene expression profiles, depending on the type of CHIP mutations.To summarize, our single-cell sequencing platform enables to detect both genetic and transcriptional heterogeneities, providing a powerful clue to understand clonal evolution and intratumor heterogeneity of MDS. DisclosuresNakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Inagaki:Sumitomo Dainippon Pharma Co., Ltd.: Employment. Yoda:Chordia Therapeutics Inc.: Research Funding.