Droplet-based Single-cell RNA Sequencing Research Articles

High-throughput single-cell RNA sequencing.

High-throughput, droplet-based single-cell RNA sequencing (scRNA-Seq) provides unprecedented resolution to examine cell–cell heterogeneity in tissues and organs. Microfluidic channels combine individual cells with uniquely barcoded beads to analyze transcriptomes of thousands of cells in parallel that can reconstruct the biological structure in silico. Individual cells can be traced back through bead/cell-specific barcodes on the oligos, and individual transcript amplification bias is accounted for with unique molecular identifiers (UMIs).

Crafting a blueprint for single-cell RNA sequencing.

Droplet-based single-cell RNA sequencing (scRNA-Seq) has moved rapidly from a technology with great potential to a method applied to ever-broader questions. The detailed information that scRNA-Seq offers has proven incredibly powerful in resolving cell responses to developmental and environmental cues. However, to maximize the potential of this technology, a panoply of upstream, practical points require consideration. Principal among these are the optimization of cell-isolation procedures, accommodating biotic/abiotic stress responses, and discerning the number of cells and sequencing reads needed. To complement excellent reviews outlining applications and data analysis tools for scRNA-Seq, we here discuss these considerations and provide practical tips to tailor experimental design and ensure the best possible outcome.

Inflammatory molecular endotypes of nasal polyps derived from White and Japanese populations

Emerging evidence suggests that chronic rhinosinusitis with nasal polyps (CRSwNP) is a highly heterogeneous disease with disparate inflammatory characteristics between different racial groups and geographies. Currently, little is known about possible underlying distinguishing factors between these inflammatory differences. Our aim was to interrogate differences in CRSwNP disease between White/non-Asian patients and Japanese patients by using whole transcriptome and single-cell RNA gene expression profiling of nasal polyps (NPs). We performed whole transcriptome RNA sequencing with endotype stratification of NPs from 8 White patients (residing in the United States) and 9 Japanese patients (residing in Japan). Reproducibility was confirmed by quantitative PCR in an independent validation set of 46 White and 31 Japanese patients. Single-cell RNA sequencing (scRNAseq) was used to stratify key cell types for contributory transcriptional signatures. Unsupervised clustering analysis identified 2 major endotypes that were present within both cohorts of patients with NPs and had previously been reported at the cytokine level: (1) type 2 endotype and (2) non-type 2 endotype. Importantly, there was a statistically significant difference in the proportion of these endotypes between these geographically distinct subgroups with NPs (P= .03). Droplet-based single-cell RNA sequencing further identified prominent type 2 inflammatory transcript expression: C-C motif chemokine ligand 13 (CCL13) and CCL18 in M2 macrophages, as well as cystatin SN (CST1) and CCL26 in basal, suprabasal, and secretory epithelial cells. NPs from both racial groups harbor the same 2 major endotypes, which we have determined to be present in differing ratios between each cohort with CRSwNP disease. Distinct inflammatory and epithelial cells contribute to the type 2 inflammatory profiles observed.

Therapeutic and Immunologic Responses Elicited By in Situ Vaccination with CpG, Ibrutinib, and Low-Dose Radiation

Introduction: In situ vaccination aims to induce an immune response locally at one tumor site that propagates systemically to all tumor sites. This approach can be effective in indolent lymphoma (Brody et al., JCO 2010, Frank et al., Cancer Discov 2018, Hammerich et al., Nat Med 2019). We designed a novel clinical strategy combining in situ vaccination with systemic ibrutinib, a kinase inhibitor that modulates B and T cells. Our preclinical work had shown that combined intratumoral CpG injection and systemic ibrutinib administration was curative of systemic disease in a mouse lymphoma model, an effect that was T cell dependent (Sagiv-Barfi, Blood, 2015). Here we report the results and correlative data from the Phase I/II clinical trial testing this combination along with local low-dose radiation in adults with recurrent low-grade B cell lymphoma (NCT02927964).Methods: Enrolled patients received intratumoral injections of CpG (SD-101, 3 mg) weekly for 5 doses and local radiation (4Gy in two fractions) to the same site. Daily oral ibrutinib (560mg) began after the second intratumoral injection. Revised Lugano criteria (Cheson et al., JCO, 2014) were used to assess overall radiographic responses to therapy. Distal responses were assessed by excluding the injected site and measuring only non-injected sites. Fine needle aspirates (FNAs) were obtained from CpG-injected and non-injected nodal tumor sites pre- and post-treatment and analyzed by flow cytometry and droplet-based single-cell RNA sequencing (scRNAseq).Results: Among the twenty patients treated on study, median age was 64, 55% were male, and all but one had a diagnosis of follicular lymphoma. All patients were previously treated with an average of 2 lines of therapy, and half had previously received chemotherapy. Adverse events (AEs) were consistent with known effects of ibrutinib (including diarrhea and rash) and of CpG (including fever and flu-like reactions). No drug-related grade 4, serious, or unexpected AEs were observed. As anticipated, all patients experienced tumor reduction at the locally treated site (median 84% reduction). Remarkably, all patients experienced some tumor reduction at non-injected non-irradiated index lesions (median 45%, range 13-100%), suggesting the generation of systemic immune responses (Figure 1A). By Cheson criteria, ten patients achieved an objective response, including one complete response (ORR 50%). Despite an overall improvement in tumor burden, three patients had new or progressing non-index lesions and scored as progressive disease. Treatment induced an expansion of naïve and effector memory T cells and reductions in T follicular helper (Tfh) and activated regulatory T cells (Tregs) at the injected site. T cells with high expression of transcripts related to oxidative phosphorylation (Toxphos) increased preferentially in patients with subsequent clinical tumor reduction (Figure 1B), implicating T cell metabolism in successful generation of immune responses. Analysis of single cell T cell receptor (TCR) sequencing data revealed>300 clones that were comprised of at least 2 cells at each timepoint and which expanded or contracted at least two-fold during treatment. Expanding clones were more likely than contracting clones to be activated or memory T cells and less likely than contracting clones to be Tfh or Tregs (Figure 1C-D). Clone dynamics were often similar at the two sampled tumor sites, reflecting systemic immune responses. Finally, in vitro assays showed treatment-induced expansion of tumor-specific T cells in the peripheral blood of all 6 evaluable patients.Conclusion: The combination of oral ibrutinib, intratumoral CpG, and local low-dose radiation is safe and can generate systemic antitumor immune responses and systemic tumor shrinkage in low-grade B cell lymphoma. [Display omitted] DisclosuresShree: Gilead: Other: Spouse's employment. Khodadoust: CRISPR Therapeutics, Nutcracker Therapeutics: Research Funding; Myeloid Therapeutics: Membership on an entity's Board of Directors or advisory committees; Alexion, AstraZeneca Rare Disease: Other: Study investigator. Frank: Kite-Gilead: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Research Funding; Allogene Therapeutics: Research Funding. Beygi: Kite/Gilead: Current Employment. Levy: GigaGen: Membership on an entity's Board of Directors or advisory committees; Teneobio: Membership on an entity's Board of Directors or advisory committees; Nurix: Membership on an entity's Board of Directors or advisory committees; Dragonfly: Membership on an entity's Board of Directors or advisory committees; Apexigen: Membership on an entity's Board of Directors or advisory committees; Viracta: Membership on an entity's Board of Directors or advisory committees; Spotlight: Membership on an entity's Board of Directors or advisory committees; Immunocore: Membership on an entity's Board of Directors or advisory committees; Walking Fish: Membership on an entity's Board of Directors or advisory committees; Kira: Membership on an entity's Board of Directors or advisory committees; Abintus Bio: Membership on an entity's Board of Directors or advisory committees; Khloris: Membership on an entity's Board of Directors or advisory committees; Virsti: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; BeiGene: Membership on an entity's Board of Directors or advisory committees; Quadriga: Membership on an entity's Board of Directors or advisory committees.

Single-cell RNA-seq of out-of-thaw mesenchymal stromal cells shows tissue-of-origin differences and inter-donor cell-cycle variations

BackgroundHuman Mesenchymal stromal cells (hMSCs) from various tissue sources are widely investigated in clinical trials. These MSCs are often administered to patients immediately after thawing the cryopreserved product (out-of-thaw), yet little is known about the single-cell transcriptomic landscape and tissue-specific differences of out-of-thaw human MSCs.Methods13 hMSC samples derived from 10 “healthy” donors were used to assess donor variability and tissue-of-origin differences in single-cell gene expression profiles. hMSCs derived and expanded from the bone marrow (BM) or cord tissue (CT) underwent controlled-rate freezing for 24 h. Cells were then transferred to the vapor phase of liquid nitrogen for cryopreservation. hMSCs cryopreserved for at least one week, were characterized immediately after thawing using a droplet-based single-cell RNA sequencing method. Data analysis was performed with SC3 and SEURAT pipelines followed by gene ontology analysis.ResultsscRNA-seq analysis of the hMSCs revealed two major clusters of donor profiles, which differ in immune-signaling, cell surface properties, abundance of cell-cycle related transcripts, and metabolic pathways of interest. Within-sample transcriptomic heterogeneity is low. We identified numerous differentially expressed genes (DEGs) that are associated with various cellular functions, such as cytokine signaling, cell proliferation, cell adhesion, cholesterol/steroid biosynthesis, and regulation of apoptosis. Gene-set enrichment analyses indicated different functional pathways in BM vs. CT hMSCs. In addition, MSC-batches showed significant variations in cell cycle status, suggesting different proliferative vs. immunomodulatory potential. Several potential transcript-markers for tissue source differences were identified for further investigation in future studies. In functional assays, both BM and CT MSCs suppressed macrophage TNFα secretion upon interferon stimulation. However, differences between donors, tissue-of-origin, and cell cycle are evident in both TNF suppression and cytokine secretion.ConclusionsThis study shows that donor differences in hMSC transcriptome are minor relative to the intrinsic differences in tissue-of-origin. hMSCs with different transcriptomic profiles showed potential differences in functional characteristics. These findings contribute to our understanding of tissue origin-based differences in out-of-thaw therapeutic hMSC products and assist in the identification of cells with immune-regulatory or survival potential from a heterogeneous MSC population. Our results form the basis of future studies in correlating single-cell transcriptomic markers with immunomodulatory functions.

Single-cell RNA sequencing reveals the epithelial cell heterogeneity and invasive subpopulation in human bladder cancer.

Bladder cancer represents a highly heterogeneous disease characterized by distinct histological, molecular and clinical phenotypes, and a detailed analysis of tumor cell invasion and crosstalks within bladder tumor cells has not been determined. Here, we applied droplet-based single-cell RNA sequencing (scRNA-seq) to acquire transcriptional profiles of 36 619 single cells isolated from seven patients. Single cell transcriptional profiles matched well with the pathological basal/luminal subtypes. Notably, in T1 tumors diagnosed as luminal subtype, basal cells displayed characteristics of epithelial-mesenchymal transition (EMT) and mainly located at the tumor-stromal interface as well as micrometastases in the lamina propria. In one T3 tumor, muscle-invasive tumor showed significantly higher expression of cancer stem cell markers SOX9 and SOX2 than the primary tumor. We additionally analyzed communications between tumor cells and demonstrated its relevance to basal/luminal phenotypes. Overall, our single-cell study provides a deeper insight into the tumor cell heterogeneity associated with bladder cancer progression.

Single-cell analysis reveals urothelial cell heterogeneity and regenerative cues following cyclophosphamide-induced bladder injury

Cyclophosphamide is a commonly used chemotherapeutic drug to treat cancer with side effects that trigger bladder injury and hemorrhagic cystitis. Although previous studies have demonstrated that certain cell subsets and communications are activated to drive the repair and regeneration of bladder, it is not well understood how distinct bladder cell subsets function synergistically in this process. Here, we used droplet-based single-cell RNA sequencing (scRNA-seq) to profile the cell types within the murine bladder mucous layer under normal and injured conditions. Our analysis showed that superficial cells are directly repaired by cycling intermediate cells. We further identified two resident mesenchymal lineages (Acta2+ myofibroblasts and Cd34+ fibroblasts). The delineation of cell-cell communications revealed that Acta2+ myofibroblasts upregulated Fgf7 expression during acute injury, which activated Fgfr signaling in progenitor cells within the basal/intermediate layers to promote urothelial cell growth and repair. Overall, our study contributes to a more comprehensive understanding of the cellular dynamics during cyclophosphamide-induced bladder injury and may help identify important niche factors contributing to the regeneration of injured bladders.

Single-cell transcriptomics reveals involution mimicry during the specification of the basal breast cancer subtype.

Basal breast cancer is associated with younger age, early relapse, and a high mortality rate. Here, we use unbiased droplet-based single-cell RNA sequencing (RNA-seq) to elucidate the cellular basis of tumor progression during the specification of the basal breast cancer subtype from the luminal progenitor population in the MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) mammary tumor model. We find that basal-like cancer cells resemble the alveolar lineage that is specified upon pregnancy and encompass the acquisition of an aberrant post-lactation developmental program of involution that triggers remodeling of the tumor microenvironment and metastatic dissemination. This involution mimicry is characterized by a highly interactive multicellular network, with involution cancer-associated fibroblasts playing a pivotal role in extracellular matrix remodeling and immunosuppression. Our results may partially explain the increased risk and poor prognosis of breast cancer associated with childbirth.

Single-cell RNA sequencing reveals developmental heterogeneity among Plasmodium berghei sporozoites

In the malaria-causing parasite’s life cycle, Plasmodium sporozoites must travel from the midgut of a mosquito to the salivary glands before they can infect a mammalian host. However, only a fraction of sporozoites complete the journey. Since salivary gland invasion is required for transmission of sporozoites, insights at the molecular level can contribute to strategies for malaria prevention. Recent advances in single-cell RNA sequencing provide an opportunity to assess sporozoite heterogeneity at a resolution unattainable by bulk RNA sequencing methods. In this study, we use a droplet-based single-cell RNA sequencing workflow to analyze the transcriptomes of over 8000 Plasmodium berghei sporozoites derived from the midguts and salivary glands of Anopheles stephensi mosquitoes. The detection of known marker genes confirms the successful capture and sequencing of samples composed of a mixed population of sporozoites. Using data integration, clustering, and trajectory analyses, we reveal differences in gene expression profiles of individual sporozoites, and identify both annotated and unannotated markers associated with sporozoite development. Our work highlights the utility of a high-throughput workflow for the transcriptomic profiling of Plasmodium sporozoites, and provides new insights into gene usage during the parasite’s development in the mosquito.

Single-Cell Profiling of Coding and Noncoding Genes in Human Dopamine Neuron Differentiation.

Dopaminergic (DA) neurons derived from human pluripotent stem cells (hPSCs) represent a renewable and available source of cells useful for understanding development, developing disease models, and stem-cell therapies for Parkinson’s disease (PD). To assess the utility of stem cell cultures as an in vitro model system of human DA neurogenesis, we performed high-throughput transcriptional profiling of ~20,000 ventral midbrain (VM)-patterned stem cells at different stages of maturation using droplet-based single-cell RNA sequencing (scRNAseq). Using this dataset, we defined the cellular composition of human VM cultures at different timepoints and found high purity DA progenitor formation at an early stage of differentiation. DA neurons sharing similar molecular identities to those found in authentic DA neurons derived from human fetal VM were the major cell type after two months in culture. We also developed a bioinformatic pipeline that provided a comprehensive long noncoding RNA landscape based on temporal and cell-type specificity, which may contribute to unraveling the intricate regulatory network of coding and noncoding genes in DA neuron differentiation. Our findings serve as a valuable resource to elucidate the molecular steps of development, maturation, and function of human DA neurons, and to identify novel candidate coding and noncoding genes driving specification of progenitors into functionally mature DA neurons.

SoupX removes ambient RNA contamination from droplet-based single-cell RNA sequencing data.

BackgroundDroplet-based single-cell RNA sequence analyses assume that all acquired RNAs are endogenous to cells. However, any cell-free RNAs contained within the input solution are also captured by these assays. This sequencing of cell-free RNA constitutes a background contamination that confounds the biological interpretation of single-cell transcriptomic data.ResultsWe demonstrate that contamination from this "soup" of cell-free RNAs is ubiquitous, with experiment-specific variations in composition and magnitude. We present a method, SoupX, for quantifying the extent of the contamination and estimating "background-corrected" cell expression profiles that seamlessly integrate with existing downstream analysis tools. Applying this method to several datasets using multiple droplet sequencing technologies, we demonstrate that its application improves biological interpretation of otherwise misleading data, as well as improving quality control metrics.ConclusionsWe present SoupX, a tool for removing ambient RNA contamination from droplet-based single-cell RNA sequencing experiments. This tool has broad applicability, and its application can improve the biological utility of existing and future datasets.

Single Cell Analysis of Serial Lymphoma Biopsies Reveals Dynamic Immune Modulation and Predictors of Response in Patients Undergoing in Situ Vaccination

Introduction: In situ vaccination aims to induce an immune response locally at one tumor site, that propagates systemically to all tumor sites. Prior clinical studies have demonstrated that this strategy can be effective in indolent lymphoma (Brody et al., JCO 2010, Frank et al., Cancer Discov 2018, Hammerich et al., Nat Med 2019). The underlying mechanisms, however, remain unclear, as immune recognition and destruction of cancer cells occurs at the level of the tumor microenvironment (TME), and the human TME has been difficult to study due to challenges in acquiring adequate tissue samples. Here, we directly investigate changes induced in the TME by in situ vaccination, applying single cell methods to serial samples from patients on a clinical trial, and relate these changes to clinical tumor response. Methods: We profiled serial specimens from 2 sites of disease in 10 indolent lymphoma patients enrolled in an in situ vaccination trial (NCT02927964). Motivated by compelling preclinical data demonstrating the efficacy of intratumoral CpG, a TLR9 agonist, and systemic ibrutinib, a BTK inhibitor (Sagiv-Barfi et al., Blood 2015), patients receive local low-dose radiation and CpG intratumorally to one site of disease and begin oral ibrutinib in the second week of treatment. Tumor fine needle aspirates (FNAs) from the injected and a distant uninjected tumor site as well as peripheral blood samples are obtained prior to, and at 1 and 6 weeks after treatment start. Single cells from the FNAs and blood samples are then subject to droplet-based single cell RNA sequencing, with approximately 3,000-10,000 cells sequenced per sample. Results: In total, we analyzed 314,827 cells from 52 tumor samples and 30 peripheral blood samples (Fig. 1A). At both the local injected and the distant uninjected tumor sites, we found significant changes in proportions and transcriptional phenotypes of tumor and immune cells, several of which tracked with clinical response. Upon treatment, the proportions of tumor cells decreased significantly at the treated site, even within the 6-week time frame (p=0.003). Increased expression of inflammation related genes at both the injected and uninjected sites (Fig. 1B) and of TGFb pathway genes at the uninjected site (Fig. 1C) were negatively associated with clinical response. Conversely, high expression of antigen presentation genes by week 6 at the injected site correlated with greater shrinkage of distant uninjected tumors (Fig. 1D). Among T and NK cells (Fig. 1E), we observed treatment-induced increases in naïve and cytotoxic CD4 and CD8 T cells, and reductions in T follicular helper and exhausted T cells (Fig. 1F). While all patients exhibited strong interferon responses in their T and NK cells, the degree of interferon response was not associated with overall clinical response (Fig. 1G). Instead, higher cytotoxic CD4 T cells at the injected site and fewer exhausted T cells at the uninjected site after treatment correlated with clinical response. Conclusions: By sampling multiple tumors over time in patients undergoing in situ vaccination, we identify significant changes in tumor and microenvironmental cells at both the injected and uninjected sites, several of which associate with clinical tumor regression. Our approach represents successful application of single-cell genomics to a clinical trial, illuminating treatment-induced cellular dynamics, and implicating tumor cell antigen presentation and CD4 T cell responses as possible determinants of clinical response. Disclosures Levy: Immunocore: Membership on an entity's Board of Directors or advisory committees; Walking Fish: Membership on an entity's Board of Directors or advisory committees; Viracta: Membership on an entity's Board of Directors or advisory committees; Quadriga: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; GigaGen: Membership on an entity's Board of Directors or advisory committees; Teneobio: Membership on an entity's Board of Directors or advisory committees; Sutro: Membership on an entity's Board of Directors or advisory committees; Checkmate: Membership on an entity's Board of Directors or advisory committees; Nurix: Membership on an entity's Board of Directors or advisory committees; Dragonfly: Membership on an entity's Board of Directors or advisory committees; Abpro: Membership on an entity's Board of Directors or advisory committees; Apexigen: Membership on an entity's Board of Directors or advisory committees; Spotlight: Membership on an entity's Board of Directors or advisory committees; 47 Inc.: Membership on an entity's Board of Directors or advisory committees; XCella: Membership on an entity's Board of Directors or advisory committees.

The Bone Marrow Microenvironment of Multiple Myeloma Long-Term Survivors at Single Cell Resolution

The Bone Marrow Microenvironment of Multiple Myeloma Long-Term Survivors at Single Cell Resolution

Abstract IA12: Single cell multi-omics to define normal and malignant differentiation topologies

Abstract Cancer progression, relapse and resistance are the result of an evolutionary optimization process. Vast intra-tumoral diversity provides the critical substrate for cancer to evolve and adapt to the selective pressures provided by effective therapy. Our previous work has shown that genetically distinct subpopulations compete and mold the genetic makeup of the malignancy. Additionally, we have shown that epigenetic changes in cancer may be similar to the process of genetic diversification, in which stochastic trial and error leads to rare fitness enhancing events. These studies demonstrate the need to integrate genetic, epigenetic and transcriptional information in the study of cancer evolution, specifically at the single-cell resolution – the atomic unit of somatic evolution. To enable this work, we have developed a single-cell multi-omics toolkit, and apply it to chart the evolutionary history and developmental topographies of normal and malignant cells. We demonstrated that epimutations serve as a molecular clock, and that heritable epimutation information therefore allows to infer high-resolution lineages with single-cell data, directly in patient samples. Multi-omic single-cell Integration of methylome sequencing with whole transcriptome and genotyping capture validated tree topology inferred solely on the basis of epimutation information. To examine potential lineage biases during therapy, we profiled serial samples, and identified clades of cells that were preferentially affected by targeted therapy. The single-cell integration of genetic, epigenetic and transcriptional information thus charts the lineage history of cancer and its evolution with therapy. Second, charting the transcriptomes of clonally mutated cells is challenging in the absence of surface markers that distinguish cancer clones from one another, or from admixed non-neoplastic cells. To tackle this challenge, we developed Genotyping of Transcriptomes (GoT), a technology to integrate genotyping with high-throughput droplet-based single-cell RNA sequencing. With GoT we profiled thousands of CD34+ cells from patients myeloproliferative neoplasms to study how somatic mutations corrupt the process of human hematopoiesis. These data allow to superimpose the two differentiation trees; the native wildtype tree and the one corrupted by mutation. High-resolution mapping of malignant versus normal progenitors showed increased fitness with myeloid differentiation with CALR mutation. We identified the unfolded protein response as a predominant outcome of CALR mutations, with dependency on cell identity. Notably, stem cells and more differentiated progenitors show distinct transcriptional programs as a result of somatic mutation, suggesting differential sensitivity to therapeutic targeting. We further extended the GoT toolkit to genotype multiple targets and loci that are distant from transcript ends. Together, these findings reveal that the transcriptional output of somatic mutations in blood neoplasms is dependent on the native cell identity. Finally, The expanding application of single cell RNA-seq to primary human samples has shown that transcriptional cell state diversity is found ubiquitously across cancer, often independent from genetic clonal diversity. Thus, tumors are composed of admixtures of cells that differ in central phenotypes such as stemness and resistance to therapy. These observation prompts key questions in cancer biology: (i) How are cell states encoded epigenetically? (ii) What is the heritability of cell states? (iii) What is the ability of cells to toggle between cell states? While the exploration of these central aspects of cancer has only begun in model organisms using artificial constructs for lineage tracing, in primary patient samples these questions remains completely unexplored. To address this fundamental knowledge gap in cancer biology, and to define cell state heritability in human somatic evolution, we leveraged our unique ability for joint profiling of DNA methylation and RNA-seq within the same single cells, and performed a direct comparison of the epigenome of transcriptionally defined stem-like cells vs. differentiated malignant cells in glioblastoma. This led to the discovery of PRC2 (Polycomb Repressive Complex 2) as a key switch for differentiation in glioblastoma. Moreover, we integrated high-resolution lineage trees with transcriptional cell states to address critical question of cell state heritability and transitions. We developed an innovative analytic framework, drawing on principles in the field of quantitative ecology, to measure for the first time heritability and transition probability of transcriptional cell states directly in clinical samples. Our findings contrast IDH-mutant gliomas that follow a traditional stem cell hierarchy model with IDH-wildtype glioblastomas that are dominated by cellular plasticity, illuminating a longstanding debate in the field. Thus, we show that in some cancers cell state transitions follow a unidirectional hierarchy akin to normal development, while in others a more plastic bidirectional hierarchy emerges that allows for significant de-differentiation. Citation Format: Dan A. Landau. Single cell multi-omics to define normal and malignant differentiation topologies [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA12.

Cellular taxonomy and spatial organization of the murine ventral posterior hypothalamus.

The ventral posterior hypothalamus (VPH) is an anatomically complex brain region implicated in arousal, reproduction, energy balance, and memory processing. However, neuronal cell type diversity within the VPH is poorly understood, an impediment to deconstructing the roles of distinct VPH circuits in physiology and behavior. To address this question, we employed a droplet-based single-cell RNA sequencing (scRNA-seq) approach to systematically classify molecularly distinct cell populations in the mouse VPH. Analysis of >16,000 single cells revealed 20 neuronal and 18 non-neuronal cell populations, defined by suites of discriminatory markers. We validated differentially expressed genes in selected neuronal populations through fluorescence in situ hybridization (FISH). Focusing on the mammillary bodies (MB), we discovered transcriptionally-distinct clusters that exhibit neuroanatomical parcellation within MB subdivisions and topographic projections to the thalamus. This single-cell transcriptomic atlas of VPH cell types provides a resource for interrogating the circuit-level mechanisms underlying the diverse functions of VPH circuits.

Abstract 6155: Pediatric rhabdomyosarcomas recapitulate the transcriptomic and epigenomic development of skeletal myogenesis

Abstract Rhabdomyosarcoma (RMS), is a pediatric tumor that histologically mimics developing skeletal muscle. Importantly, RMS tumors feature heterogeneous expression of the skeletal muscle transcription factor myogenin (MYOG). However, the degree by which RMS tumors model the development of normal skeletal muscle remains unclear. Here, we have utilized droplet-based single-cell RNA-sequencing (scRNA-seq) of 158,070 cells or nuclei isolated from 20 patient RMS tumors (14 embryonal RMS [ERMS], 6 alveolar RMS [ARMS]). Surprisingly, we found that cells from ERMS tumors recapitulate the entire developmental hierarchy of muscle development, with a subset of cells expressing the presomitic mesoderm homeobox gene, MEOX2. In contrast, cells from ARMS tumors were more narrowly restricted to expression of the myoblast- and myocyte-specific transcription factors MYF5 and MYOG. We extended this observation using a developmental indexing approach that leverages single-nuclear transcriptomic data from the Mouse Organogenesis Cell Atlas. By identifying latent cellular states within developing non-malignant embryonal muscle and projecting those latent states onto malignant cells, our developmental index estimates the equivalent myogenic developmental state of each individual malignant cell. ERMS tumors consistently displayed a broad diversity of development indices, even in relapsed tumors; alveolar RMS tumors, however, were restricted to late myoblast and myocyte developmental states. We applied our developmental index to an additional 310,937 cells isolated from 19 orthotopic patient-derived xenografts (O-PDXs), which preserved the developmental diversity of their originating tumor. Next, we used lentiviral barcoding with a high-complexity library to demonstrate that developmental states are interrelated rather than isolated subclones. Additionally, we transduced O-PDX cell suspensions with RFP-encoding retrovirus to lineage trace proliferative cells to show that ERMS tumors transition from immature to mature myogenic states in a unidirectional manner. Finally, we utilized single-cell assay of transposase-accessible chromatin with sequencing (scATAC-seq) in combination with scRNA-seq to show that core regulatory circuit superenhancer accessibility is heterogeneously specific to developmental state within both ERMS and ARMS. Taken together, our findings reveal that pediatric RMS consist of transformed cells that recapitulate the transcriptomic and epigenomic diversity of embryonic muscle. Citation Format: Anand G. Patel, Xiang Chen, Michael Clay, Brittney Gordon, Jim Houston, Asa Karlstrom, Jackie Norrie, Elizabeth Stewart, Michael Dyer. Pediatric rhabdomyosarcomas recapitulate the transcriptomic and epigenomic development of skeletal myogenesis [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 6155.

Abstract PR05: Genome-wide screening identifies novel YAP modulators

Abstract The transcriptional coactivator YAP is the key downstream effector of the Hippo pathway, which is a central regulator of organ size, stem cell/progenitor cell expansion, and tissue regeneration. Diverse studies have shown the importance of a precise regulation of this pathway and its key downstream regulator YAP for tissue homeostasis. To achieve new insights in the Hippo-independent transcriptional regulation of YAP in breast cancer, we set out to identify new YAP modulators using a genome-wide CRISPR screen and Perturb-Seq. We identified the transcriptional repressor protein Trichorhinophalangeal Syndrome 1 (TRPS1) as a potent repressor of YAP-dependent transactivation, using a genome-wide CRISPR screen. TRPS1 globally regulates YAP-dependent transcription by binding to a large set of joint genomic sites, mainly enhancers. Moreover, TRPS1 antagonizes activating YAP/TEAD complexes by recruiting corepressor complexes to joint sites. Loss of TRPS1 leads to activation of enhancers due to increased H3K27 acetylation and an altered promoter-enhancer interaction landscape. TRPS1 is commonly amplified in breast cancer, suggesting that restrained YAP activity favors tumor growth. Combined, we uncover TRPS1 as an epigenetic regulator of YAP activity in breast cancer. Additionally, we developed a Trps1-deficient mouse line that we will use to test the in vivo role of Trps1 in mammary gland physiology and breast cancer. Furthermore, we combined CRISPR-based gene perturbation with droplet-based single-cell RNA sequencing (Perturb-Seq), to perform a second large-scale analysis on potential YAP modulators. To this end, we used a CRISPR-library containing 158 previously from us in a Bio-ID assay identified YAP interactors. Together, we uncover several novel YAP modulators, contributing to a better understanding of the Hippo-independent transcriptional regulation of YAP. This abstract is also being presented as Poster B40. Citation Format: Dana Elster, Marie Tollot, Karin Schlegelmilch, Alessandro Ori, Andreas Rosenwald, Erik Sahai, Björn von Eyss. Genome-wide screening identifies novel YAP modulators [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr PR05.

Single-cell deconstruction of post-sepsis skeletal muscle and adipose tissue microenvironments.

BackgroundPersistent loss of skeletal muscle mass and function as well as altered fat metabolism are frequently observed in severe sepsis survivors. Studies examining sepsis‐associated tissue dysfunction from the perspective of the tissue microenvironment are scarce. In this study, we comprehensively assessed transcriptional changes in muscle and fat at single‐cell resolution following experimental sepsis induction.MethodsSkeletal muscle and visceral white adipose tissue from control mice or mice 1 day or 1 month following faecal slurry‐induced sepsis were used. Single cells were mechanically and enzymatically prepared from whole tissue, and viable cells were further isolated by fluorescence activated cell sorting. Droplet‐based single‐cell RNA‐sequencing (scRNA‐seq; 10× Genomics) was used to generate single‐cell gene expression profiles of thousands of muscle and fat‐resident cells. Bioinformatics analyses were performed to identify and compare individual cell populations in both tissues.ResultsIn skeletal muscle, scRNA‐seq analysis classified 1438 single cells into myocytes, endothelial cells, fibroblasts, mesenchymal stem cells, macrophages, neutrophils, T‐cells, B‐cells, and dendritic cells. In adipose tissue, scRNA‐seq analysis classified 2281 single cells into adipose stem cells, preadipocytes, endothelial cells, fibroblasts, macrophages, dendritic cells, B‐cells, T‐cells, NK cells, and gamma delta T‐cells. One day post‐sepsis, the proportion of most non‐immune cell populations was decreased, while immune cell populations, particularly neutrophils and macrophages, were highly enriched. Proportional changes of endothelial cells, neutrophils, and macrophages were validated using faecal slurry and cecal ligation and puncture models. At 1 month post‐sepsis, we observed persistent enrichment/depletion of cell populations and further uncovered a cell‐type and tissue‐specific ability to return to a baseline transcriptomic state. Differential gene expression analyses revealed key genes and pathways altered in post‐sepsis muscle and fat and highlighted the engagement of infection/inflammation and tissue damage signalling. Finally, regulator analysis identified gonadotropin‐releasing hormone and Bay 11‐7082 as targets/compounds that we show can reduce sepsis‐associated loss of lean or fat mass.ConclusionsThese data demonstrate persistent post‐sepsis muscle and adipose tissue disruption at the single‐cell level and highlight opportunities to combat long‐term post‐sepsis tissue wasting using bioinformatics‐guided therapeutic interventions.

CB2 improves power of cell detection in droplet-based single-cell RNA sequencing data

An important challenge in pre-processing data from droplet-based single-cell RNA sequencing protocols is distinguishing barcodes associated with real cells from those binding background reads. Existing methods test barcodes individually and consequently do not leverage the strong cell-to-cell correlation present in most datasets. To improve cell detection, we introduce CB2, a cluster-based approach for distinguishing real cells from background barcodes. As demonstrated in simulated and case study datasets, CB2 has increased power for identifying real cells which allows for the identification of novel subpopulations and improves the precision of downstream analyses.

A new protocol for single-cell RNA-seq reveals stochastic gene expression during lag phase in budding yeast.

Current methods for single-cell RNA sequencing (scRNA-seq) of yeast cells do not match the throughput and relative simplicity of the state-of-the-art techniques that are available for mammalian cells. In this study, we report how 10x Genomics' droplet-based single-cell RNA sequencing technology can be modified to allow analysis of yeast cells. The protocol, which is based on in-droplet spheroplasting of the cells, yields an order-of-magnitude higher throughput in comparison to existing methods. After extensive validation of the method, we demonstrate its use by studying the dynamics of the response of isogenic yeast populations to a shift in carbon source, revealing the heterogeneity and underlying molecular processes during this shift. The method we describe opens new avenues for studies focusing on yeast cells, as well as other cells with a degradable cell wall.