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Rozenblatt-Rosen, O.

Publications and source records attributed to Rozenblatt-Rosen, O..

53 records · Page 3Linked to original sources
bioRxiv2019

Multiplexed single-cell profiling of post-perturbation transcriptional responses to define cancer vulnerabilities and therapeutic mechanism of action

Assays to study cancer cell responses to pharmacologic or genetic perturbations are typically restricted to using simple phenotypic readouts such as proliferation rate or the expression of a marker gene. Information-rich assays, such as gene-expression profiling, are generally not amenable to efficient profiling of a given perturbation across multiple cellular contexts. Here, we developed MIX-Seq, a method for multiplexed transcriptional profiling of post-perturbation responses across a mixture of samples with single-cell resolution, using SNP-based computational demultiplexing of single-cell RNA-sequencing data. We show that MIX-Seq can be used to profile responses to chemical or genetic perturbations across pools of 100 or more cancer cell lines, and combine it with Cell Hashing to further multiplex additional experimental conditions, such as multiple post-treatment time points or drug doses. Analyzing the high-content readout of scRNA-seq reveals both shared and context-specific transcriptional response components that can identify drug mechanism of action and can be used to predict long-term cell viability from short-term transcriptional responses to treatment.

cancer biology↗
bioRxiv2019

Cumulus: a cloud-based data analysis framework for large-scale single-cell and single-nucleus RNA-seq

Massively parallel single-cell and single-nucleus RNA-seq (sc/snRNA-seq) have opened the way to systematic tissue atlases in health and disease, but as the scale of data generation is growing, so does the need for computational pipelines for scaled analysis. Here, we developed Cumulus, a cloud-based framework for analyzing large scale sc/snRNA-seq datasets. Cumulus combines the power of cloud computing with improvements in algorithm implementations to achieve high scalability, low cost, user-friendliness, and integrated support for a comprehensive set of features. We benchmark Cumulus on the Human Cell Atlas Census of Immune Cells dataset of bone marrow cells and show that it substantially improves efficiency over conventional frameworks, while maintaining or improving the quality of results, enabling large-scale studies.

bioinformatics↗
bioRxiv2019

Pan-cancer single cell RNA-seq uncovers recurring programs of cellular heterogeneity

Cultured cell lines are the workhorse of cancer research, but it is unclear to what extent they recapitulate the cellular heterogeneity observed among malignant cells in tumors, given the absence of a native tumor microenvironment. Here, we used multiplexed single cell RNA-seq to profile ~200 cancer cell lines. We uncovered expression programs that are recurrently heterogeneous within many cancer cell lines and are largely independent of observed genetic diversity. These programs of heterogeneity are associated with diverse biological processes, including cell cycle, senescence, stress and interferon responses, epithelial-to-mesenchymal transition, and protein maturation and degradation. Notably, some of these recurrent programs recapitulate those seen in human tumors, suggesting a prominent role of intrinsic plasticity in generating intra-tumoral heterogeneity. Moreover, the data allowed us to prioritize specific cell lines as model systems of cellular plasticity. We used two such models to demonstrate the dynamics, regulation and drug sensitivities associated with a cancer senescence program also observed in human tumors. Our work describes the landscape of cellular heterogeneity in diverse cancer cell lines, and identifies recurrent patterns of expression heterogeneity that are shared between tumors and specific cell lines and can thus be further explored in follow up studies.

cancer biology↗
bioRxiv2019

An endogenous glucocorticoid-cytokine signaling circuit promotes CD8+ T cell dysfunction in the tumor microenvironment

Identifying signals in the tumor microenvironment (TME) that promote CD8+ T cell dysfunction can inform improved therapeutic approaches for cancer. Here, we identify that Nr3c1, the gene encoding the glucocorticoid receptor (GR), is highly expressed in dysfunctional CD8+ tumor-infiltrating lymphocytes (TILs). The GR transactivates expression of multiple checkpoint receptors and loss of GR in CD8+ T cells limits dysfunctional phenotype in CD8+ TILs resulting in improved tumor growth control. We show that glucocorticoids can be produced in the TME and that they co-operate with the immunosuppressive cytokine IL-27 to promote the dysfunction gene program in CD8+ T cells. The presence of the glucocorticoid + IL27 signature in CD8+ TILs correlates with failure to respond to checkpoint blockade in melanoma patients, highlighting the relevance of this immunoregulatory glucocorticoid-cytokine circuit in tumor tissue.

immunology↗
bioRxiv2019

The enteric nervous system of the human and mouse colon at a single-cell resolution

As the largest branch of the autonomic nervous system, the enteric nervous system (ENS) controls the entire gastrointestinal tract, but remains incompletely characterized. Here, we develop RAISIN RNA-seq, which enables the capture of intact single nuclei along with ribosome-bound mRNA, and use it to profile the adult mouse and human colon to generate a reference map of the ENS at a single-cell resolution. This map reveals an extraordinary diversity of neuron subsets across intestinal locations, ages, and circadian phases, with conserved transcriptional programs that are shared between human and mouse. These data suggest possible revisions to the current model of peristalsis and molecular mechanisms that may allow enteric neurons to orchestrate tissue homeostasis, including immune regulation and stem cell maintenance. Human enteric neurons specifically express risk genes for neuropathic, inflammatory, and extra-intestinal diseases with concomitant gut dysmotility. Our study therefore provides a roadmap to understanding the ENS in health and disease.

genomics↗
bioRxiv2019

Opposing immune and genetic forces shape oncogenic programs in synovial sarcoma

Synovial sarcoma is an aggressive mesenchymal neoplasm, driven by the SS18-SSX fusion, and characterized by immunogenic antigens expression and exceptionally low T cell infiltration levels. To study the cancer-immune interplay in this disease, we profiled 16,872 cells from 12 human synovial sarcoma tumors using single-cell RNA-sequencing (scRNA-Seq). Synovial sarcoma manifests antitumor immunity, high cellular plasticity and a core oncogenic program, which is predictive of low immune levels and poor clinical outcomes. Using genetic and pharmacological perturbations, we demonstrate that the program is controlled by the SS18-SSX driver and repressed by cytokines secreted by macrophages and T cells in the tumor microenvironment. Network modeling predicted that SS18-SSX promotes the program through HDAC1 and CDK6. Indeed, the combination of HDAC and CDK4/6 inhibitors represses the program, induces immunogenic cell states, and selectively targets synovial sarcoma cells. Our study demonstrates that immune evasion, cellular plasticity, and cell cycle are co-regulated and can be co-targeted in synovial sarcoma and potentially in other malignancies.

cancer biology↗
bioRxiv2019

Decoding the development of the blood and immune systems during human fetal liver haematopoiesis

Definitive haematopoiesis in the fetal liver supports self-renewal and differentiation of haematopoietic stem cells/multipotent progenitors (HSC/MPPs), yet remains poorly defined in humans. Using single cell transcriptome profiling of ~133,000 fetal liver and ~65,000 fetal skin and kidney cells, we identify the repertoire of blood and immune cells in first and early second trimesters of development. From this data, we infer differentiation trajectories from HSC/MPPs, and evaluate the impact of tissue microenvironment on blood and immune cell development. We predict coupling of mast cell differentiation with erythro-megakaryopoiesis and identify physiological erythropoiesis in fetal skin. We demonstrate a shift in fetal liver haematopoietic composition during gestation away from being erythroid-predominant, accompanied by a parallel change in HSC/MPP differentiation potential, which we functionally validate. Our integrated map of fetal liver haematopoiesis provides a blueprint for the study of paediatric blood and immune disorders, and a valuable reference for understanding and harnessing the therapeutic potential of HSC/MPPs.

developmental biology↗
bioRxiv2019

Systematic comparative analysis of single cell RNA-sequencing methods

A multitude of single-cell RNA sequencing methods have been developed in recent years, with dramatic advances in scale and power, and enabling major discoveries and large scale cell mapping efforts. However, these methods have not been systematically and comprehensively benchmarked. Here, we directly compare seven methods for single cell and/or single nucleus profiling from three types of samples - cell lines, peripheral blood mononuclear cells and brain tissue - generating 36 libraries in six separate experiments in a single center. To analyze these datasets, we developed and applied scumi, a flexible computational pipeline that can be used for any scRNA-seq method. We evaluated the methods for both basic performance and for their ability to recover known biological information in the samples. Our study will help guide experiments with the methods in this study as well as serve as a benchmark for future studies and for computational algorithm development.

genomics↗
bioRxiv2019

A cellular and spatial map of the choroid plexus across brain ventricles and ages

The choroid plexus (ChP), located in each brain ventricle, produces cerebrospinal fluid (CSF) and forms the blood-CSF barrier, but is under-characterized. Here, we combine single cell RNA-Seq and spatial mapping of RNA and proteins to construct an atlas of each ChP in the developing and adult mouse brain. Each ChP comprises of epithelial, endothelial, mesenchymal, immune, neuronal, and glial cells, with distinct subtypes, differentiation states and anatomical locations. Epithelial, fibroblast, and macrophage populations had ventricle-specific, regionalized gene expression programs across the developing brain. Key cell types are retained in adult, with loss of developmental signatures and maturation of ventricle-specific regionalization in the epithelial cells. Expression of cognate ligand-receptor pairs across cell subtypes suggests substantial cell-cell interactions within the ChP. Our atlas sheds new light on the development and function of the ChP brain barrier system, and will facilitate future studies on its role in brain development, homeostasis and disease.

neuroscience↗
bioRxiv2019

A cellular taxonomy of the bone marrow stroma in homeostasis and leukemia demonstrates cancer-crosstalk with stroma to impair normal tissue function

Stroma is a poorly defined non-parenchymal component of virtually every organ with key roles in organ development, homeostasis and repair. Studies of the bone marrow stroma have defined individual populations in the stem cell niche regulating hematopoietic regeneration and capable of initiating leukemia. Here, we use single-cell RNA-seq to define a cellular taxonomy of the mouse bone marrow stroma and its perturbation by malignancy. We identified seventeen stromal subsets expressing distinct hematopoietic regulatory genes, spanning new fibroblastic, and osteoblastic subpopulations. Emerging acute myeloid leukemia resulted in impaired osteogenic differentiation and reduced production of hematopoietic regulatory molecules necessary for normal hematopoiesis. Thus, cancer can affect tissue stroma in which they reside to disadvantage normal parenchymal cells. Our taxonomy of the regulatory stromal compartment provides experimental support for a model where malignant clone is not a destroyer of normal tissue but an architect of it, remodeling tissue stroma to enable emergent cancer.

cell biology↗
bioRxiv2019

Kidney organoid reproducibility across multiple human iPSC lines and diminished off target cells after transplantation revealed by single cell transcriptomics

Human iPSC-derived kidney organoids have the potential to revolutionize discovery, but assessing their consistency and reproducibility across iPSC lines, and reducing the generation of off-target cells remain an open challenge. Here, we used single cell RNA-Seq (scRNA-Seq) to profile 415,775 cells to show that organoid composition and development are comparable to human fetal and adult kidneys. Although cell classes were largely reproducible across iPSC lines, time points, protocols, and replicates, cell proportions were variable between different iPSC lines. Off-target cell proportions were the most variable. Prolonged in vitro culture did not alter cell types, but organoid transplantation under the mouse kidney capsule diminished off-target cells. Our work shows how scRNA-seq can help score organoids for reproducibility, faithfulness and quality, that kidney organoids derived from different iPSC lines are comparable surrogates for human kidney, and that transplantation enhances their formation by diminishing off-target cells.

genomics↗
bioRxiv2019

Longitudinal single cell profiling of regulatory T cells identifies IL-33 as a driver of tumor immunosuppression

Regulatory T cells (Tregs) can impair anti-tumor immune responses and are associated with poor prognosis in multiple cancer types. Tregs in human tumors span diverse transcriptional states distinct from those of peripheral Tregs, but their contribution to tumor development remains unknown. Here, we used single cell RNA-Seq to longitudinally profile conventional CD4+ T cells (Tconv) and Tregs in a genetic mouse model of lung adenocarcinoma. Tissue-infiltrating and peripheral CD4+ T cells differed, highlighting divergent pathways of activation during tumorigenesis. Longitudinal shifts in Treg heterogeneity suggested increased terminal differentiation and stabilization of an effector phenotype over time. In particular, effector Tregs had enhanced expression of the interleukin 33 receptor ST2. Treg-specific deletion of ST2 reduced effector Tregs, increased infiltration of CD8+ T cells into tumors, and decreased tumor burden. Our study shows that ST2 plays a critical role in Treg-mediated immunosuppression in cancer, highlighting new potential paths for therapeutic intervention.

cancer biology↗
bioRxiv2018

Nuclei multiplexing with barcoded antibodies for single-nucleus genomics

Single-nucleus RNA-Seq (snRNA-seq) enables the interrogation of cellular states in complex tissues that are challenging to dissociate, including frozen clinical samples. This opens the way, in principle, to large studies, such as those required for human genetics, clinical trials, or precise cell atlases of large organs. However, such applications are currently limited by batch effects, sequential processing, and costs. To address these challenges, we present an approach for multiplexing snRNA-seq, using sample-barcoded antibodies against the nuclear pore complex to uniquely label nuclei from distinct samples. Comparing human brain cortex samples profiled in multiplex with or without hashing antibodies, we demonstrate that nucleus hashing does not significantly alter the recovered transcriptome profiles. We further developed demuxEM, a novel computational tool that robustly detects inter-sample nucleus multiplets and assigns singlets to their samples of origin by antibody barcodes, and validated its accuracy using gender-specific gene expression, species-mixing and natural genetic variation. Nucleus hashing significantly reduces cost per nucleus, recovering up to about 5 times as many single nuclei per microfluidc channel. Our approach provides a robust technique for diverse studies including tissue atlases of isogenic model organisms or from a single larger human organ, multiple biopsies or longitudinal samples of one donor, and large-scale perturbation screens.

genomics↗
bioRxiv2017

T helper cells modulate intestinal stem cell renewal and differentiation

In the small intestine, a cellular niche of diverse accessory cell types supports the rapid generation of mature epithelial cell types through self-renewal, proliferation, and differentiation of intestinal stem cells (ISCs). However, not much is known about interactions between immune cells and ISCs, and it is unclear if and how immune cell dynamics affect eventual ISC fate or the balance between self-renewal and differentiation. Here, we used single-cell RNA-seq (scRNA-Seq) of intestinal epithelial cells (IECs) to identify new mechanisms for ISC-immune cell interactions. Surprisingly, MHC class II (MHCII) is enriched in two distinct subsets of Lgr5+ crypt base columnar ISCs, which are also distinguished by higher proliferation rates. Using co-culture of T cells with intestinal organoids, cytokine stimulations, and in vivo mouse models, we confirm that CD4+ T helper (Th) cells communicate with ISCs and affect their differentiation, in a manner specific to the Th subtypes and their signature cytokines and dependent on MHCII expression by ISCs. Specific inducible knockout of MHCII in intestinal epithelial cells in mice in vivo results in expansion of the ISC pool. Mice lacking T cells have expanded ISC pools, whereas specific depletion of Treg cells in vivo results in substantial reduction of ISC numbers. Our findings show that interactions between Th cells and ISCs mediated via MHCII expressed in intestinal epithelial stem cells help orchestrate tissue-wide responses to external signals.

immunology↗
bioRxiv2017

The Human Cell Atlas

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body, by undertaking a Human Cell Atlas Project as an international collaborative effort. The aim would be to define all human cell types in terms of distinctive molecular profiles (e.g., gene expression) and connect this information with classical cellular descriptions (e.g., location and morphology). A comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, as well as provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas.

cell biology↗
bioRxiv2017

DroNc-Seq: Deciphering cell types in human archived brain tissues by massively-parallel single nucleus RNA-seq

Single nucleus RNA-Seq (sNuc-Seq) profiles RNA from tissues that are preserved or cannot be dissociated, but does not provide the throughput required to analyse many cells from complex tissues. Here, we develop DroNc-Seq, massively parallel sNuc-Seq with droplet technology. We profile 29,543 nuclei from mouse and human archived brain samples to demonstrate sensitive, efficient and unbiased classification of cell types, paving the way for charting systematic cell atlases.

genomics↗