RESUMEN
Glioma contains malignant cells in diverse states. Here, we combine spatial transcriptomics, spatial proteomics, and computational approaches to define glioma cellular states and uncover their organization. We find three prominent modes of organization. First, gliomas are composed of small local environments, each typically enriched with one major cellular state. Second, specific pairs of states preferentially reside in proximity across multiple scales. This pairing of states is consistent across tumors. Third, these pairwise interactions collectively define a global architecture composed of five layers. Hypoxia appears to drive the layers, as it is associated with a long-range organization that includes all cancer cell states. Accordingly, tumor regions distant from any hypoxic/necrotic foci and tumors that lack hypoxia such as low-grade IDH-mutant glioma are less organized. In summary, we provide a conceptual framework for the organization of cellular states in glioma, highlighting hypoxia as a long-range tissue organizer.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Glioblastoma/patología , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Análisis Espacial , Transcriptoma/genética , Microambiente Tumoral , Proteómica , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Regulación Neoplásica de la Expresión GénicaRESUMEN
T cells are critical effectors of cancer immunotherapies, but little is known about their gene expression programs in diffuse gliomas. Here, we leverage single-cell RNA sequencing (RNA-seq) to chart the gene expression and clonal landscape of tumor-infiltrating T cells across 31 patients with isocitrate dehydrogenase (IDH) wild-type glioblastoma and IDH mutant glioma. We identify potential effectors of anti-tumor immunity in subsets of T cells that co-express cytotoxic programs and several natural killer (NK) cell genes. Analysis of clonally expanded tumor-infiltrating T cells further identifies the NK gene KLRB1 (encoding CD161) as a candidate inhibitory receptor. Accordingly, genetic inactivation of KLRB1 or antibody-mediated CD161 blockade enhances T cell-mediated killing of glioma cells in vitro and their anti-tumor function in vivo. KLRB1 and its associated transcriptional program are also expressed by substantial T cell populations in other human cancers. Our work provides an atlas of T cells in gliomas and highlights CD161 and other NK cell receptors as immunotherapy targets.
Asunto(s)
Glioma/inmunología , Subfamilia B de Receptores Similares a Lectina de Células NK/genética , Linfocitos T/inmunología , Animales , Antígenos de Neoplasias , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Glioma/genética , Células Asesinas Naturales/inmunología , Lectinas Tipo C/genética , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Receptores de Superficie Celular/genética , Análisis de la Célula Individual , Subgrupos de Linfocitos T/inmunología , Linfocitos T/citología , Escape del TumorRESUMEN
In the small intestine, a niche of accessory cell types supports the generation of mature epithelial cell types from intestinal stem cells (ISCs). It is unclear, however, if and how immune cells in the niche affect ISC fate or the balance between self-renewal and differentiation. Here, we use single-cell RNA sequencing (scRNA-seq) to identify MHC class II (MHCII) machinery enrichment in two subsets of Lgr5+ ISCs. We show that MHCII+ Lgr5+ ISCs are non-conventional antigen-presenting cells in co-cultures with CD4+ T helper (Th) cells. Stimulation of intestinal organoids with key Th cytokines affects Lgr5+ ISC renewal and differentiation in opposing ways: pro-inflammatory signals promote differentiation, while regulatory cells and cytokines reduce it. In vivo genetic perturbation of Th cells or MHCII expression on Lgr5+ ISCs impacts epithelial cell differentiation and IEC fate during infection. These interactions between Th cells and Lgr5+ ISCs, thus, orchestrate tissue-wide responses to external signals.
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Diferenciación Celular , Autorrenovación de las Células , Interleucina-10/metabolismo , Células Madre/citología , Linfocitos T Colaboradores-Inductores/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Autorrenovación de las Células/efectos de los fármacos , Citocinas/farmacología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Antígenos de Histocompatibilidad Clase II/metabolismo , Sistema Inmunológico/metabolismo , Intestinos/citología , Intestinos/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Organoides/citología , Organoides/efectos de los fármacos , Organoides/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Salmonella enterica/patogenicidad , Células Madre/metabolismo , Linfocitos T Colaboradores-Inductores/citologíaRESUMEN
Immune checkpoint inhibitors (ICIs) produce durable responses in some melanoma patients, but many patients derive no clinical benefit, and the molecular underpinnings of such resistance remain elusive. Here, we leveraged single-cell RNA sequencing (scRNA-seq) from 33 melanoma tumors and computational analyses to interrogate malignant cell states that promote immune evasion. We identified a resistance program expressed by malignant cells that is associated with T cell exclusion and immune evasion. The program is expressed prior to immunotherapy, characterizes cold niches in situ, and predicts clinical responses to anti-PD-1 therapy in an independent cohort of 112 melanoma patients. CDK4/6-inhibition represses this program in individual malignant cells, induces senescence, and reduces melanoma tumor outgrowth in mouse models in vivo when given in combination with immunotherapy. Our study provides a high-resolution landscape of ICI-resistant cell states, identifies clinically predictive signatures, and suggests new therapeutic strategies to overcome immunotherapy resistance.
Asunto(s)
Antineoplásicos/uso terapéutico , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Melanoma/inmunología , Inhibidores de Proteínas Quinasas/uso terapéutico , Linfocitos T/inmunología , Escape del Tumor , Anciano , Anciano de 80 o más Años , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Femenino , Humanos , Inmunoterapia/métodos , Masculino , Melanoma/tratamiento farmacológico , Melanoma/terapia , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
The diverse malignant, stromal, and immune cells in tumors affect growth, metastasis, and response to therapy. We profiled transcriptomes of â¼6,000 single cells from 18 head and neck squamous cell carcinoma (HNSCC) patients, including five matched pairs of primary tumors and lymph node metastases. Stromal and immune cells had consistent expression programs across patients. Conversely, malignant cells varied within and between tumors in their expression of signatures related to cell cycle, stress, hypoxia, epithelial differentiation, and partial epithelial-to-mesenchymal transition (p-EMT). Cells expressing the p-EMT program spatially localized to the leading edge of primary tumors. By integrating single-cell transcriptomes with bulk expression profiles for hundreds of tumors, we refined HNSCC subtypes by their malignant and stromal composition and established p-EMT as an independent predictor of nodal metastasis, grade, and adverse pathologic features. Our results provide insight into the HNSCC ecosystem and define stromal interactions and a p-EMT program associated with metastasis.
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Carcinoma de Células Escamosas/patología , Neoplasias de Cabeza y Cuello/patología , Metástasis de la Neoplasia/patología , Carcinoma de Células Escamosas/genética , Células Cultivadas , Transición Epitelial-Mesenquimal , Perfilación de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Humanos , Masculino , Análisis de la Célula Individual , Microambiente TumoralRESUMEN
Homeostatic programs balance immune protection and self-tolerance. Such mechanisms likely impact autoimmunity and tumor formation, respectively. How homeostasis is maintained and impacts tumor surveillance is unknown. Here, we find that different immune mononuclear phagocytes share a conserved steady-state program during differentiation and entry into healthy tissue. IFNγ is necessary and sufficient to induce this program, revealing a key instructive role. Remarkably, homeostatic and IFNγ-dependent programs enrich across primary human tumors, including melanoma, and stratify survival. Single-cell RNA sequencing (RNA-seq) reveals enrichment of homeostatic modules in monocytes and DCs from human metastatic melanoma. Suppressor-of-cytokine-2 (SOCS2) protein, a conserved program transcript, is expressed by mononuclear phagocytes infiltrating primary melanoma and is induced by IFNγ. SOCS2 limits adaptive anti-tumoral immunity and DC-based priming of T cells in vivo, indicating a critical regulatory role. These findings link immune homeostasis to key determinants of anti-tumoral immunity and escape, revealing co-opting of tissue-specific immune development in the tumor microenvironment.
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Interferón gamma/inmunología , Melanoma/inmunología , Monocitos/inmunología , Metástasis de la Neoplasia/patología , Neoplasias Cutáneas/inmunología , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Microambiente Tumoral , Animales , Diferenciación Celular , Células Dendríticas/inmunología , Homeostasis , Humanos , Melanoma/genética , Melanoma/patología , Ratones , Monocitos/patología , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , TranscriptomaRESUMEN
Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell's RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts' cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution. VIDEO ABSTRACT.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Estudio de Asociación del Genoma Completo , Técnicas Analíticas Microfluídicas , Retina/citología , Análisis de la Célula Individual , Animales , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Análisis de Secuencia de ARNRESUMEN
Finding the components of cellular circuits and determining their functions systematically remains a major challenge in mammalian cells. Here, we introduced genome-wide pooled CRISPR-Cas9 libraries into dendritic cells (DCs) to identify genes that control the induction of tumor necrosis factor (Tnf) by bacterial lipopolysaccharide (LPS), a key process in the host response to pathogens, mediated by the Tlr4 pathway. We found many of the known regulators of Tlr4 signaling, as well as dozens of previously unknown candidates that we validated. By measuring protein markers and mRNA profiles in DCs that are deficient in known or candidate genes, we classified the genes into three functional modules with distinct effects on the canonical responses to LPS and highlighted functions for the PAF complex and oligosaccharyltransferase (OST) complex. Our findings uncover new facets of innate immune circuits in primary cells and provide a genetic approach for dissection of mammalian cell circuits.
Asunto(s)
Sistemas CRISPR-Cas , Técnicas Genéticas , Inmunidad Innata , Animales , Células de la Médula Ósea/inmunología , Diferenciación Celular , Supervivencia Celular , Células Dendríticas/citología , Células Dendríticas/inmunología , Técnicas de Inactivación de Genes , Redes Reguladoras de Genes , Hexosiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Receptor Toll-Like 4/inmunología , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
The mouse small intestine shows profound variability in gene expression along the crypt-villus axis1,2. Whether similar spatial heterogeneity exists in the adult human gut remains unclear. Here we use spatial transcriptomics, spatial proteomics and single-molecule fluorescence in situ hybridization to reconstruct a comprehensive spatial expression atlas of the adult human proximal small intestine. We describe zonated expression and cell type representation for epithelial, mesenchymal and immune cell types. We find that migrating enterocytes switch from lipid droplet assembly and iron uptake at the villus bottom to chylomicron biosynthesis and iron release at the tip. Villus tip cells are pro-immunogenic, recruiting γδ T cells and macrophages to the tip, in contrast to their immunosuppressive roles in mouse. We also show that the human small intestine contains abundant serrated and branched villi that are enriched at the tops of circular folds. Our study presents a detailed resource for understanding the biology of the adult human small intestine.
RESUMEN
A broad range of brain pathologies critically relies on the vasculature, and cerebrovascular disease is a leading cause of death worldwide. However, the cellular and molecular architecture of the human brain vasculature remains incompletely understood1. Here we performed single-cell RNA sequencing analysis of 606,380 freshly isolated endothelial cells, perivascular cells and other tissue-derived cells from 117 samples, from 68 human fetuses and adult patients to construct a molecular atlas of the developing fetal, adult control and diseased human brain vasculature. We identify extensive molecular heterogeneity of the vasculature of healthy fetal and adult human brains and across five vascular-dependent central nervous system (CNS) pathologies, including brain tumours and brain vascular malformations. We identify alteration of arteriovenous differentiation and reactivated fetal as well as conserved dysregulated genes and pathways in the diseased vasculature. Pathological endothelial cells display a loss of CNS-specific properties and reveal an upregulation of MHC class II molecules, indicating atypical features of CNS endothelial cells. Cell-cell interaction analyses predict substantial endothelial-to-perivascular cell ligand-receptor cross-talk, including immune-related and angiogenic pathways, thereby revealing a central role for the endothelium within brain neurovascular unit signalling networks. Our single-cell brain atlas provides insights into the molecular architecture and heterogeneity of the developing, adult/control and diseased human brain vasculature and serves as a powerful reference for future studies.
Asunto(s)
Neoplasias Encefálicas , Encéfalo , Malformaciones Vasculares del Sistema Nervioso Central , Células Endoteliales , Feto , RNA-Seq , Análisis de Expresión Génica de una Sola Célula , Femenino , Humanos , Masculino , Encéfalo/irrigación sanguínea , Encéfalo/patología , Encéfalo/embriología , Encéfalo/metabolismo , Neoplasias Encefálicas/irrigación sanguínea , Neoplasias Encefálicas/patología , Comunicación Celular , Células Endoteliales/metabolismo , Células Endoteliales/patología , Células Endoteliales/citología , Feto/irrigación sanguínea , Feto/citología , Feto/embriología , Malformaciones Vasculares del Sistema Nervioso Central/patología , Antígenos HLA-D/metabolismo , Adulto , SaludRESUMEN
Developmental fate decisions are dictated by master transcription factors (TFs) that interact with cis-regulatory elements to direct transcriptional programs. Certain malignant tumors may also depend on cellular hierarchies reminiscent of normal development but superimposed on underlying genetic aberrations. In glioblastoma (GBM), a subset of stem-like tumor-propagating cells (TPCs) appears to drive tumor progression and underlie therapeutic resistance yet remain poorly understood. Here, we identify a core set of neurodevelopmental TFs (POU3F2, SOX2, SALL2, and OLIG2) essential for GBM propagation. These TFs coordinately bind and activate TPC-specific regulatory elements and are sufficient to fully reprogram differentiated GBM cells to "induced" TPCs, recapitulating the epigenetic landscape and phenotype of native TPCs. We reconstruct a network model that highlights critical interactions and identifies candidate therapeutic targets for eliminating TPCs. Our study establishes the epigenetic basis of a developmental hierarchy in GBM, provides detailed insight into underlying gene regulatory programs, and suggests attendant therapeutic strategies. PAPERCLIP:
Asunto(s)
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Glioblastoma/genética , Glioblastoma/patología , Células Madre Neoplásicas/patología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias Encefálicas/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Células Cultivadas , Proteínas Co-Represoras/metabolismo , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/metabolismo , Humanos , Células Madre Neoplásicas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Factor de Transcripción 2 de los Oligodendrocitos , Elementos Reguladores de la Transcripción , Factores de Transcripción/metabolismoRESUMEN
Each tumour contains diverse cellular states that underlie intratumour heterogeneity (ITH), a central challenge of cancer therapeutics1. Dozens of recent studies have begun to describe ITH by single-cell RNA sequencing, but each study typically profiled only a small number of tumours and provided a narrow view of transcriptional ITH2. Here we curate, annotate and integrate the data from 77 different studies to reveal the patterns of transcriptional ITH across 1,163 tumour samples covering 24 tumour types. Among the malignant cells, we identify 41 consensus meta-programs, each consisting of dozens of genes that are coordinately upregulated in subpopulations of cells within many tumours. The meta-programs cover diverse cellular processes including both generic (for example, cell cycle and stress) and lineage-specific patterns that we map into 11 hallmarks of transcriptional ITH. Most meta-programs of carcinoma cells are similar to those identified in non-malignant epithelial cells, suggesting that a large fraction of malignant ITH programs are variable even before oncogenesis, reflecting the biology of their cell of origin. We further extended the meta-program analysis to six common non-malignant cell types and utilize these to map cell-cell interactions within the tumour microenvironment. In summary, we have assembled a comprehensive pan-cancer single-cell RNA-sequencing dataset, which is available through the Curated Cancer Cell Atlas website, and leveraged this dataset to carry out a systematic characterization of transcriptional ITH.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Heterogeneidad Genética , Neoplasias , Análisis de Expresión Génica de una Sola Célula , Humanos , Células Epiteliales/citología , Células Epiteliales/metabolismo , Neoplasias/clasificación , Neoplasias/genética , Neoplasias/patología , Microambiente TumoralRESUMEN
The role of the nervous system in the regulation of cancer is increasingly appreciated. In gliomas, neuronal activity drives tumour progression through paracrine signalling factors such as neuroligin-3 and brain-derived neurotrophic factor1-3 (BDNF), and also through electrophysiologically functional neuron-to-glioma synapses mediated by AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors4,5. The consequent glioma cell membrane depolarization drives tumour proliferation4,6. In the healthy brain, activity-regulated secretion of BDNF promotes adaptive plasticity of synaptic connectivity7,8 and strength9-15. Here we show that malignant synapses exhibit similar plasticity regulated by BDNF. Signalling through the receptor tropomyosin-related kinase B16 (TrkB) to CAMKII, BDNF promotes AMPA receptor trafficking to the glioma cell membrane, resulting in increased amplitude of glutamate-evoked currents in the malignant cells. Linking plasticity of glioma synaptic strength to tumour growth, graded optogenetic control of glioma membrane potential demonstrates that greater depolarizing current amplitude promotes increased glioma proliferation. This potentiation of malignant synaptic strength shares mechanistic features with synaptic plasticity17-22 that contributes to memory and learning in the healthy brain23-26. BDNF-TrkB signalling also regulates the number of neuron-to-glioma synapses. Abrogation of activity-regulated BDNF secretion from the brain microenvironment or loss of glioma TrkB expression robustly inhibits tumour progression. Blocking TrkB genetically or pharmacologically abrogates these effects of BDNF on glioma synapses and substantially prolongs survival in xenograft models of paediatric glioblastoma and diffuse intrinsic pontine glioma. Together, these findings indicate that BDNF-TrkB signalling promotes malignant synaptic plasticity and augments tumour progression.
Asunto(s)
Adaptación Fisiológica , Glioma , Plasticidad Neuronal , Sinapsis , Animales , Niño , Humanos , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proliferación Celular , Progresión de la Enfermedad , Glioma/metabolismo , Glioma/patología , Ácido Glutámico/metabolismo , Neuronas/citología , Neuronas/metabolismo , Receptor trkB/genética , Receptor trkB/metabolismo , Receptores AMPA/metabolismo , Transducción de Señal , Sinapsis/metabolismo , Microambiente Tumoral , OptogenéticaRESUMEN
Bulk genomic analyses and expression profiling of clinical specimens have shaped much of our understanding of cancer in patients. However, human tumors are intricate ecosystems composed of diverse cells, including malignant, immune, and stromal subsets, whose precise characterization is masked by bulk genomic methods. Single-cell genomic techniques have emerged as powerful approaches to dissect human tumors at the resolution of individual cells, providing a compelling approach to deciphering cancer biology. Here, we discuss some of the common themes emerging from initial studies of single-cell RNA sequencing in cancer and then highlight challenges in cancer biology for which emerging single-cell genomics methods may provide a compelling approach.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Neoplasias/genética , Células Neoplásicas Circulantes/metabolismo , ARN Neoplásico/genética , Análisis de la Célula Individual/métodos , Antineoplásicos/uso terapéutico , Comunicación Celular , Línea Celular Tumoral , Linaje de la Célula , Perfilación de la Expresión Génica , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Células Neoplásicas Circulantes/patología , ARN Neoplásico/metabolismoRESUMEN
Neoantigens, which are derived from tumour-specific protein-coding mutations, are exempt from central tolerance, can generate robust immune responses1,2 and can function as bona fide antigens that facilitate tumour rejection3. Here we demonstrate that a strategy that uses multi-epitope, personalized neoantigen vaccination, which has previously been tested in patients with high-risk melanoma4-6, is feasible for tumours such as glioblastoma, which typically have a relatively low mutation load1,7 and an immunologically 'cold' tumour microenvironment8. We used personalized neoantigen-targeting vaccines to immunize patients newly diagnosed with glioblastoma following surgical resection and conventional radiotherapy in a phase I/Ib study. Patients who did not receive dexamethasone-a highly potent corticosteroid that is frequently prescribed to treat cerebral oedema in patients with glioblastoma-generated circulating polyfunctional neoantigen-specific CD4+ and CD8+ T cell responses that were enriched in a memory phenotype and showed an increase in the number of tumour-infiltrating T cells. Using single-cell T cell receptor analysis, we provide evidence that neoantigen-specific T cells from the peripheral blood can migrate into an intracranial glioblastoma tumour. Neoantigen-targeting vaccines thus have the potential to favourably alter the immune milieu of glioblastoma.
Asunto(s)
Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/inmunología , Glioblastoma/inmunología , Glioblastoma/terapia , Linfocitos T/inmunología , Adulto , Anciano , Metilación de ADN , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/genética , Dexametasona/administración & dosificación , Glioblastoma/diagnóstico , Glioblastoma/genética , Humanos , Persona de Mediana Edad , Regiones Promotoras Genéticas/genética , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Supresoras de Tumor/genética , Adulto JovenRESUMEN
Intestinal epithelial cells absorb nutrients, respond to microbes, function as a barrier and help to coordinate immune responses. Here we report profiling of 53,193 individual epithelial cells from the small intestine and organoids of mice, which enabled the identification and characterization of previously unknown subtypes of intestinal epithelial cell and their gene signatures. We found unexpected diversity in hormone-secreting enteroendocrine cells and constructed the taxonomy of newly identified subtypes, and distinguished between two subtypes of tuft cell, one of which expresses the epithelial cytokine Tslp and the pan-immune marker CD45, which was not previously associated with non-haematopoietic cells. We also characterized the ways in which cell-intrinsic states and the proportions of different cell types respond to bacterial and helminth infections: Salmonella infection caused an increase in the abundance of Paneth cells and enterocytes, and broad activation of an antimicrobial program; Heligmosomoides polygyrus caused an increase in the abundance of goblet and tuft cells. Our survey highlights previously unidentified markers and programs, associates sensory molecules with cell types, and uncovers principles of gut homeostasis and response to pathogens.
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Células Epiteliales/citología , Epitelio/metabolismo , Intestino Delgado/citología , Análisis de la Célula Individual , Animales , Diferenciación Celular , Citocinas/metabolismo , Enterocitos/metabolismo , Células Epiteliales/metabolismo , Femenino , Perfilación de la Expresión Génica , Homeostasis , Antígenos Comunes de Leucocito/metabolismo , Masculino , Ratones , Organoides/citología , Organoides/metabolismo , Células de Paneth/metabolismo , Transcripción Genética , Linfopoyetina del Estroma TímicoRESUMEN
Although human tumours are shaped by the genetic evolution of cancer cells, evidence also suggests that they display hierarchies related to developmental pathways and epigenetic programs in which cancer stem cells (CSCs) can drive tumour growth and give rise to differentiated progeny. Yet, unbiased evidence for CSCs in solid human malignancies remains elusive. Here we profile 4,347 single cells from six IDH1 or IDH2 mutant human oligodendrogliomas by RNA sequencing (RNA-seq) and reconstruct their developmental programs from genome-wide expression signatures. We infer that most cancer cells are differentiated along two specialized glial programs, whereas a rare subpopulation of cells is undifferentiated and associated with a neural stem cell expression program. Cells with expression signatures for proliferation are highly enriched in this rare subpopulation, consistent with a model in which CSCs are primarily responsible for fuelling the growth of oligodendroglioma in humans. Analysis of copy number variation (CNV) shows that distinct CNV sub-clones within tumours display similar cellular hierarchies, suggesting that the architecture of oligodendroglioma is primarily dictated by developmental programs. Subclonal point mutation analysis supports a similar model, although a full phylogenetic tree would be required to definitively determine the effect of genetic evolution on the inferred hierarchies. Our single-cell analyses provide insight into the cellular architecture of oligodendrogliomas at single-cell resolution and support the cancer stem cell model, with substantial implications for disease management.
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Células Madre Neoplásicas/patología , Oligodendroglioma/genética , Oligodendroglioma/patología , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Diferenciación Celular , Proliferación Celular , Variaciones en el Número de Copia de ADN/genética , Humanos , Isocitrato Deshidrogenasa/genética , Células Madre Neoplásicas/metabolismo , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Neuroglía/metabolismo , Neuroglía/patología , Filogenia , Mutación PuntualRESUMEN
The application of RNA interference (RNAi) to mammalian cells has provided the means to perform phenotypic screens to determine the functions of genes. Although RNAi has revolutionized loss-of-function genetic experiments, it has been difficult to systematically assess the prevalence and consequences of off-target effects. The Connectivity Map (CMAP) represents an unprecedented resource to study the gene expression consequences of expressing short hairpin RNAs (shRNAs). Analysis of signatures for over 13,000 shRNAs applied in 9 cell lines revealed that microRNA (miRNA)-like off-target effects of RNAi are far stronger and more pervasive than generally appreciated. We show that mitigating off-target effects is feasible in these datasets via computational methodologies to produce a consensus gene signature (CGS). In addition, we compared RNAi technology to clustered regularly interspaced short palindromic repeat (CRISPR)-based knockout by analysis of 373 single guide RNAs (sgRNAs) in 6 cells lines and show that the on-target efficacies are comparable, but CRISPR technology is far less susceptible to systematic off-target effects. These results will help guide the proper use and analysis of loss-of-function reagents for the determination of gene function.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Perfilación de la Expresión Génica , Redes Reguladoras de Genes/genética , Genómica/métodos , Interferencia de ARN/fisiología , Células Cultivadas , Regulación Neoplásica de la Expresión Génica , Genómica/normas , Células HT29 , Células Hep G2 , Humanos , Células MCF-7 , ARN Interferente Pequeño/genética , TranscriptomaRESUMEN
Gene expression shows a significant variation (noise) between genetically identical cells. Noise depends on the gene expression process regulated by the chromatin environment. We screened for chromatin factors that modulate noise in S. cerevisiae and analyzed the results using a theoretical model that infers regulatory mechanisms from the noise versus mean relationship. Distinct activities of the Rpd3(L) and Set3 histone deacetylase complexes were predicted. Both HDACs repressed expression. Yet, Rpd3(L)C decreased the frequency of transcriptional bursts, while Set3C decreased the burst size, as did H2B monoubiquitination (ubH2B). We mapped the acetylation of H3 lysine 9 (H3K9ac) upon deletion of multiple subunits of Set3C and Rpd3(L)C and of ubH2B effectors. ubH2B and Set3C appear to function in the same pathway to reduce the probability that an elongating PolII produces a functional transcript (PolII processivity), while Rpd3(L)C likely represses gene expression at a step preceding elongation.