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
T and B cells are the two known lineages of adaptive immune cells. Here, we describe a previously unknown lymphocyte that is a dual expresser (DE) of TCR and BCR and key lineage markers of both B and T cells. In type 1 diabetes (T1D), DEs are predominated by one clonotype that encodes a potent CD4 T cell autoantigen in its antigen binding site. Molecular dynamics simulations revealed that this peptide has an optimal binding register for diabetogenic HLA-DQ8. In concordance, a synthetic version of the peptide forms stable DQ8 complexes and potently stimulates autoreactive CD4 T cells from T1D patients, but not healthy controls. Moreover, mAbs bearing this clonotype are autoreactive against CD4 T cells and inhibit insulin tetramer binding to CD4 T cells. Thus, compartmentalization of adaptive immune cells into T and B cells is not absolute, and violators of this paradigm are likely key drivers of autoimmune diseases.
Asunto(s)
Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Adolescente , Adulto , Autoantígenos/inmunología , Niño , Preescolar , Diabetes Mellitus Tipo 1/metabolismo , Epítopos/inmunología , Femenino , Células HEK293 , Antígenos HLA-DQ/inmunología , Antígenos HLA-DQ/ultraestructura , Humanos , Activación de Linfocitos/inmunología , Linfocitos/inmunología , Linfocitos/metabolismo , Masculino , Persona de Mediana Edad , Simulación de Dinámica Molecular , Péptidos , Unión Proteica/inmunologíaRESUMEN
In situ transgenesis methods such as viruses and electroporation can rapidly create somatic transgenic mice but lack control over copy number, zygosity, and locus specificity. Here we establish mosaic analysis by dual recombinase-mediated cassette exchange (MADR), which permits stable labeling of mutant cells expressing transgenic elements from precisely defined chromosomal loci. We provide a toolkit of MADR elements for combination labeling, inducible and reversible transgene manipulation, VCre recombinase expression, and transgenesis of human cells. Further, we demonstrate the versatility of MADR by creating glioma models with mixed reporter-identified zygosity or with "personalized" driver mutations from pediatric glioma. MADR is extensible to thousands of existing mouse lines, providing a flexible platform to democratize the generation of somatic mosaic mice. VIDEO ABSTRACT.
Asunto(s)
Neoplasias Encefálicas/genética , Modelos Animales de Enfermedad , Marcación de Gen/métodos , Sitios Genéticos/genética , Glioma/genética , Mutagénesis Insercional/métodos , Transgenes/genética , Animales , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células-Madre Neurales/metabolismo , Recombinasas/metabolismo , TransfecciónRESUMEN
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
Development of effective targeted cancer therapies is fundamentally limited by our molecular understanding of disease pathogenesis. Diffuse intrinsic pontine glioma (DIPG) is a fatal malignancy of the childhood pons characterized by a unique substitution to methionine in histone H3 at lysine 27 (H3K27M) that results in globally altered epigenetic marks and oncogenic transcription. Through primary DIPG tumor characterization and isogenic oncohistone expression, we show that the same H3K27M mutation displays distinct modes of oncogenic reprogramming and establishes distinct enhancer architecture depending upon both the variant of histone H3 and the cell context in which the mutation occurs. Compared with non-malignant pediatric pontine tissue, we identify and functionally validate both shared and variant-specific pathophysiology. Altogether, we provide a powerful resource of epigenomic data in 25 primary DIPG samples and 5 rare normal pediatric pontine tissue samples, revealing clinically relevant functional distinctions previously unidentified in DIPG.
Asunto(s)
Glioma Pontino Intrínseco Difuso/genética , Histonas/genética , Encéfalo/patología , Neoplasias Encefálicas/genética , Reprogramación Celular/genética , Glioma Pontino Intrínseco Difuso/metabolismo , Elementos de Facilitación Genéticos/genética , Epigénesis Genética/genética , Epigenómica , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Glioma/genética , Glioma/metabolismo , Humanos , Lisina/genética , Mutación/genética , Puente/metabolismo , Transducción de Señal , Transcriptoma/fisiologíaRESUMEN
ABSTRACT: The CD161 inhibitory receptor is highly upregulated by tumor-infiltrating T cells in multiple human solid tumor types, and its ligand, CLEC2D, is expressed by both tumor cells and infiltrating myeloid cells. Here, we assessed the role of the CD161 receptor in hematological malignancies. Systematic analysis of CLEC2D expression using the Cancer Cell Line Encyclopedia revealed that CLEC2D messenger RNA was most abundant in hematological malignancies, including B-cell and T-cell lymphomas as well as lymphocytic and myelogenous leukemias. CLEC2D protein was detected by flow cytometry on a panel of cell lines representing a diverse set of hematological malignancies. We, therefore, used yeast display to generate a panel of high-affinity, fully human CD161 monoclonal antibodies (mAbs) that blocked CLEC2D binding. These mAbs were specific for CD161 and had a similar affinity for human and nonhuman primate CD161, a property relevant for clinical translation. A high-affinity CD161 mAb enhanced key aspects of T-cell function, including cytotoxicity, cytokine production, and proliferation, against B-cell lines originating from patients with acute lymphoblastic leukemia, diffuse large B-cell lymphoma, and Burkitt lymphoma. In humanized mouse models, this CD161 mAb enhanced T-cell-mediated immunity, resulting in a significant survival benefit. Single cell RNA-seq data demonstrated that CD161 mAb treatment enhanced expression of cytotoxicity genes by CD4 T cells as well as a tissue-residency program by CD4 and CD8 T cells that is associated with favorable survival outcomes in multiple human cancer types. These fully human mAbs, thus, represent potential immunotherapy agents for hematological malignancies.
Asunto(s)
Neoplasias Hematológicas , Neoplasias , Animales , Ratones , Humanos , Linfocitos T CD4-Positivos , Inmunidad Celular , Linfocitos T CD8-positivos , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/terapia , Subfamilia B de Receptores Similares a Lectina de Células NK/genéticaRESUMEN
Immune checkpoint blockers (ICBs) have failed in all phase III glioblastoma trials. Here, we found that ICBs induce cerebral edema in some patients and mice with glioblastoma. Through single-cell RNA sequencing, intravital imaging, and CD8+ T cell blocking studies in mice, we demonstrated that this edema results from an inflammatory response following antiprogrammed death 1 (PD1) antibody treatment that disrupts the blood-tumor barrier. Used in lieu of immunosuppressive corticosteroids, the angiotensin receptor blocker losartan prevented this ICB-induced edema and reprogrammed the tumor microenvironment, curing 20% of mice which increased to 40% in combination with standard of care treatment. Using a bihemispheric tumor model, we identified a "hot" tumor immune signature prior to losartan+anti-PD1 therapy that predicted long-term survival. Our findings provide the rationale and associated biomarkers to test losartan with ICBs in glioblastoma patients.
Asunto(s)
Glioblastoma , Animales , Ratones , Glioblastoma/patología , Losartán/farmacología , Losartán/uso terapéutico , Inhibidores de Puntos de Control Inmunológico/efectos adversos , Linfocitos T CD8-positivos , Edema , Microambiente TumoralRESUMEN
High-grade gliomas are lethal brain cancers whose progression is robustly regulated by neuronal activity. Activity-regulated release of growth factors promotes glioma growth, but this alone is insufficient to explain the effect that neuronal activity exerts on glioma progression. Here we show that neuron and glioma interactions include electrochemical communication through bona fide AMPA receptor-dependent neuron-glioma synapses. Neuronal activity also evokes non-synaptic activity-dependent potassium currents that are amplified by gap junction-mediated tumour interconnections, forming an electrically coupled network. Depolarization of glioma membranes assessed by in vivo optogenetics promotes proliferation, whereas pharmacologically or genetically blocking electrochemical signalling inhibits the growth of glioma xenografts and extends mouse survival. Emphasizing the positive feedback mechanisms by which gliomas increase neuronal excitability and thus activity-regulated glioma growth, human intraoperative electrocorticography demonstrates increased cortical excitability in the glioma-infiltrated brain. Together, these findings indicate that synaptic and electrical integration into neural circuits promotes glioma progression.
Asunto(s)
Encéfalo/fisiopatología , Sinapsis Eléctricas/patología , Fenómenos Electrofisiológicos , Glioma/fisiopatología , Animales , Encéfalo/citología , Membrana Celular/patología , Proliferación Celular , Uniones Comunicantes/patología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Ratones , Ratones Endogámicos NOD , Neuronas/patología , Optogenética , Potasio/metabolismo , Transmisión Sináptica , Células Tumorales CultivadasRESUMEN
Medulloblastoma is a malignant childhood cerebellar tumour type that comprises distinct molecular subgroups. Whereas genomic characteristics of these subgroups are well defined, the extent to which cellular diversity underlies their divergent biology and clinical behaviour remains largely unexplored. Here we used single-cell transcriptomics to investigate intra- and intertumoral heterogeneity in 25 medulloblastomas spanning all molecular subgroups. WNT, SHH and Group 3 tumours comprised subgroup-specific undifferentiated and differentiated neuronal-like malignant populations, whereas Group 4 tumours consisted exclusively of differentiated neuronal-like neoplastic cells. SHH tumours closely resembled granule neurons of varying differentiation states that correlated with patient age. Group 3 and Group 4 tumours exhibited a developmental trajectory from primitive progenitor-like to more mature neuronal-like cells, the relative proportions of which distinguished these subgroups. Cross-species transcriptomics defined distinct glutamatergic populations as putative cells-of-origin for SHH and Group 4 subtypes. Collectively, these data provide insights into the cellular and developmental states underlying subtype-specific medulloblastoma biology.
Asunto(s)
Genómica , Meduloblastoma/genética , Meduloblastoma/patología , Análisis de la Célula Individual , Transcriptoma , Adolescente , Adulto , Animales , Linaje de la Célula , Cerebelo/metabolismo , Cerebelo/patología , Niño , Preescolar , Variaciones en el Número de Copia de ADN , Regulación Neoplásica de la Expresión Génica , Ácido Glutámico/metabolismo , Humanos , Lactante , Meduloblastoma/clasificación , Ratones , Neuronas/metabolismo , Neuronas/patologíaRESUMEN
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
The longitudinal transition of phenotypes is pivotal in glioblastoma treatment resistance and DNA methylation emerged as an important tool for classifying glioblastoma phenotypes. We aimed to characterize DNA methylation subclass heterogeneity during progression and assess its clinical impact. Matched tissues from 47 glioblastoma patients were subjected to DNA methylation profiling, including CpG-site alterations, tissue and serum deconvolution, mass spectrometry, and immunoassay. Effects of clinical characteristics on temporal changes and outcomes were studied. Among 47 patients, 8 (17.0%) had non-matching classifications at recurrence. In the remaining 39 cases, 28.2% showed dominant DNA methylation subclass transitions, with 72.7% being a mesenchymal subclass. In general, glioblastomas with a subclass transition showed upregulated metabolic processes. Newly diagnosed glioblastomas with mesenchymal transition displayed increased stem cell-like states and decreased immune components at diagnosis and exhibited elevated immune signatures and cytokine levels in serum. In contrast, tissue of recurrent glioblastomas with mesenchymal transition showed increased immune components but decreased stem cell-like states. Survival analyses revealed comparable outcomes for patients with and without subclass transitions. This study demonstrates a temporal heterogeneity of DNA methylation subclasses in 28.2% of glioblastomas, not impacting patient survival. Changes in cell state composition associated with subclass transition may be crucial for recurrent glioblastoma targeted therapies.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/genética , Glioblastoma/terapia , Metilación de ADN , Recurrencia Local de Neoplasia/genética , Análisis de SupervivenciaRESUMEN
Gain-of-function IDH mutations are initiating events that define major clinical and prognostic classes of gliomas. Mutant IDH protein produces a new onco-metabolite, 2-hydroxyglutarate, which interferes with iron-dependent hydroxylases, including the TET family of 5'-methylcytosine hydroxylases. TET enzymes catalyse a key step in the removal of DNA methylation. IDH mutant gliomas thus manifest a CpG island methylator phenotype (G-CIMP), although the functional importance of this altered epigenetic state remains unclear. Here we show that human IDH mutant gliomas exhibit hypermethylation at cohesin and CCCTC-binding factor (CTCF)-binding sites, compromising binding of this methylation-sensitive insulator protein. Reduced CTCF binding is associated with loss of insulation between topological domains and aberrant gene activation. We specifically demonstrate that loss of CTCF at a domain boundary permits a constitutive enhancer to interact aberrantly with the receptor tyrosine kinase gene PDGFRA, a prominent glioma oncogene. Treatment of IDH mutant gliomaspheres with a demethylating agent partially restores insulator function and downregulates PDGFRA. Conversely, CRISPR-mediated disruption of the CTCF motif in IDH wild-type gliomaspheres upregulates PDGFRA and increases proliferation. Our study suggests that IDH mutations promote gliomagenesis by disrupting chromosomal topology and allowing aberrant regulatory interactions that induce oncogene expression.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Glioma/enzimología , Glioma/genética , Elementos Aisladores/genética , Isocitrato Deshidrogenasa/genética , Mutación/genética , Oncogenes/genética , Secuencia de Bases , Sitios de Unión , Factor de Unión a CCCTC , Sistemas CRISPR-Cas/genética , Proteínas de Ciclo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/efectos de los fármacos , Células Cultivadas , Cromatina/efectos de los fármacos , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Islas de CpG/genética , Metilación de ADN/efectos de los fármacos , Metilación de ADN/genética , Regulación hacia Abajo/efectos de los fármacos , Elementos de Facilitación Genéticos/genética , Epigénesis Genética/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glioma/tratamiento farmacológico , Glioma/patología , Glutaratos/metabolismo , Humanos , Elementos Aisladores/efectos de los fármacos , Isocitrato Deshidrogenasa/química , Isocitrato Deshidrogenasa/metabolismo , Fenotipo , Unión Proteica , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Proteínas Represoras/metabolismo , Regulación hacia Arriba , CohesinasRESUMEN
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.
Asunto(s)
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
Brain tumor-initiating cells (BTICs) have been identified as key contributors to therapy resistance, recurrence, and progression of diffuse gliomas, particularly glioblastoma (GBM). BTICs are elusive therapeutic targets that reside across the blood-brain barrier, underscoring the urgent need to develop novel therapeutic strategies. Additionally, intratumoral heterogeneity and adaptations to therapeutic pressure by BTICs impede the discovery of effective anti-BTIC therapies and limit the efficacy of individual gene targeting. Recent discoveries in the genetic and epigenetic determinants of BTIC tumorigenesis offer novel opportunities for RNAi-mediated targeting of BTICs. Here we show that BTIC growth arrest in vitro and in vivo is accomplished via concurrent siRNA knockdown of four transcription factors (SOX2, OLIG2, SALL2, and POU3F2) that drive the proneural BTIC phenotype delivered by multiplexed siRNA encapsulation in the lipopolymeric nanoparticle 7C1. Importantly, we demonstrate that 7C1 nano-encapsulation of multiplexed RNAi is a viable BTIC-targeting strategy when delivered directly in vivo in an established mouse brain tumor. Therapeutic potential was most evident via a convection-enhanced delivery method, which shows significant extension of median survival in two patient-derived BTIC xenograft mouse models of GBM. Our study suggests that there is potential advantage in multiplexed targeting strategies for BTICs and establishes a flexible nonviral gene therapy platform with the capacity to channel multiplexed RNAi schemes to address the challenges posed by tumor heterogeneity.
Asunto(s)
Glioblastoma/patología , Nanopartículas/uso terapéutico , Interferencia de ARN , Animales , Carcinogénesis/genética , Resistencia a Antineoplásicos , Femenino , Terapia Genética/métodos , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Humanos , Masculino , Ratones , Ratones Desnudos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Growth of numerous cancer types is believed to be driven by a subpopulation of poorly differentiated cells, often referred to as cancer stem cells (CSCs), that have the capacity for self-renewal, tumor initiation, and generation of nontumorigenic progeny. Despite their potentially key role in tumor establishment and maintenance, the energy requirements of these cells and the mechanisms that regulate their energy production are unknown. Here, we show that the oncofetal insulin-like growth factor 2 mRNA-binding protein 2 (IMP2, IGF2BP2) regulates oxidative phosphorylation (OXPHOS) in primary glioblastoma (GBM) sphere cultures (gliomaspheres), an established in vitro model for CSC expansion. We demonstrate that IMP2 binds several mRNAs that encode mitochondrial respiratory chain complex subunits and that it interacts with complex I (NADH:ubiquinone oxidoreductase) proteins. Depletion of IMP2 in gliomaspheres decreases their oxygen consumption rate and both complex I and complex IV activity that results in impaired clonogenicity in vitro and tumorigenicity in vivo. Importantly, inhibition of OXPHOS but not of glycolysis abolishes GBM cell clonogenicity. Our observations suggest that gliomaspheres depend on OXPHOS for their energy production and survival and that IMP2 expression provides a key mechanism to ensure OXPHOS maintenance by delivering respiratory chain subunit-encoding mRNAs to mitochondria and contributing to complex I and complex IV assembly.