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Tumors may contain billions of cells, including distinct malignant clones and nonmalignant cell types. Clarifying the evolutionary histories, prevalence, and defining molecular features of these cells is essential for improving clinical outcomes, since intratumoral heterogeneity provides fuel for acquired resistance to targeted therapies. Here we present a statistically motivated strategy for deconstructing intratumoral heterogeneity through multiomic and multiscale analysis of serial tumor sections (MOMA). By combining deep sampling of IDH-mutant astrocytomas with integrative analysis of single-nucleotide variants, copy-number variants, and gene expression, we reconstruct and validate the phylogenies, spatial distributions, and transcriptional profiles of distinct malignant clones. By genotyping nuclei analyzed by single-nucleus RNA-seq for truncal mutations, we further show that commonly used algorithms for identifying cancer cells from single-cell transcriptomes may be inaccurate. We also demonstrate that correlating gene expression with tumor purity in bulk samples can reveal optimal markers of malignant cells and use this approach to identify a core set of genes that are consistently expressed by astrocytoma truncal clones, including AKR1C3, whose expression is associated with poor outcomes in several types of cancer. In summary, MOMA provides a robust and flexible strategy for precisely deconstructing intratumoral heterogeneity and clarifying the core molecular properties of distinct cellular populations in solid tumors.
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Neurogenesis and gliogenesis continue in the Ventricular-Subventricular Zone (V-SVZ) of the adult rodent brain. B1 cells are astroglial cells derived from radial glia that function as primary progenitors or neural stem cells (NSCs) in the V-SVZ. B1 cells, which have a small apical contact with the ventricle, decline in numbers during early postnatal life, yet neurogenesis continues into adulthood. Here we found that a second population of V-SVZ astroglial cells (B2 cells), that do not contact the ventricle, function as NSCs in the adult brain. B2 cell numbers increase postnatally, remain constant in 12-month-old mice and decrease by 18 months. Transcriptomic analysis of ventricular-contacting and non-contacting B cells revealed key molecular differences to distinguish B1 from B2 cells. Transplantation and lineage tracing of B2 cells demonstrate their function as primary progenitors for adult neurogenesis. This study reveals how NSC function is relayed from B1 to B2 progenitors to maintain adult neurogenesis.
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Tumors may contain billions of cells including distinct malignant clones and nonmalignant cell types. Clarifying the evolutionary histories, prevalence, and defining molecular features of these cells is essential for improving clinical outcomes, since intratumoral heterogeneity provides fuel for acquired resistance to targeted therapies. Here we present a statistically motivated strategy for deconstructing intratumoral heterogeneity through multiomic and multiscale analysis of serial tumor sections (MOMA). By combining deep sampling of IDH-mutant astrocytomas with integrative analysis of single-nucleotide variants, copy-number variants, and gene expression, we reconstruct and validate the phylogenies, spatial distributions, and transcriptional profiles of distinct malignant clones. By genotyping nuclei analyzed by single-nucleus RNA-seq for truncal mutations, we further show that commonly used algorithms for identifying cancer cells from single-cell transcriptomes may be inaccurate. We also demonstrate that correlating gene expression with tumor purity in bulk samples can reveal optimal markers of malignant cells and use this approach to identify a core set of genes that is consistently expressed by astrocytoma truncal clones, including AKR1C3, whose expression is associated with poor outcomes in several types of cancer. In summary, MOMA provides a robust and flexible strategy for precisely deconstructing intratumoral heterogeneity and clarifying the core molecular properties of distinct cellular populations in solid tumors.
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Adeno-associated virus (AAV)-based gene therapies, exemplified by the approved therapy for spinal muscular atrophy, have the potential to deliver disease-course-altering treatments for central nervous system (CNS) indications. However, several clinical trials have reported severe adverse events, including patient deaths following high-dose systemic administration for muscle-directed gene transfer, highlighting the need to explore approaches utilizing lower doses when targeting the CNS. Animal models of disease provide insight into the response to new AAV therapies. However, translation from small to larger animals and eventually to humans is hampered by anatomical and biological differences across the species and their impact on AAV delivery. We performed a literature review of preclinical studies of AAV gene therapy biodistribution following cerebrospinal fluid (CSF) delivery (intracerebroventricular, intra-cisterna magna, and intrathecal lumbar). The reviewed literature varies greatly in the reported biodistribution of AAV following administration into the CSF. Differences between studies, including animal model, vector serotype used, method used to assess biodistribution, and route of administration, among other variables, contribute to differing outcomes and difficulties in translating these preclinical results. For example, only half of the published AAV-based gene therapy studies report vector copy number, the most direct readout following administration of a vector; none of these studies reported details such as the empty:full capsid ratio and quality of encapsidated genome. Analysis of the last decade's literature focusing on AAV-based gene therapies targeting the CNS underscores limitations of the body of knowledge and room for continued research. In particular, there is a need to understand the biodistribution achieved by different CSF-directed routes of administration and determining if specific cell types/structures of interest will be transduced. Our findings point to a clear need for a more systematic approach across the field to align the assessments and elements reported in preclinical research to enable more reliable translation across animal models and into human studies.
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Dependovirus , Terapia Genética , Animais , Humanos , Dependovirus/genética , Distribuição Tecidual , Terapia Genética/métodos , Sistema Nervoso Central , Modelos Animais , Vetores Genéticos/genética , Técnicas de Transferência de GenesRESUMO
The chromatin remodeler CHD8 represents a high-confidence risk factor in autism, a multistage progressive neurologic disorder, however the underlying stage-specific functions remain elusive. In this study, by analyzing Chd8 conditional knock-out mice (male and female), we find that CHD8 controls cortical neural stem/progenitor cell (NSC) proliferation and survival in a stage-dependent manner. Strikingly, inducible genetic deletion reveals that CHD8 is required for the production and fitness of transit-amplifying intermediate progenitors (IPCs) essential for upper-layer neuron expansion in the embryonic cortex. p53 loss of function partially rescues apoptosis and neurogenesis defects in the Chd8-deficient brain. Further, transcriptomic and epigenomic profiling indicates that CHD8 regulates the chromatin accessibility landscape to activate neurogenesis-promoting factors including TBR2, a key regulator of IPC neurogenesis, while repressing DNA damage- and p53-induced apoptotic programs. In the adult brain, CHD8 depletion impairs forebrain neurogenesis by impeding IPC differentiation from NSCs in both subventricular and subgranular zones; however, unlike in embryos, it does not affect NSC proliferation and survival. Treatment with an antidepressant approved by the Federal Drug Administration (FDA), fluoxetine, partially restores adult hippocampal neurogenesis in Chd8-ablated mice. Together, our multistage functional studies identify temporally specific roles for CHD8 in developmental and adult neurogenesis, pointing to a potential strategy to enhance neurogenesis in the CHD8-deficient brain.SIGNIFICANCE STATEMENT The role of the high-confidence autism gene CHD8 in neurogenesis remains incompletely understood. Here, we identify a stage-specific function of CHD8 in development of NSCs in developing and adult brains by conserved, yet spatiotemporally distinct, mechanisms. In embryonic cortex, CHD8 is critical for the proliferation, survival, and differentiation of both NSC and IPCs during cortical neurogenesis. In adult brain, CHD8 is required for IPC generation but not the proliferation and survival of adult NSCs. Treatment with FDA-approved antidepressant fluoxetine partially rescues the adult neurogenesis defects in CHD8 mutants. Thus, our findings help resolve CHD8 functions throughout life during embryonic and adult neurogenesis and point to a potential avenue to promote neurogenesis in CHD8 deficiency.
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Transtorno Autístico , Cromatina , Proteínas de Ligação a DNA , Neurogênese , Animais , Feminino , Masculino , Camundongos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fluoxetina , Hipocampo/metabolismo , Camundongos Knockout , Neurogênese/fisiologia , Proteína Supressora de Tumor p53 , ProsencéfaloRESUMO
Metastasis is a major contributor to cancer-associated deaths. It is characterized by a multistep process that occurs through the acquisition of molecular and phenotypic changes enabling cancer cells from a primary tumour to disseminate and colonize at distant organ sites. Over the past decade, the discovery and characterization of long noncoding RNAs (lncRNAs) have revealed the diversity of their regulatory roles, including key contributions throughout the metastatic cascade. Here, we review how lncRNAs promote metastasis by functioning in discrete pro-metastatic steps including the epithelial-mesenchymal transition, invasion and migration and organotrophic colonization, and by influencing the metastatic tumour microenvironment, often by interacting within ribonucleoprotein complexes or directly with other nucleic acid entities. We discuss well-characterized lncRNAs with in vivo phenotypes and highlight mechanistic commonalities such as convergence with the TGFß-ZEB1/ZEB2 axis or the nuclear factor-κB pathway, in addition to lncRNAs with controversial mechanisms and the influence of methodologies on mechanistic interpretation. Furthermore, some lncRNAs can help identify tumours with increased metastatic risk and spur novel therapeutic strategies, with several lncRNAs having shown potential as novel targets for antisense oligonucleotide therapy in animal models. In addition to well-characterized examples of lncRNAs functioning in metastasis, we discuss controversies and ongoing challenges in lncRNA biology. Finally, we present areas for future study for this rapidly evolving field.
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Metástase Neoplásica/genética , RNA Longo não Codificante/fisiologia , Animais , Movimento Celular , Transição Epitelial-Mesenquimal , Humanos , Invasividade Neoplásica , Microambiente TumoralRESUMO
Among the large, diverse set of mammalian long noncoding RNAs (lncRNAs), long noncoding primary microRNAs (lnc-pri-miRNAs) are those that host miRNAs. Whether lnc-pri-miRNA loci have important biological function independent of their cognate miRNAs is poorly understood. From a genome-scale lncRNA screen, lnc-pri-miRNA loci were enriched for function in cell proliferation, and in glioblastoma (i.e., GBM) cells with DGCR8 or DROSHA knockdown, lnc-pri-miRNA screen hits still regulated cell growth. To molecularly dissect the function of a lnc-pri-miRNA locus, we studied LOC646329 (also known as MIR29HG), which hosts the miR-29a/b1 cluster. In GBM cells, LOC646329 knockdown reduced miR-29a/b1 levels, and these cells exhibited decreased growth. However, genetic deletion of the miR-29a/b1 cluster (LOC646329-miR29Δ) did not decrease cell growth, while knockdown of LOC646329-miR29Δ transcripts reduced cell proliferation. The miR-29a/b1-independent activity of LOC646329 corresponded to enhancer-like activation of a neighboring oncogene (MKLN1), regulating cell propagation. The LOC646329 locus interacts with the MKLN1 promoter, and antisense oligonucleotide knockdown of the lncRNA disrupts these interactions and reduces the enhancer-like activity. More broadly, analysis of genome-wide data from multiple human cell types showed that lnc-pri-miRNA loci are significantly enriched for DNA looping interactions with gene promoters as well as genomic and epigenetic characteristics of transcriptional enhancers. Functional studies of additional lnc-pri-miRNA loci demonstrated cognate miRNA-independent enhancer-like activity. Together, these data demonstrate that lnc-pri-miRNA loci can regulate cell biology via both miRNA-dependent and miRNA-independent mechanisms.
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Proliferação de Células/genética , Loci Gênicos , RNA Longo não Codificante/metabolismo , Apoptose/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Longo não Codificante/genética , RNA-SeqRESUMO
INTRODUCTION: During deep brain stimulation (DBS) surgery, computed tomography (CT) and magnetic resonance imaging (MRI) scans need to be co-registered or fused. Image fusion is associated with the error that can distort the location of anatomical structures. Co-registration in DBS surgery is usually performed automatically by proprietary software; the amount of error during this process is not well understood. Here, our goal is to quantify the error during automated image co-registration with FrameLink™, a commonly used software for DBS planning and clinical research. METHODS: This is a single-center retrospective study at a quaternary care referral center, comparing CT and MR imaging co-registration for a consecutive series of patients over a 12-month period. We collected CT images and MRI scans for 22 patients with Parkinson's disease requiring placement of DBS. Anatomical landmarks were located on CT images and MRI scans using a novel image analysis algorithm that included a method for capturing the potential error inherent in the image standardization step of the analysis. The distance between the anatomical landmarks was measured, and the error was found by averaging the distances across all patients. RESULTS: The average error during co-registration was 1.25 mm. This error was significantly larger than the error resulting from image standardization (0.19 mm) and was worse in the anterior-posterior direction. CONCLUSIONS: The image fusion errors found in this analysis were nontrivial. Although the estimated error may be inflated, it is sig-nificant enough that users must be aware of this potential inaccuracy, and developers of proprietary software should provide details about the magnitude and direction of co-registration errors.
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Estimulação Encefálica Profunda , Humanos , Imageamento por Ressonância Magnética , Estudos Retrospectivos , Software , Tomografia Computadorizada por Raios XRESUMO
CRISPR-mediated interference (CRISPRi), a robust and specific system for programmably repressing transcription, provides a versatile tool for systematically characterizing the function of long noncoding RNAs (lncRNAs). When used with highly parallel, lentiviral pooled screening approaches, CRISPRi enables the targeted knockdown of tens of thousands of lncRNA-expressing loci in a single screen. Here we describe the use of CRISPRi to target lncRNA loci in a pooled screen, using cell growth and proliferation as an example of a phenotypic readout. Considerations for custom lncRNA-targeting libraries, alternative phenotypic readouts, and orthogonal validation approaches are also discussed.
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Técnicas de Silenciamento de Genes/métodos , Lentivirus/fisiologia , RNA Longo não Codificante/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proliferação de Células , Células HEK293 , Humanos , Lentivirus/genética , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
Meningiomas are the most common primary intracranial tumors, but the molecular drivers of meningioma tumorigenesis are poorly understood. We hypothesized that investigating intratumor heterogeneity in meningiomas would elucidate biologic drivers and reveal new targets for molecular therapy. To test this hypothesis, here we perform multiplatform molecular profiling of 86 spatially-distinct samples from 13 human meningiomas. Our data reveal that regional alterations in chromosome structure underlie clonal transcriptomic, epigenomic, and histopathologic signatures in meningioma. Stereotactic co-registration of sample coordinates to preoperative magnetic resonance images further suggest that high apparent diffusion coefficient (ADC) distinguishes meningioma regions with proliferating cells enriched for developmental gene expression programs. To understand the function of these genes in meningioma, we develop a human cerebral organoid model of meningioma and validate the high ADC marker genes CDH2 and PTPRZ1 as potential targets for meningioma therapy using live imaging, single cell RNA sequencing, CRISPR interference, and pharmacology.
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Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Perfilação da Expressão Gênica/métodos , Heterogeneidade Genética , Imageamento por Ressonância Magnética/métodos , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/metabolismo , Idoso , Antígenos CD/genética , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/patologia , Caderinas/genética , Imagem de Difusão por Ressonância Magnética/métodos , Epigenômica , Feminino , Marcadores Genéticos , Genômica , Humanos , Neoplasias Meníngeas/diagnóstico por imagem , Neoplasias Meníngeas/patologia , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/genética , TranscriptomaRESUMO
Neural stem cells (NSCs) in the developing and postnatal brain have distinct positional identities that dictate the types of neurons they generate. Although morphogens initially establish NSC positional identity in the neural tube, it is unclear how such regional differences are maintained as the forebrain grows much larger and more anatomically complex. We found that the maintenance of NSC positional identity in the murine brain requires a mixed-lineage leukemia 1 (Mll1)-dependent epigenetic memory system. After establishment by sonic hedgehog, ventral NSC identity became independent of this morphogen. Even transient MLL1 inhibition caused a durable loss of ventral identity, resulting in the generation of neurons with the characteristics of dorsal NSCs in vivo. Thus, spatial information provided by morphogens can be transitioned to epigenetic mechanisms that maintain regionally distinct developmental programs in the forebrain.
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Impressão Genômica , Histona-Lisina N-Metiltransferase/fisiologia , Proteína de Leucina Linfoide-Mieloide/fisiologia , Células-Tronco Neurais/fisiologia , Neurogênese/genética , Prosencéfalo/citologia , Prosencéfalo/embriologia , Fator Nuclear 1 de Tireoide/genética , Animais , Proteínas Hedgehog/metabolismo , Histona-Lisina N-Metiltransferase/genética , Camundongos , Camundongos Mutantes , Proteína de Leucina Linfoide-Mieloide/genética , Células-Tronco Neurais/citologia , TranscriptomaRESUMO
BACKGROUND: Long non-coding RNAs (lncRNAs) exhibit highly cell type-specific expression and function, making this class of transcript attractive for targeted cancer therapy. However, the vast majority of lncRNAs have not been tested as potential therapeutic targets, particularly in the context of currently used cancer treatments. Malignant glioma is rapidly fatal, and ionizing radiation is part of the current standard-of-care used to slow tumor growth in both adult and pediatric patients. RESULTS: We use CRISPR interference (CRISPRi) to screen 5689 lncRNA loci in human glioblastoma (GBM) cells, identifying 467 hits that modify cell growth in the presence of clinically relevant doses of fractionated radiation. Thirty-three of these lncRNA hits sensitize cells to radiation, and based on their expression in adult and pediatric gliomas, nine of these hits are prioritized as lncRNA Glioma Radiation Sensitizers (lncGRS). Knockdown of lncGRS-1, a primate-conserved, nuclear-enriched lncRNA, inhibits the growth and proliferation of primary adult and pediatric glioma cells, but not the viability of normal brain cells. Using human brain organoids comprised of mature neural cell types as a three-dimensional tissue substrate to model the invasive growth of glioma, we find that antisense oligonucleotides targeting lncGRS-1 selectively decrease tumor growth and sensitize glioma cells to radiation therapy. CONCLUSIONS: These studies identify lncGRS-1 as a glioma-specific therapeutic target and establish a generalizable approach to rapidly identify novel therapeutic targets in the vast non-coding genome to enhance radiation therapy.
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Neoplasias Encefálicas/terapia , Sistemas CRISPR-Cas , Glioblastoma/terapia , RNA Longo não Codificante/antagonistas & inibidores , Adulto , Astrócitos , Encéfalo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/radioterapia , Linhagem Celular Tumoral , Terapia Combinada , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/radioterapia , Humanos , Oligonucleotídeos Antissenso , Organoides , Tolerância a RadiaçãoRESUMO
Four boys with Pelizaeus-Merzbacher disease, an X-linked leukodystrophy, underwent transplantation with human allogeneic central nervous system stem cells (HuCNS-SC). Subsequently, all subjects were followed for an additional 4 years in this separate follow-up study to evaluate safety, neurologic function, magnetic resonance imaging (MRI) data, and immunologic response. The neurosurgical procedure, immunosuppression, and HuCNS-SC transplantation were well tolerated and all four subjects were alive at the conclusion of the study period. At year 2, all subjects exhibited diffusion MRI changes at the implantation sites as well as in more distant brain regions. There were persistent, increased signal changes in the three patients who were studied up to year 5. Two of four subjects developed donor-specific HLA alloantibodies, demonstrating that neural stem cells can elicit an immune response when injected into the CNS, and suggesting the importance of monitoring immunologic parameters and identifying markers of engraftment in future studies.
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Encéfalo/diagnóstico por imagem , Células-Tronco Neurais/transplante , Doença de Pelizaeus-Merzbacher/terapia , Encéfalo/fisiologia , Pré-Escolar , Seguimentos , Antígenos HLA/imunologia , Humanos , Lactente , Isoanticorpos/sangue , Imageamento por Ressonância Magnética , Masculino , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Doença de Pelizaeus-Merzbacher/imunologia , Doença de Pelizaeus-Merzbacher/patologia , Índice de Gravidade de Doença , Transplante de Células-Tronco/efeitos adversos , Transplante Homólogo , Resultado do TratamentoRESUMO
Motivation: Single-cell RNA-sequencing (scRNA-seq) has enabled studies of tissue composition at unprecedented resolution. However, the application of scRNA-seq to clinical cancer samples has been limited, partly due to a lack of scRNA-seq algorithms that integrate genomic mutation data. Results: To address this, we present. CONICS: COpy-Number analysis In single-Cell RNA-Sequencing. CONICS is a software tool for mapping gene expression from scRNA-seq to tumor clones and phylogenies, with routines enabling: the quantitation of copy-number alterations in scRNA-seq, robust separation of neoplastic cells from tumor-infiltrating stroma, inter-clone differential-expression analysis and intra-clone co-expression analysis. Availability and implementation: CONICS is written in Python and R, and is available from https://github.com/diazlab/CONICS. Supplementary information: Supplementary data are available at Bioinformatics online.
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Perfilação da Expressão Gênica/métodos , Neoplasias/genética , RNA Citoplasmático Pequeno , Análise de Célula Única/métodos , Software , Algoritmos , Humanos , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodosRESUMO
Noncoding mutations in cancer genomes are frequent but challenging to interpret. PVT1 encodes an oncogenic lncRNA, but recurrent translocations and deletions in human cancers suggest alternative mechanisms. Here, we show that the PVT1 promoter has a tumor-suppressor function that is independent of PVT1 lncRNA. CRISPR interference of PVT1 promoter enhances breast cancer cell competition and growth in vivo. The promoters of the PVT1 and the MYC oncogenes, located 55 kb apart on chromosome 8q24, compete for engagement with four intragenic enhancers in the PVT1 locus, thereby allowing the PVT1 promoter to regulate pause release of MYC transcription. PVT1 undergoes developmentally regulated monoallelic expression, and the PVT1 promoter inhibits MYC expression only from the same chromosome via promoter competition. Cancer genome sequencing identifies recurrent mutations encompassing the human PVT1 promoter, and genome editing verified that PVT1 promoter mutation promotes cancer cell growth. These results highlight regulatory sequences of lncRNA genes as potential disease-associated DNA elements.
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Neoplasias da Mama/genética , Regulação Neoplásica da Expressão Gênica , Genes myc , RNA Longo não Codificante/genética , Animais , Neoplasias da Mama/metabolismo , Sistemas CRISPR-Cas , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Cromatina , DNA de Neoplasias/genética , Elementos Facilitadores Genéticos , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Mutação , Transplante de Neoplasias , Regiões Promotoras Genéticas , RNA Longo não Codificante/metabolismo , Transcrição GênicaRESUMO
BACKGROUND: Tumor-associated macrophages (TAMs) are abundant in gliomas and immunosuppressive TAMs are a barrier to emerging immunotherapies. It is unknown to what extent macrophages derived from peripheral blood adopt the phenotype of brain-resident microglia in pre-treatment gliomas. The relative proportions of blood-derived macrophages and microglia have been poorly quantified in clinical samples due to a paucity of markers that distinguish these cell types in malignant tissue. RESULTS: We perform single-cell RNA-sequencing of human gliomas and identify phenotypic differences in TAMs of distinct lineages. We isolate TAMs from patient biopsies and compare them with macrophages from non-malignant human tissue, glioma atlases, and murine glioma models. We present a novel signature that distinguishes TAMs by ontogeny in human gliomas. Blood-derived TAMs upregulate immunosuppressive cytokines and show an altered metabolism compared to microglial TAMs. They are also enriched in perivascular and necrotic regions. The gene signature of blood-derived TAMs, but not microglial TAMs, correlates with significantly inferior survival in low-grade glioma. Surprisingly, TAMs frequently co-express canonical pro-inflammatory (M1) and alternatively activated (M2) genes in individual cells. CONCLUSIONS: We conclude that blood-derived TAMs significantly infiltrate pre-treatment gliomas, to a degree that varies by glioma subtype and tumor compartment. Blood-derived TAMs do not universally conform to the phenotype of microglia, but preferentially express immunosuppressive cytokines and show an altered metabolism. Our results argue against status quo therapeutic strategies that target TAMs indiscriminately and in favor of strategies that specifically target immunosuppressive blood-derived TAMs.
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Glioma/genética , Glioma/patologia , Ativação de Macrófagos/genética , Macrófagos/metabolismo , Macrófagos/patologia , Microambiente Tumoral/genética , Animais , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Glioma/imunologia , Glioma/terapia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imunoterapia/métodos , Ativação de Macrófagos/imunologia , Camundongos , Prognóstico , Análise de Célula Única , Análise de Sobrevida , Transcriptoma , Microambiente Tumoral/imunologiaRESUMO
Neural stem cells (B1 astrocytes; NSCs) in the adult ventricular-subventricular-zone (V-SVZ) originate in the embryo. Surprisingly, recent work has shown that B1 cells remain largely quiescent. They are reactivated postnatally to function as primary progenitors for neurons destined for the olfactory bulb and some corpus callosum oligodendrocytes. The cellular and molecular properties of quiescent B1 cells remain unknown. Here we found that a subpopulation of B1 cells has a unique nuclear envelope invagination specialization similar to envelope-limited chromatin sheets (ELCS), reported in certain lymphocytes and some cancer cells. Using molecular markers, [3H]thymidine birth-dating, and Ara-C, we found that B1 cells with ELCS correspond to quiescent NSCs. ELCS begin forming in embryonic radial glia cells and represent a specific nuclear compartment containing particular epigenetic modifications and telomeres. These results reveal a unique nuclear compartment in quiescent NSCs, which is useful for identifying these primary progenitors and study their gene regulation.
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Ventrículos Laterais/citologia , Células-Tronco Neurais/citologia , Membrana Nuclear/ultraestrutura , Células-Tronco Adultas/citologia , Animais , Astrócitos/citologia , Ciclo Celular , Células Cultivadas , Cromatina/química , CamundongosRESUMO
Glioblastoma multiforme (GBM) is the most common and aggressive type of primary brain tumor. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) receptors are frequently amplified and/or possess gain-of-function mutations in GBM However, clinical trials of tyrosine-kinase inhibitors have shown disappointing efficacy, in part due to intra-tumor heterogeneity. To assess the effect of clonal heterogeneity on gene expression, we derived an approach to map single-cell expression profiles to sequentially acquired mutations identified from exome sequencing. Using 288 single cells, we constructed high-resolution phylogenies of EGF-driven and PDGF-driven GBMs, modeling transcriptional kinetics during tumor evolution. Descending the phylogenetic tree of a PDGF-driven tumor corresponded to a progressive induction of an oligodendrocyte progenitor-like cell type, expressing pro-angiogenic factors. In contrast, phylogenetic analysis of an EGFR-amplified tumor showed an up-regulation of pro-invasive genes. An in-frame deletion in a specific dimerization domain of PDGF receptor correlates with an up-regulation of growth pathways in a proneural GBM and enhances proliferation when ectopically expressed in glioma cell lines. In-frame deletions in this domain are frequent in public GBM data.
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Receptores ErbB/genética , Perfilação da Expressão Gênica/métodos , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Neoplasias Encefálicas , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Heterogeneidade Genética , Glioblastoma , Humanos , MutaçãoRESUMO
A large population of neural stem/precursor cells (NSCs) persists in the ventricular-subventricular zone (V-SVZ) located in the walls of the lateral brain ventricles. V-SVZ NSCs produce large numbers of neuroblasts that migrate a long distance into the olfactory bulb (OB) where they differentiate into local circuit interneurons. Here, we review a broad range of discoveries that have emerged from studies of postnatal V-SVZ neurogenesis: the identification of NSCs as a subpopulation of astroglial cells, the neurogenic lineage, new mechanisms of neuronal migration, and molecular regulators of precursor cell proliferation and migration. It has also become evident that V-SVZ NSCs are regionally heterogeneous, with NSCs located in different regions of the ventricle wall generating distinct OB interneuron subtypes. Insights into the developmental origins and molecular mechanisms that underlie the regional specification of V-SVZ NSCs have also begun to emerge. Other recent studies have revealed new cell-intrinsic molecular mechanisms that enable lifelong neurogenesis in the V-SVZ. Finally, we discuss intriguing differences between the rodent V-SVZ and the corresponding human brain region. The rapidly expanding cellular and molecular knowledge of V-SVZ NSC biology provides key insights into postnatal neural development, the origin of brain tumors, and may inform the development regenerative therapies from cultured and endogenous human neural precursors.
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Células-Tronco Neurais/citologia , Bulbo Olfatório/citologia , Animais , Diferenciação Celular/genética , Linhagem da Célula , Epigênese Genética , Humanos , Interneurônios/citologia , Camundongos , Modelos Biológicos , Neurogênese/genética , Transdução de SinaisRESUMO
In the past decade, thousands of long noncoding RNAs (lncRNAs) have been identified, and emerging data indicate that lncRNAs can have important biological functions and roles in human diseases including cancer. Many lncRNAs appear to be expressed specifically in the brain, and the roles of lncRNAs in neural stem cells (NSCs) and brain development are now beginning to be discovered. Here we review recent advances in understanding the diversity of lncRNA structure and functions in NSCs and brain development. NSCs in the adult mouse ventricular-subventricular zone (V-SVZ) generate new neurons throughout life, and we discuss how key elements of this adult neurogenic system have facilitated the discovery and functional characterization of known and novel lncRNAs. A review of lncRNAs described in other NSC systems reveals a variety of molecular mechanisms, including binding and recruitment of transcription factors, epigenetic modifiers, and RNA-splicing factors. Finally, we review emerging evidence indicating that specific lncRNAs can be key drivers of glial tumors, and discuss next steps towards an in vivo understanding of lncRNA function in development and disease.