RESUMEN
Glioblastoma (GBM) is an immunologically "cold" tumor that does not respond to current immunotherapy. Here, we demonstrate a fundamental role for the α-isoform of the catalytic subunit of protein phosphatase-2A (PP2Ac) in regulating glioma immunogenicity. Genetic ablation of PP2Ac in glioma cells enhanced double-stranded DNA (dsDNA) production and cGAS-type I IFN signaling, MHC-I expression, and tumor mutational burden. In coculture experiments, PP2Ac deficiency in glioma cells promoted dendritic cell (DC) cross-presentation and clonal expansion of CD8+ T cells. In vivo, PP2Ac depletion sensitized tumors to immune-checkpoint blockade and radiotherapy treatment. Single-cell analysis demonstrated that PP2Ac deficiency increased CD8+ T-cell, natural killer cell, and DC accumulation and reduced immunosuppressive tumor-associated macrophages. Furthermore, loss of PP2Ac increased IFN signaling in myeloid and tumor cells and reduced expression of a tumor gene signature associated with worse patient survival in The Cancer Genome Atlas. Collectively, this study establishes a novel role for PP2Ac in inhibiting dsDNA-cGAS-STING signaling to suppress antitumor immunity in glioma. SIGNIFICANCE: PP2Ac deficiency promotes cGAS-STING signaling in glioma to induce a tumor-suppressive immune microenvironment, highlighting PP2Ac as a potential therapeutic target to enhance tumor immunogenicity and improve response to immunotherapy.
Asunto(s)
Glioblastoma , Glioma , Interferón Tipo I , Humanos , Inmunidad Innata , Interferón Tipo I/metabolismo , Nucleotidiltransferasas/genética , Microambiente TumoralRESUMEN
Glioblastoma is believed to originate from nervous system cells; however, a putative origin from vessel-associated progenitor cells has not been considered. We deeply single-cell RNA-sequenced glioblastoma progenitor cells of 18 patients and integrated 710 bulk tumors and 73,495 glioma single cells of 100 patients to determine the relation of glioblastoma cells to normal brain cell types. A novel neural network-based projection of the developmental trajectory of normal brain cells uncovered two principal cell-lineage features of glioblastoma, neural crest perivascular and radial glia, carrying defining methylation patterns and survival differences. Consistently, introducing tumorigenic alterations in naïve human brain perivascular cells resulted in brain tumors. Thus, our results suggest that glioblastoma can arise from the brains' vasculature, and patients with such glioblastoma have a significantly poorer outcome.
RESUMEN
Intratumoral heterogeneity is a characteristic of glioblastomas that contain an intermixture of cell populations displaying different glioblastoma subtype gene expression signatures. Proportions of these populations change during tumor evolution, but the occurrence and regulation of glioblastoma subtype transition is not well described. To identify regulators of glioblastoma subtypes we utilized a combination of in vitro experiments and in silico analyses, using experimentally generated as well as publicly available data. Through this combined approach SOX2 was identified to confer a proneural glioblastoma subtype gene expression signature. SFRP2 was subsequently identified as a SOX2-antagonist, able to induce a mesenchymal glioblastoma subtype signature. A subset of patient glioblastoma samples with high SFRP2 and low SOX2 expression was particularly enriched with mesenchymal subtype samples. Phenotypically, SFRP2 decreased tumor sphere formation, stemness as assessed by limiting dilution assay, and overall cell proliferation but increased cell motility, whereas SOX2 induced the opposite effects. Furthermore, an SFRP2/non-canonical-WNT/KLF4/PDGFR/phospho-AKT/SOX2 signaling axis was found to be involved in the mesenchymal transition. Analysis of human tumor tissue spatial gene expression patterns showed distinct expression of SFRP2- and SOX2-correlated genes in vascular and cellular areas, respectively. Finally, conditioned media from SFRP2 overexpressing cells increased CD206 on macrophages. Together, these findings present SFRP2 as a SOX2-antagonist with the capacity to induce a mesenchymal subtype transition in glioma cells located in vascular tumor areas, highlighting its role in glioblastoma tumor evolution and intratumoral heterogeneity.
Asunto(s)
Transición Epitelial-Mesenquimal/genética , Regulación Neoplásica de la Expresión Génica , Glioblastoma/etiología , Glioblastoma/metabolismo , Proteínas de la Membrana/genética , Factores de Transcripción SOXB1/genética , Proteínas Portadoras , Línea Celular Tumoral , Perfilación de la Expresión Génica , Glioblastoma/patología , Humanos , Factor 4 Similar a Kruppel/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Proteínas de la Membrana/metabolismo , Especificidad de Órganos , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factores de Transcripción SOXB1/metabolismo , Transducción de SeñalRESUMEN
The complexity of glioblastoma multiforme, the most common and lethal variant of gliomas, is reflected by cellular and molecular heterogeneity at both the inter- and intra-tumoural levels. Molecular subtyping has arisen in the past two decades as a promising strategy to give better predictions of glioblastoma multiforme evolution, common disease pathways, and rational treatment options. The Cancer Genome Atlas network initially identified four molecular subtypes of glioblastoma multiforme: proneural, neural, mesenchymal and classical. However, further studies, also investigated glioma stem cells, have only identified two to three subtypes: proneural, mesenchymal and classical. The proneural-mesenchymal transition upon tumour recurrence has been suggested as a mechanism of tumour resistance to radiation and chemotherapy treatment. Glioblastoma multiforme patients with the mesenchymal subtype tend to survive shorter than other subtypes when analysis is restricted to samples with low transcriptional heterogeneity. Although the mesenchymal signature in malignant glioma may seem at odds with the common idea of the ectodermal origin of neural-glial lineages, the presence of the mesenchymal signature in glioma is supported by several studies suggesting that it can result from: (i) intrinsic expression of tumour cells affected with accumulated genetic mutations and cell of origin; (ii) tumour micro-environments with recruited macrophages or microglia, mesenchymal stem cells or pericytes, and other progenitors; (iii) resistance to tumour treatment, including radiotherapy, antiangiogenic therapy and possibly chemotherapy. Genetic abnormalities, mainly NF1 mutations, together with NF-κB transcriptional programs, are the main driver of acquiring mesenchymal-signature. This signature is far from being simply tissue artefacts, as it has been identified in single cell glioma, circulating tumour cells, and glioma stem cells that are released from the tumour micro-environment. All these together suggest that the mesenchymal signature in glioblastoma multiforme is induced and sustained via cell intrinsic mechanisms and tumour micro-environment factors. Although patients with the mesenchymal subtype tend to have poorer prognosis, they may have favourable response to immunotherapy and intensive radio- and chemotherapy.
Asunto(s)
Glioblastoma/genética , Glioblastoma/metabolismo , Células Madre Mesenquimatosas/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Regulación Neoplásica de la Expresión Génica/genética , Glioblastoma/clasificación , Glioma/genética , Humanos , Recurrencia Local de Neoplasia , Células Madre Neoplásicas/patología , Transducción de Señal , Microambiente TumoralRESUMEN
Adult neural progenitor cells (aNPCs) are a potential source for cell based therapy for neurodegenerative diseases and traumatic brain injuries. These cells have been traditionally isolated from hippocampus, subventricular zone and white matter. However, there is still a need for an easily accessible source with better yield to counter the limitations of small surgical samples of previously characterized aNPCs. Here we show that ultrasonic aspirate (UA) samples currently considered as 'biological waste after surgery,' offer a good source for aNPCs. Furthermore, we show that culture conditions dictated the phenotype of cells across patients. The neurosphere-enriched cells were more similar to freshly isolated brain cells, while cells expanded adherently in serum conditions were similar to mesenchymal stem cells. However, cells expanded in these adherent conditions expressed some NPC and glial markers in addition to active canonical Wnt signaling. This suggests a mesenchymal-neuroectodermal hybrid nature of these cells. Finally, we show that UA-NPCs are comparable to those from neurogenic regions. Our findings suggest that UA samples can be used as a source for fresh and in vitro propagated aNPCs that could have various clinical applications.
Asunto(s)
Células Madre Adultas/citología , Células-Madre Neurales/citología , Células Madre Adultas/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Citometría de Flujo , Hipocampo/citología , Humanos , Ventrículos Laterales/citología , Células-Madre Neurales/metabolismo , Neurogénesis/fisiología , Sustancia Blanca/citología , Vía de Señalización Wnt/fisiologíaRESUMEN
Glioma stem cells (GSCs) are thought to be the source of tumor growth and therapy resistance. To understand the biology of GSCs, and target these tumors therapeutically, we need robust strategies for in vitro expansion of primary GSCs. To date, tumor core biopsies have been the main established source of GSCs. Since these samples are used for diagnostic purposes, the available tissue for cell culture and therapeutic targeting can be limited. In addition, a core biopsy is usually taken from one part of the tumor, thus would be unlikely to represent intra-tumor heterogeneity. To overcome these problems, tissue fragments from all over the tumor can be collected using an ultrasonic aspirator during surgery, thus assembling a "global tumor biopsy". Usually, this ultrasonic aspirate (UA) sample is considered as biological waste after operations. Here, we show that UA samples offer a large and reliable source of live cells. Similar to core biopsies, UA samples enriched for GSCs that differentiated into neural lineages, showed inter-individual variation of GSC markers, and induced tumors. Molecular profiling showed that UA samples cover tumor heterogeneity better than core biopsies. These results suggest that UA samples can be used to establish large scale cultures for therapeutic applications.
Asunto(s)
Biopsia con Aguja/métodos , Neoplasias Encefálicas/patología , Glioblastoma/patología , Células Madre Neoplásicas/patología , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/cirugía , Diferenciación Celular/genética , Supervivencia Celular , Análisis Mutacional de ADN , Glioblastoma/genética , Glioblastoma/cirugía , Humanos , Biopsia Guiada por Imagen/métodos , Ratones SCID , Células Madre Neoplásicas/fisiología , Transcriptoma , Células Tumorales Cultivadas , Ultrasonografía Intervencional/métodos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The biology of glioblastoma invasion and its mechanisms are poorly understood. We demonstrate using time-lapse microscopy that grafting of glioblastoma (GBM) tumorspheres into rodent brain slices results in experimental ex vivo tumors with invasive properties that recapitulate the invasion observed after orthotopic transplantation into the rodent brain. The migratory movements and mitotic patterns were clearly modified by signals extrinsic to the invading cells. The cells migrated away from the tumorspheres, and removal of the spheres reduced the directed invasive movement. The cell cultures contained different populations of invasive cells that had distinct morphology and invasive behavior patterns. Grafts of the most invasive GBM culture contained 91±8% cells with an invasive phenotype, characterized by small soma with a distinct leading process. Conversely, the majority of cells in less invasive GBM grafts were phenotypically heterogeneous: only 6.3±4.1% of the cells had the invasive phenotype. Grafts of highly and moderately invasive cultures had different proportions of cells that advanced into the brain slice parenchyma during the observation period: 89.2±2.2% and 23.1±6.8%, respectively. In grafts with moderately invasive properties, most of the cells (76.8±6.8%) invading the surrounding brain tissue returned to the tumor bulk or stopped centrifugal migration. Our data suggest that the invasion of individual GBM tumors can be conditioned by the prevalence of a cell fraction with particular invasive morphology and by signaling between the tumor core and invasive cells. These findings can be important for the development of new therapeutic strategies that target the invasive GBM cells.
Asunto(s)
Neoplasias Encefálicas/patología , Movimiento Celular/fisiología , Regulación Neoplásica de la Expresión Génica , Glioma/patología , Invasividad Neoplásica/patología , Transducción de Señal/fisiología , Línea Celular Tumoral , Proliferación Celular/fisiología , Humanos , Invasividad Neoplásica/genética , Fenotipo , Transducción de Señal/genética , Factores de TiempoRESUMEN
BACKGROUND: Glioblastomas are invasive therapy resistant brain tumors with extremely poor prognosis. The Glioma initiating cell (GIC) population contributes to therapeutic resistance and tumor recurrence. Targeting GIC-associated gene candidates could significantly impact GBM tumorigenicity. Here, we investigate a protein kinase, PBK/TOPK as a candidate for regulating growth, survival and in vivo tumorigenicity of GICs. METHODS: PBK is highly upregulated in GICs and GBM tissues as shown by RNA and protein analyses. We knocked down PBK using shRNA vectors and inhibited the function of PBK protein with a pharmacological PBK inhibitor, HITOPK-032. We assessed viability, tumorsphere formation and apoptosis in three patient derived GIC cultures. RESULTS: Gene knockdown of PBK led to decreased viability and sphere formation and in one culture an increase in apoptosis. Treatment of cells with inhibitor HITOPK-032 (5 µM and 10 µM) almost completely abolished growth and elicited a large increase in apoptosis in all three cultures. HI-TOPK-032 treatment (5 mg/kg and 10 mg/kg bodyweight) in vivo resulted in diminished growth of experimentally induced subcutaneous GBM tumors in mice. We also carried out multi-culture assays of cell survival to investigate the relative effects on GICs compared with the normal neural stem cells (NSCs) and their differentiated counterparts. Normal NSCs seemed to withstand treatment slightly better than the GICs. CONCLUSION: Our study of identification and functional validation of PBK suggests that this candidate can be a promising molecular target for GBM treatment.
Asunto(s)
Glioblastoma/metabolismo , Glioblastoma/patología , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Células Madre Neoplásicas/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Técnicas de Silenciamiento del Gen , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Humanos , Indolizinas/farmacología , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Inhibidores de Proteínas Quinasas/farmacología , Quinoxalinas/farmacología , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Esferoides Celulares , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
There is a great potential for the development of new cell replacement strategies based on adult human neural stem-like cells. However, little is known about the hierarchy of cells and the unique molecular properties of stem- and progenitor cells of the nervous system. Stem cells from the adult human brain can be propagated and expanded in vitro as free floating neurospheres that are capable of self-renewal and differentiation into all three cell types of the central nervous system. Here we report the first global gene expression study of adult human neural stem-like cells originating from five human subventricular zone biopsies (mean age 42, range 33-60). Compared to adult human brain tissue, we identified 1,189 genes that were significantly up- and down-regulated in adult human neural stem-like cells (1% false discovery rate). We found that adult human neural stem-like cells express stem cell markers and have reduced levels of markers that are typical of the mature cells in the nervous system. We report that the genes being highly expressed in adult human neural stem-like cells are associated with developmental processes and the extracellular region of the cell. The calcium signaling pathway and neuroactive ligand-receptor interactions are enriched among the most differentially regulated genes between adult human neural stem-like cells and adult human brain tissue. We confirmed the expression of 10 of the most up-regulated genes in adult human neural stem-like cells in an additional sample set that included adult human neural stem-like cells (nâ=â6), foetal human neural stem cells (nâ=â1) and human brain tissues (nâ=â12). The NGFR, SLITRK6 and KCNS3 receptors were further investigated by immunofluorescence and shown to be heterogeneously expressed in spheres. These receptors could potentially serve as new markers for the identification and characterisation of neural stem- and progenitor cells or as targets for manipulation of cellular fate.
Asunto(s)
Células Madre Adultas/metabolismo , Encéfalo/citología , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/fisiología , Marcadores Genéticos/genética , Células-Madre Neurales/metabolismo , Adulto , Encéfalo/metabolismo , Cartilla de ADN/genética , Técnica del Anticuerpo Fluorescente , Humanos , Proteínas de la Membrana/metabolismo , Análisis por Micromatrices , Persona de Mediana Edad , Proteínas del Tejido Nervioso/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Factor de Crecimiento Nervioso/metabolismoRESUMEN
The identity of the cells that contribute to brain tumor structure and progression remains unclear. Mesenchymal stem cells (MSCs) have recently been isolated from normal mouse brain. Here, we report the infiltration of MSC-like cells into the GL261 murine glioma model. These brain tumor-derived mesenchymal stem cells (BT-MSCs) are defined with the phenotype (Lin-Sca-1+CD9+CD44+CD166+/-) and have multipotent differentiation capacity. We show that the infiltration of BT-MSCs correlates to tumor progression; furthermore, BT-MSCs increased the proliferation rate of GL261 cells in vitro. For the first time, we report that the majority of GL261 cells expressed mesenchymal phenotype under both adherent and sphere culture conditions in vitro and that the non-MSC population is nontumorigenic in vivo. Although the GL261 cell line expressed mesenchymal phenotype markers in vitro, most BT-MSCs are recruited cells from host origin in both wild-type GL261 inoculated into green fluorescent protein (GFP)-transgenic mice and GL261-GFP cells inoculated into wild-type mice. We show the expression of chemokine receptors CXCR4 and CXCR6 on different recruited cell populations. In vivo, the GL261 cells change marker profile and acquire a phenotype that is more similar to cells growing in sphere culture conditions. Finally, we identify a BT-MSC population in human glioblastoma that is CD44+CD9+CD166+ both in freshly isolated and culture-expanded cells. Our data indicate that cells with MSC-like phenotype infiltrate into the tumor stroma and play an important role in tumor cell growth in vitro and in vivo. Thus, we suggest that targeting BT-MSCs could be a possible strategy for treating glioblastoma patients.