Project description:Most clinical trials for glioblastoma (GBM) enroll patients at recurrence. However, most pre-clinical animal models are of primary disease. Most studies have used tissues from primary tumors. Knowledge of GBM cellular composition at recurrence is limited. We profiled 80 human IDH-wild-type GBM specimens via single-nucleus RNA sequencing, 40 primary tumors and 40 patient-matched recurrent tumors. Select cohorts were also profiled via single-cell assay for transposase-accessible chromatin, single-cell digital-spatial, and single-cell spatial-transcriptomic assays. All patients were treated only with standard-of-care therapy: temozolomide, radiation and surgical resection. The cohort had a 1.2 male/female ratio, ages 35–76. We found a significant increase at recurrence in cells with the Verhaak mesenchymal phenotype and fewer proneural cells. Although there was an increase in the percentage of cycling cells overall, the fraction of cycling proneural cells decreased while the fraction of cycling mesenchymal cells increased. These changes were concomitant with a significant increase in the percentage of tumor-infiltrating monocytic-lineage cells derived from the periphery. Taken together, these findings support a proneural-to-mesenchymal shift at recurrence due to an increase in the birth rate of mesenchymal cells, supported by an expansion of myeloid-derived cells from peripheral blood. Although the percentages of glioblastoma-infiltrating T-cells are generally low (~1% on average in our data), we found a statistically significant increase in T-cell abundance at recurrence. We identified a cohort of T-cell outliers that demonstrate 2-to-8-fold increases in T-cells over average levels. We validated that T-cells in these tumors have extravasated beyond the perivascular space and into the cellular tumor via immunohistochemistry and spatial profiling. We present our ongoing analysis of the paracrine signals, upstream transcription-factor expression, and cis-regulatory grammars utilized in immune-outlier GBMs. We describe how these programs change under therapy. These studies shed light on the effect of standard therapy in shaping GBM composition at recurrence.

Project description:Recent single-cell transcriptomic studies report that IDH-mutant gliomas share a common hierarchy of cellular phenotypes, independent of genetic subtype. However, the genetic differences between IDH-mutant glioma subtypes are prognostic, predictive of response to chemotherapy, and correlate with distinct tumor microenvironments. To reconcile these findings, we profiled 22 human IDH-mutant gliomas via single-cell assay for transposase-accessible chromatin (scATAC-seq). We determined the cell-type specific differences in transcription-factor expression and associated regulatory grammars between IDH-mutant glioma subtypes. We find that while IDH-mutant gliomas do share a common distribution of cell types, there are significant differences in the expression and targeting of transcription factors that regulate glial identity and cytokine elaboration. We knocked out the chromatin-remodeler ATRX, which suffers loss-of-function alterations in most IDH-mutant astrocytomas, in an IDH-mutant immunocompetent intracranial murine model. We find that both human ATRX-mutant gliomas and murine ATRX-knockout gliomas are more heavily infiltrated by immunosuppressive monocytic-lineage cells derived from circulation than ATRX-intact gliomas, in an IDH-mutant background. ATRX knockout in murine glioma recapitulates gene expression and open-chromatin signatures that are specific to human ATRX-mutant astrocytomas, including drivers of astrocytic lineage and immune-cell chemotaxis. ATRX knockout in murine glioma recapitulates gene expression and open chromatin signatures that are specific to human ATRX-mutant astrocytomas, including drivers of astrocytic lineage and immune-cell chemotaxis. Through single-cell cleavage under targets and tagmentation assays and meta-analysis of public data, we show that ATRX loss leads to a global depletion in CCCTC-binding factor association with DNA, gene dysregulation along associated chromatin loops, and protection from therapy-induced senescence.

Project description:INTRODUCTION: Neuronal activity regulated by synaptic communication exerts an important role in tumorigenesis and progression in brain tumors. Genes for soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) annotated with the function ‘vesicle’ about synaptic connectivity were identified and one of these proteins, synaptosomal-associated protein 25 (SNAP25), was found to have discrepant expression levels in neuropathies. However, the specific mechanism and prognostic value of SNAP25 during glioma progression remain unclear. METHODS: Using RNA sequencing data from The Cancer Genome Atlas (TCGA) database, the differential synaptosis-related genes between LGG and GBM were identified as highly correlated. Cox proportional hazards regression analysis and survival analysis indicated that the candidate gene SNAP25 could differentiate the outcome of low- and high-risk patients, and the Chinese Glioma Genome Atlas (CGGA) cohort was used for validation of the data set. RT-qPCR, western blot, and immunohistochemistry assays were performed to examine the expression level of SNAP25 in glioma cells and samples. Functional assays were performed to identify the effects of SNAP25 knockdown and overexpression on cell viability, migration, and invasion. Then, an immunofluorescence assay of the xenograft tissue was applied to evaluate the expression of the neuronal dendron formation marker-MAP2. Liquid chromatography-high re solution mass spectrometry (LC-MS)-based metabolomics approach was presented for identifying crucial metabolic disturbances in glioma cells. In situ mouse xenograft model was used to investigate the role of SNAP25 in vivo.RESULTS: SNAP25 was down expressed in glioma tissues and cell lines and low-level SNAP25 indicated an unfavorable prognosis of glioma patients. SNAP25 inhibited cell proliferation, migration, invasion and fostered glutamate metabolism of glioma cells, exerting a tumor suppressor role. SNAP25 overexpression expressed lower expression of MAP2, indicating poor neuronal plasticity and connectivity. SNAP25 could interact with glutaminase（GLS）and GLS knockdown could rescue the anti-tumor effect of SNAP25 in glioma cells. Moreover, upregulation of SNAP25 also decreased tumor volume and prolonged the overall survival (OS) of the xenograft mouse.CONCLUSION: SNAP25 inhibited carcinogenesis of glioma via sponging glutamate metabolism by regulating GLS expression, as well as inhibiting dendritic formation, which could be considered as a molecular target for glioma diagnosis and therapy.

Project description:Gliomas are the most frequent type of primary tumor of the central nervous system in adults with significant morbidity and mortality. Despite the development of novel, complex, multidisciplinary, and targeted therapies, glioma therapy has not progressed much over the last decades. Therefore, there is an urgent need to develop novel patient-adjusted immunotherapies that actively stimulate antitumor T cells generating long-term memory and thus resulting in significant clinical benefits. This work aimed to investigate the efficacy and molecular mechanism of dendritic cell (DC) vaccines loaded with glioma cells undergoing immunogenic cell death (ICD) induced by photosens based photodynamic therapy (PS-PDT) and to identify reliable prognostic genes’ signatures for predicting patients’ overall survival (OS). The analysis of the transcriptional program of the ICD-based DC vaccine led to the identification of robust induction of Th17 signature when used as a vaccine, these DCs demonstrate retinoic acid receptor-related orphan receptor-γt dependent efficacy in orthotopic mouse model. Moreover, by comparative analysis of transcriptome program of the ICD-based DC vaccine with transcriptome data from the TCGA-low grade glioma (LGG) dataset the four-gene signature (CFH, GALNT3, SMC4, VAV3) associated with OS of glioma patients have been identified. This model has been validated on OS of CGGA-LGG, TCGA-glioblastoma multiforme (GBM) and CGGA-GBM datasets to determine whether it has a similar prognostic value. For this the sensitivity and specificity of the prognostic model for predicting the OS were evaluated by calculating the area under curve (AUC) of the time-dependent receiver operating characteristic (ROC) curve. The values of AUC for TCGA-LGG, CGGA-LGG, TCGA-GBM and CGGA-GBM were, respectively, 0.75, 0.73, 0.9 and 0.69 for predicting 5-year survival rates. These data open attractive prospects for improving glioma therapy by employing ICD and PS-PDT-based DC vaccines to induce Th17 immunity and to use this prognostic model to predict the OS of glioma patients.

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A single cell atlas of human glioma under therapy

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