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DC vaccines loaded with glioma killed by photodynamic therapy induce Th17 anti-tumor immunity and provide a 4-gene signature for glioma prognosis

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE218081 | GEO | 2022/12/21

REPOSITORIES: GEO

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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:Glioblastoma (GBM) is a highly aggressive type of glioma with poor prognosis. However, a small number of patients live much longer than the median survival. A better understanding of these long-term survivors (LTS) may provide important insight into the biology of GBM. We identified 7 patients with GBM treated at Memorial Sloan-Kettering Cancer Center (MSKCC) with survival greater than 48 months. We characterized the transcriptome of each patient and determined rates of MGMT promoter methylation and IDH1 and IDH2 mutational status. We identified LTS in two independent cohorts (TCGA and REMBRANDT) and analyzed the transcriptomal characteristics of these LTS. The median overall survival of our cohort was 62.5 months. LTS were distributed between the proneural (n=2), neural (n=2), classical (n=2) and mesenchymal (n=1) subtypes. Similarly, LTS in the TCGA and REMBRANDT cohorts demonstrated diverse transcriptomal subclassification identity. The majority of the MSKCC LTS (71%) were found to have methylation of the MGMT promoter. None of the patients had an IDH1 or IDH2 mutations, and IDH mutation occurred in a minority of the TCGA LTS as well. A set of 42 genes was found to be differentially expressed in the MSKCC and TCGA LTS. While IDH mutant proneural tumors impart a better prognosis in the short-term, survival beyond 4 years does not require IDH mutation and is not dictated by a single transcriptional subclass. In contrast, MGMT methylation continues to have strong prognostic value for survival beyond 4 years. These findings have substantial impact for understanding GBM biology and progression. All tumors (n = 7) were obtained following surgical resection at the MSKCC as part of routine clinical care, and snap frozen. Tumors were obtained in accordance with Institutional Review Board policies at the MSKCC. Each sample was examined histologically by 3 independent neuropathologists and confirmed to be World Health Organization (WHO) grade IV glioma (GBM). Before analysis, tumors were sectioned and microdissected. Genomic DNA or RNA was extracted using the DNeasy kit (Qiagen) or RNeasy Lipid Tissue Mini Kit (Qiagen) as per the manufacturer’s instructions. Expression analysis of tumors was performed using the Affymetrics U133 2.0 microarray (Affymetrix). Affymetrix CEL files were imported into the Partek Genomics Suite (Partek). The TCGA gene expression data (HT-HG-U133A) was accessed from the TCGA data repositories (http://cancergenome.nih.gov, date of download 12/2013).

Project description:Background: Glioma-associated macrophages (GAMs) are one of the most important cellular components in the tumor immune microenvironment (TIME). GAMs display a mostly M2-like phenotype with anti-inflammatory features, which is closely related to the malignancy and progression of cancers. Extracellular vesicles derived from GAMs (GAM-EVs), which are important components of the TIME, contain a variety of microRNAs (miRNAs). These miRNAs play a crucial role in communication between tumor cells and GAMs. In this study, we aimed to explore the effect of GAM-EVs on the malignancy and progression of glioblastoma multiforme (GBM) through miRNA-mediated communication and its possible signaling pathway. Methods: The GBM and LGG data from The Cancer Genome Atlas (TCGA) were analyzed, and the immunological features of 12 clinical glioma samples, including GBM and low-grade glioma (LGG) samples, were estimated by immunohistochemistry. To evaluate the changes induced by M2-EVs compared to M1-EVs, the human GBM cell lines U87MG and A172 were cocultured with M2-EVs and M1-EVs, and their epithelial-mesenchymal transition (EMT) behaviors were investigated. The contents of M2-EVs and M1-EVs were determined by miRNA sequencing, and the differences were analyzed. Then, the EMT signatures were evaluated after a miRNA mimic was transfected into GBM cells. Six databases were used to predict the targets of the miRNA. Human GBM cell lines with interference and overexpression of the most valuable candidate miRNA targets were established. The related signaling pathways were further explored. Finally, an orthotopic homograft mouse model was employed to verify the results of the in vitro experiments. Results: The immunosuppressive GAM genes were upregulated in GBM compared with LGG, showing that the TIME of GBM is more immunosuppressive. To explore the effect of the immunosuppressive GAMs and TIME on the progression of GBM, we established immunostimulatory (M1-like) and immunosuppressive (M2-like) GAMs and compared the differences in their EVs. The migration and invasion of U87MG and A172 cells were enhanced under coculture with M2-EVs. MiR-146a-5p was deficient in M2-EVs compared with M1-EVs. Upon treatment with the miR-146a-5p mimic, the migration and invasion abilities of GBM cells were weakened, and the EMT signature was correspondingly decreased. According to the database prediction, tumor necrosis factor receptor-associated factor 6 (TRAF6) and interleukin 1 receptor-associated kinase 1 (IRAK1) are the targets of miR-146a-5p. TRAF6 is an E3 ubiquitin ligase, and IRAK1 is the substrate. Bimolecular fluorescence complementation (BiFC) and coimmunoprecipitation assays confirmed interactions between TRAF6 and IRAK1. Interference with both TRAF6 and IRAK1 expression weakened but overexpression of both TRAF6 and IRAK1 promoted the EMT behaviors of GBM cells. The TRAF6-Glioma-associated macrophages (GAMs) are pivotal chains in the tumor immune microenvironment (TIME). GAMs mostly display M2-like phenotypes with anti-inflammatory features related to the malignancy and progression of cancers. Extracellular vesicles derived from immunosuppressive GAMs (M2-EVs), the essential components of the TIME, greatly impact the malignant behavior of GBM cells. M1- or M2-EVs were isolated in vitro, and human GBM cell invasion and migration were reinforced under M2-EV treatment. Signatures of the epithelial-mesenchymal transition (EMT) were also enhanced by M2-EVs. Compared with M1-EVs, miR-146a-5p, considered the key factor in TIME regulation, was deficient in M2-EVs according to miRNA-sequencing. When the miR-146a-5p mimic was added, EMT signatures and the invasive and migratory abilities of GBM cells were correspondingly weakened. Public databases predicted the miRNA binding targets and interleukin 1 receptor-associated kinase 1 (IRAK1) and tumor necrosis factor receptor-associated factor 6 (TRAF6) were screened as miR-146a-5p binding genes. Bimolecular fluorescent complementation and coimmunoprecipitation confirmed interactions between TRAF6 and IRAK1. The correlation between TRAF6 and IRAK1 was evaluated with immunofluorescence (IF)-stained clinical glioma samples. The TRAF6-IRAK1 complex is the switch and the brake that modulates IKK complex phosphorylation and NF-κB pathway activation, as well as the EMT behaviors of GBM cells. Furthermore, a homograft nude mouse model was explored and mice transplanted with TRAF6/IRAK1-overexpressing glioma cells had shorter survival times while mice transplanted with glioma cells with miR-146a-5p overexpression or TRAF6/IRAK1 knockdown lived longer. This work indicated that in the TIME of GBM, the deficiency of miR-146a-5p in M2-EVs enhances tumor EMT through disinhibition of the TRAF6-IRAK1 complex and IKK-dependent NF-κB signaling pathway providing a novel therapeutic strategy targeting the TIME of GBM.

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DC vaccines loaded with glioma killed by photodynamic therapy induce Th17 anti-tumor immunity and provide a 4-gene signature for glioma prognosis

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