Project description:We hypothesized that the potential ability of the 18 kDa mitochondrial translocator protein (TSPO) to regulate gene expression may underlie its numerous reported functions, ranging from mitochondrial activity till mental disorders. Therefore, we applied microarray analysis of gene expression to U118MG glioblastoma cells. Within 1 hr the TSPO ligand PK 11195 induced changes in expression of immediate early genes and transcription factors. After 24 hrs primarily gene expression for enzymes and other proteins is changed. The associated gene products are known to affect various cellular functions: programmed cell death, cell cycle, viability, proliferation, migration, differentiation, development, tumorigenesis, and inflammatory / immune responses. Our microscopic studies showed intense TSPO mitochondrial labeling but no TSPO signal in the cell nuclei. Thus, it appears that the TSPO is part of the retrograde mitochondrial-nuclear signaling pathway for modulation of gene expression and thereby may exert its numerous effects on cell function, phenotype, and disease. U118MG cells (1.255 Ã 106) were seeded in Petri dishes (i.e. 28.5 Ã 103 cells / cm2) and allowed to proliferate for 3 days in full medium. Experiments for gene expression assayed with microarray typically consisted of three experimental groups and a vehicle control group (n = 3 for each group). Serum deprived medium was applied for 24 hrs, in the experimental groups ending with the inclusion a choice of various PK 11195 exposures i.e. either 1 hr, 3 hrs, or 24 hrs. Exposure to PK 11195 implies serum deprived medium with 1% alcohol (vehicle) and PK 11195 (25 ÂµM final concentration) for the required time period. 25 ÂµM of PK 11195 is the optimal concentration for these types of experiments with U118MG cells (Kugler et al., 2008). The cells were collected by trypsinization, washed by centrifugation in phosphate buffered saline (PBS) (400 Ã g, 5 min), and lysed [RLT lysis buffer provided with the RNeasy Mini Kit diluted with Î²-mercaptoethanol (1 : 100)], according to the manufacturer's instructions. Lysates were stored at -70oC.

Project description:Glioblastoma is the most malignant and common type of primary brain tumor with a median survival of less than 21 months. It is particularly resistant to current immunotherapies. Thus, yet unknown mechanisms of immune resistance may be active in glioblastoma. Translocator protein 18 kDa (TSPO) expression is upregulated in glioblastoma and correlates with malignancy and poor prognosis but also with increased immune infiltration. An immunomodulatory role of TSPO in glioblastoma has not been reported. Here, we studied the role of TSPO in the regulation of immune resistance of human glioblastoma cells. We used public bulk and single-cell gene expression data to identify cell types, genes and signalling pathways correlating with TSPO expression. To validate inflammatory triggers and the role of TSPO experimentally, we co-cultured tumor-infiltrating T cells and cytotoxic T cell lines with autologous and antigen-loaded allogeneic, primary brain tumor initiating cells (BTICs). TSPO proficient and deficient BTIC lines and clones were generated through genetic manipulation. Inflammatory factors triggering TSPO expression were determined by ELISA and validated experimentally using blocking antibodies and recombinant proteins. The impact of TSPO on glioblastoma cell resistance against cytotoxic T cells was studied by cytotoxicity assays and by activity analysis of components of the apoptotic cascade. Individual death-inducing pathways suppressed by TSPO were determined using recombinant proteins and TSPO-regulated genes associated with apoptosis resistance were identified by gene expression analysis in TSPO proficient and deficient BTICs and subsequent functional analyses. TSPO expression in glioblastoma cells correlated with cytotoxic T cell infiltration, expression of TNFR and IFNGR, the activity of their downstream pathways, expression of TRAIL receptors and with PD-L1. Coculture of BTICs with tumor-reactive cytotoxic T cells or with T cell-derived factors induced TSPO up-regulation through TNFα and IFNγ. Silencing of TSPO sensitized BTICs against T cell-mediated cytotoxicity. TSPO protected BTICs selectively against TRAIL-induced apoptosis by regulating both intrinsic and extrinsic apoptosis pathways. TSPO regulated the expression of multiple genes associated with apoptosis-resistance of tumors. Among them, we studied the peptidase inhibitors PI3 and SLPI, which sensitized BTICs towards TRAIL-induced apoptosis. Our data revealed that TSPO expression in glioblastoma cells is induced through T cell derived cytokines IFNγ and TNFα. TSPO expression protects glioblastoma cells against cytotoxic T cell attack through the death inducing ligand TRAIL. Our data provided an indication that therapeutic targeting TSPO may be a suitable approach to sensitize glioblastoma to immune cell-mediated cytotoxicity by circumventing tumor intrinsic TRAIL-resistance.

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