Glioblastoma multiforme (GBM) is one of the extremely fatal brain tumors. The main reason that makes it so lethal is its capability to invade and spread to other parts of CNS producing secondary tumors. Among other factors hypoxia, reduced oxygen availability, is linked to higher metastatic potential of cancers. Hypoxia causes numerous changes in genome and proteome of the cell. These changes help a normal cell to adapt to nutritional deficiency, but the same changes can increase the malignancy and metastasis in tumor cells. Extensive research by a number of curious scientists reveal that various pathways involving numerous proteins cross-talk and interact with each other and execute a response to hypoxia. We are trying to establish the link between two such pathways HIF1-alpha pathway and Notch pathway. Both, HIF1-alpha, which is a transcription factor that becomes active in hypoxic conditions and Notch, which is an evolutionarily conserved cell-fate determinant, are implicated in hypoxia-induced metastasis of cancer. In this given project, we confirm the cross talk between Notch and HIF1-alpha pathway and further continue our study to show that TrpC6 is the downstream mediator of this pathway, leading to metastasis of GBM. Expression analysis of hypoxia-induced U373 cells (Grade 3 glioblastoma cells), using Real-time PCR, western blot and immunocytochemistry, revealed elevated levels of Notch, Hif1 and TrpC6 indicating that these proteins might be important for the cellular response to hypoxia. Blocking Notch and/or HIF1-alpha, either by DAPT or HIF1-inhibitor, confirmed the communication between these two pathways. Role of TrpC6 in metastasis was demonstrated by knocking down this gene using siRNA against TrpC6. Inhibition of TrpC6 markedly decreased cell proliferation, migration, angiogenesis and tumorigenesis in these hypoxia-induced Glioblastoma cells. In summary, all these results reveal that TrpC6 is indeed an important member of the Notch-mediated metastasis of Glioblastoma under hypoxic conditions. This role of TrpC6 can therefore be utilized for pharmacological intervention to prevent hypoxia-induced metastasis in GBM.

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OBJECTIVE: The overarching goal of this study was to examine the immunostimulatory potential of oHSV-1 rQNestin34.5v2 in syngeneic murine GBM models, perform in vitro screens for upregulation of immune checkpoint molecules in infected glioma cells, and evaluate the antitumor activity of the most promising combination immunovirotherapies. METHODS: The oncolytic activity of HSV-1 rQNestin34.5 was evaluated in CT-2A and GL261 syngeneic murine glioma models. Immunoassays were conducted to assess secretion of damage associated molecular patterns including ATP, HMGB1, Calreticulin, HSP70 and other proinflammatory mediators by infected glioma cells. In vitro screens for expression of inhibitory ligands by glioma cells following HSV-1 rQNestin34.5v2 infection at various doses were analyzed by flow cytometry. Intratumoral HSV-1 rQNestin34.5v2 administration and/or intraperitoneal anti-TIGIT (clone 1B4)/anti-NK1.1 treatments were performed in C57BL/6 mice bearing orthotopic CT-2A glioma to determine effect on overall survival. RESULTS: HSV-1 rQNestin34.5v2 exhibited greater capacity to infect CT-2A and minimal capacity to infect GL261 cells suggesting differences in permissiveness in HSV- 1 replication between the two GBM models. Infection stimulated immunogenic cell death as evidenced by surface expression of calreticulin and HSP70 and elevated extracellular release of ATP and HMGB1 in the GL261 model. CD155 and CD112 (both ligands of TIGIT) as well as PD-L1 were significantly highly expressed in glioma cells. TIGIT was found to be overexpressed in tumor infiltrating NK, CD4 and CD8 T cells suggesting systemic therapy with TIGIT blockade antibodies could have therapeutic utility in combination with HSV-1 rQNestin34.5v2 in GBM. Benefit in overall survival was not observed by anti-TIGIT monotherapy, and combination treatment with HSV-1 rQNestin34.5v2 exhibited modest therapeutic effect with a cure rate 25% in mice bearing intracranial CT-2A tumors. Depletion of NK cells prior to HSV-1 rQNestin34.5v2 administration attenuated brain edema and synergized with rQNestin34.5v2 virotherapy. CONCLUSION: Our findings show that the combination of HSV-1 rQNestin34.5v2 virotherapy with anti-TIGIT checkpoint blockade immunotherapy and/or NK cell inhibition represents a promising strategy to overcome primary resistance to immune checkpoint inhibitors in GBM. / 2025-02-03T00:00:00Z

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