TMIC-20. UNLOCKING THE POTENTIAL OF HYPOXIA REDUCTION IN RESHAPING THE GLIOBLASTOMA TUMOR MICROENVIRONMENT FOR ENHANCED THERAPEUTIC SYNERGY

Abstract Glioblastoma (GBM) tumors respond poorly to nearly all cancer therapies due to their highly immunosuppressive tumor microenvironment (TME). Hypoxia exacerbates this immunosuppression by upregulating the hypoxia-inducible factor (HIF) pathway in glioma-associated myeloid cells (GAMs) which suppressively polarizes them to maintain this immune “cold” state. This hypoxia-driven programming inhibits CD8+ T cell infiltration and effector function by inducing metabolic dysfunction, thereby increasing resistance to chemotherapy and T cell immune checkpoint blockade (ICB). Targeting hypoxia thus emerges as a promising strategy to overcome such resistance. Our study suggests that GBM hypoxia ablation can convert the immunosuppressive tumor microenvironment into a more pro-inflammatory one. Tumor hypoxia ablation using the hypoxia-activated prodrug Evofosfamide significantly enhances survival in both GL261 (p<0.0001) and the checkpoint blockade-resistant CT2A murine GBM models (p<0.01). Evofosfamide treatment boosts tumor infiltrating Ki67+ proliferative CD8+ T cells (p<0.001) and heightens cytotoxicity, indicated by increased Granzyme B expression (p<0.01). It also reduces the co-expression of exhaustion markers LAG-3 and TIM-3 (p<0.001), signaling decreased T cell exhaustion. Antigen-specific CD8+ T cells from Evofosfamide-treated primary GBM tumors exhibit enhanced ex vivo killing capacity when co-cultured with GL261-OVA cells (p<0.001). Furthermore, Evofosfamide enhances microglia proliferation and repolarizes them towards a pro-inflammatory state, marked by upregulation of M1 markers such as CD80 (p<0.001) and downregulation of M2 markers like Arginase (p<0.05). Hypoxia reduction also increases chemotherapy sensitivity, synergistically improving survival when combined with the blood-brain barrier-penetrating, immunogenic chemotherapy Berubicin (CT2A; vehicle MS=26d, Evo MS=36d, Berubicin MS=55d, Combo MS=70.5d). Future research will investigate the mechanisms underlying this shift towards a pro-inflammatory state, focusing on metabolic changes in CD8+ T cells and the impact of altered HIF pathway signaling on GAM polarization following hypoxia ablation. Understanding the therapeutic potential of reversing hypoxia-mediated immunosuppression could lead to novel combination therapies capable of overcoming resistance to both chemotherapy and immunotherapy in GBM.