Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults, characterized by poor prognosis, high recurrence rates, and low survival. Its tumor microenvironment (TME) is highly heterogeneous, with immune infiltrates dominated by tumor-associated microglia/macrophages (TAMs) and regulatory T cells (Tregs). Their presence is associated with resistance to chemotherapy and radiotherapy and an impediment to successful immunotherapy. Hypoxia in GBM, associated with an immunosuppressive gene signature and poor prognosis, activates hypoxia-inducible factors (HIFs). HIF-2a has emerged as a critical regulator of tumor progression, particularly in VHL-related cancers; however, its role in glioma immunosuppression remains unknown. Here, we report the first steps in elucidating this issue. Using the GL261 glioma mouse model, we show that administering a specific HIF-2a inhibitor (PT2385) significantly prolonged overall survival and decreased tumor volume. Additionally, PT2385 shaped the composition of the TME by reducing the frequency of macrophages, increasing the presence of microglial cells, and reducing their pro-tumoral signature. Notably, PT2385 significantly decreased Tregs within the glioma microenvironment.

HIF-2a inhibition combined with immune checkpoint blockade (ICB) (αPD-1/αTIM-3) resulted in long-term survival of GL261-bearing mice. This combination altered the heterogeneity of the glioma microenvironment in a multifaceted manner, enhancing the anti-tumor immune response. Through scRNA-seq and flow cytometry analysis, we found that HIF-2a inhibition combined with ICB reduced macrophage numbers and increased microglial cells. Moreover, this combination led to a predominance of pro-inflammatory macrophages and homeostatic microglia, known for their anti-tumor activities. Additionally, HIF-2a inhibition enhanced the ICB response, decreasing Treg infiltration and promoting T cell subpopulations associated with anti-tumor functions (Th1, gd T cells, effector memory CD8 T cells). Overall, our results support HIF-2a targeting as a rational therapeutic opportunity for GBM, potentially regulating the immunosuppressive nature of the GBM microenvironment and emphasizing the necessity of a personalized immunotherapeutic approach based on the TME composition. This encourages further investigation to elucidate how HIF-2a targeting could shape the tumor microenvironment to ultimately overcome GBM immunosuppression.