Hypoxia shapes tumor immune microenvironment through cell-type dependent responses in diffuse astrocytomas

Background Hypoxia is a critical driver of tumor aggressiveness in high-grade gliomas, yet its cell-type-specific effects on immune cell populations within the tumor microenvironment (TME) remain poorly understood. Materials and methods We have investigated how hypoxia shapes the spatial distribution and functional states of monocyte-derived macrophages (MDMs) and brain-resident microglia (MG) in diffuse astrocytomas and glioblastomas (GB) by using cyclic immunohistochemistry (cIHC), single-cell RNA sequencing, spatial transcriptomics, and in vitro cell culture models in controlled oxygen and pH conditions. Results Hypoxia induces divergent responses in these myeloid subsets, driving spatial immune patterning. In GB, CD163⁻ MDMs dominate hypoxic niches, while MG populations are excluded from these regions, correlating with hypoxia-induced TNF upregulation, stress response signatures, and dampened interferon responses. Conversely, MDMs exhibit a shift towards hypoxia-associated immunosuppressive traits, altered metabolism and upregulated cell survival. GBs display elevated hypoxic intensity compared to diffuse astrocytomas, as supported by hypoxia-response gene expression in the TCGA dataset. In vitro, MG are characterized by heightened sensitivity to hypoxia-associated acidity compared to MDMs, suggesting that their exclusion from hypoxic zones results from intrinsic vulnerability. Conclusion Our findings reveal that hypoxia and hypoxia-associated acidity remodel the TME by promoting immunosuppressive MDM accumulation and depletion of MG, creating spatially distinct immune landscapes that may underlie GB progression. These results highlight hypoxia-driven immune dysregulation as a therapeutic target and underscore the importance of cell-type-specific strategies to counteract TME-driven immunosuppression in malignant gliomas.