Hypoxia-Induced Osteopontin-Positive Glioma-Associated Macrophages Facilitate Glioma Mesenchymal Transition via NF-κB Pathway Activation

<b>Background:</b> Hypoxia is a prevalent, characteristic feature of the tumor microenvironment (TME) in glioblastomas (GBMs). As dominant immune cells within the TME, glioma-associated macrophages (GAMs) crucially regulate tumor progression. A comprehensive understanding of the effect of hypoxia on the behavior of GAMs is essential for elucidating the immune landscape and developing innovative therapeutic strategies. This study aimed to elucidate the mechanisms by which GAMs facilitate GBM progression under hypoxic conditions. <b>Methods:</b> Transcriptome sequencing, single-cell RNA sequencing, and spatial transcriptomic analyses were performed to explore the correlation between hypoxia and GAMs. Clinical samples were used to validate the findings. The underlying molecular mechanisms were examined via chromatin immunoprecipitation, quantitative real-time polymerase chain reaction, Western blotting analysis, and immunofluorescence assays. The therapeutic effectiveness was assessed via the use of in vivo models. <b>Results:</b> A subset of GAMs with elevated osteopontin (OPN) expression accumulates in response to hypoxic stimulation. Hypoxia induces OPN expression in macrophages via the histone 3 lysine 4 trimethylation-WD40 repeat-containing protein 5 (H3K4me3-WDR5) epigenetic axis. These OPN-positive GAMs (OPN⁺ GAMs) enhance the mesenchymal transition in GBMs by secreting OPN into the TME. Mechanistically, OPN activates nuclear factor κB (NF-κB) signaling through cluster of differentiation 44 (CD44), subsequently leading to increased programmed cell death ligand 1 (PD-L1) expression. The inhibition of OPN increased GBM sensitivity to temozolomide (TMZ) in orthotopic models. <b>Conclusions:</b> This study revealed the potential mechanism by which hypoxia-induced OPN⁺ GAMs promote the mesenchymal transition in GBM cells and demonstrated the therapeutic potential of targeting OPN to enhance TMZ treatment effectiveness.