Tumor-associated Macrophages protect Glioblastoma cells from ferroptosis by inducing the release of Ferritin-bound iron via exosomes

Tumor-associated macrophages (TAMs) are the most abundant non-tumor cell type in glioblastoma (GBM) and act as the pivotal cell type in regulating iron metabolism in the GBM tumor microenvironment. High TAM infiltration into the TME is also associated with increased resistance to ferroptosis, an iron-dependent cell death. However, the exact mechanism by which TAMs make the cancer cells resistant to ferroptosis remains relatively unexplored. Here, we have investigated how TAMs modify iron metabolism in GBM cells to make them more resistant to ferroptotic stress. We utilized GL261 cells, a GBM cell line derived from C57BL6 mice, and syngeneic primary murine bone marrow-derived macrophages (BMDM) to study GBM-TAM interactions in vitro. We found that male macrophages exhibited higher iron uptake, greater iron storage, and a larger labile iron pool compared to female macrophages, indicating intrinsic sex biases in macrophage iron metabolism. Subsequently, we used co-culture experiments to study how macrophages regulate the iron and ferroptotic status of GL261 cells. We discovered that GL261 cells cocultured with BMDMs showed higher resistance to RSL3-induced ferroptotic stress. Mechanistically, BMDMs caused a decrease in total cellular iron in GL261 cells by inducing increased H-ferritin-bound iron release via CD63-positive exosomes; thus, limiting the amount of iron that is available for lipid peroxidation during ferroptosis. This process was moderately sex biased in favor of male macrophages. Finally, we show that this mechanism of BMDM-induced resistance to ferroptosis is independent of Hepcidin regulation and can act as a possible pathway by which GBM cells escape ferroptotic stress during proinflammatory conditions.