Tumor-associated macrophages promote ferroptosis resistance in glioblastoma by stimulating iron-loaded extracellular vesicle release.

PURPOSE: Glioblastoma (GBM) cells have a higher iron requirement than normal cells and therefore upregulate iron uptake. However, elevated intracellular iron levels can render cancer cells susceptible to ferroptosis, a non-apoptotic, iron-dependent form of cell death. Tumor-associated macrophages (TAMs) are the most abundant non-tumor cell type in GBM and play a pivotal role in GBM iron homeostasis. Although the role of TAMs in regulating iron availability in the TME is well established, their role in regulating ferroptosis in the GBM cells remains unexplored. METHODS: A transwell coculture system was used involving GL261 cells and bone marrow–derived macrophages (BMDMs) from C57BL/6J mice to study cancer cell- TAM interaction in GBM. In vitro analyses including immunoblot, cell death assay, lipid peroxidation assay and labile iron pool measurements were done to examine the iron and ferroptotic status of GL261 cells. Extracellular Vesicles (EVs) were isolated by ultracentrifugation. Pharmacological inhibition of EVs was done to confirm the mechanism of action of TAM-induced ferroptosis resistance. RESULTS: TAMs rendered GL261 cells more resistant to RSL3-induced ferroptotic stress. Mechanistically, TAMs reduced Gl261 cellular iron levels by increasing the release of H-ferritin–bound iron via CD63-positive EVs, with a male sex bias. TAM-secreted TNF-α played a key role in promoting this ferritin-bound iron release. Treatment with GW4869, a potent inhibitor of EV formation, in the presence of hepcidin resensitized TAM-cocultured GL261 cells to RSL3-induced ferroptosis. CONCLUSIONS: These findings indicate that TAMs protect GBM cells from ferroptotic stress by inducing EV-mediated ferritin-bound iron release from the GBM cells through TNF-α signaling. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11060-026-05556-w.