Physioxia Reprograms Glioblastoma Cells Enhancing Migration and Altering Therapeutic Sensitivity

Background/Objectives: Glioblastoma is an aggressive primary brain tumor that develops within a chronically low-oxygen microenvironment, yet most preclinical studies are performed under atmospheric oxygen conditions that poorly reflect in vivo physiology. This study investigated how sustained culture under physiological oxygen tension (physioxia) influences glioblastoma cell behavior, signaling, and therapeutic response. Methods: Multiple patient-derived glioblastoma models were cultured under normoxia (21% O2) or sustained physioxia (5% O2) for at least seven days before experimentation. Cell migration, proliferation, cell cycle distribution, expression of the epithelial-to-mesenchymal transition-associated transcription factor Slug (SNAI2), PDGFRβ-associated signaling, and sensitivity to 5-fluorouracil were evaluated using transwell migration assays, cell counting, flow cytometry, RT-qPCR, immunoblotting, and BrdU incorporation assays. To examine the effects of oxygen history, we established and maintained additional patient-derived cultures grown under physioxia. Results: Sustained physioxia consistently increased migration across all glioblastoma models while reducing proliferation in normoxia-adapted cell lines through increased G0/G1 cell cycle arrest. Physioxia significantly increased Slug expression in all models and enhanced PDGFRβ, AKT, and ERK phosphorylation in a cell line-dependent manner. Therapeutic sensitivity to 5-fluorouracil was altered, with physioxia conferring increased resistance in some glioblastoma models but not universally. Patient-derived cell lines cultured continuously under physioxia retained enhanced migratory capacity and exhibited increased proliferation compared to cells grown in normoxia, suggesting that prior oxygen exposure influences proliferative responses while the pro-migratory phenotype remains conserved. Conclusions: Physiological oxygen tension is a major regulator of glioblastoma cell behavior, influencing migration, proliferation, signaling, and therapeutic response. These findings demonstrate that conventional normoxic culture conditions can obscure biologically relevant phenotypes and support the consideration of physioxia in experimental design. Such technical changes may improve the physiological and translational relevance of preclinical glioblastoma research.