Chronic IFN-γ Exposure Drives Divergent Cellular and Molecular Responses in Glioblastoma Subtypes

Background: Glioblastoma (GBM) is an aggressive primary brain tumor with poor prognosis and limited treatments. Interferon-gamma (IFN-γ) plays a complex role in modulating immune and tumor responses, and its long-term impact on GBM remains unclear. Methods: Human GBM cell lines U87 and U251 were subjected to up to 28 days of chronic IFN-γ exposure and assessed for morphology, gene expression, protein dynamics, and cytokine secretion. RNA sequencing, automated Western blotting, and single-cell proteomics were employed to uncover distinct cellular and molecular adaptations to prolonged IFN-γ signaling. Results: Chronic IFN-γ exposure induced significantly divergent phenotypic changes in U87 and U251 cells. U87 cells formed neurosphere-like structures and upregulated STAT1/STAT3 signaling, suggesting an immunogenic response. In contrast, U251 cells displayed elongated morphologies and activated PI3K-Akt and Erk pathways, promoting survival and immune evasion. Differential gene expression analysis highlighted unique temporal profiles, with U251 cells exhibiting higher expression of immune resistance genes such as PD-L1 and VEGF. Secretome profiling further demonstrated contrasting cytokine landscapes: U87 cells secreted pro-inflammatory cytokines, while U251 cells produced immunosuppressive factors. Conclusions: Chronic IFN-γ exposure drives distinct molecular and phenotypic changes in GBM subtypes, reflecting their intrinsic heterogeneity. While IFN-γ enhances immune activation, prolonged exposure may also promote immune escape. These findings emphasize the need for therapeutic strategies combining IFN-γ with immune checkpoint inhibitors and targeted pathway modulation to optimize GBM treatment outcomes.