Investigating TMZ-induced cell death and immunogenic responses in murine syngeneic glioblastoma models

Glioblastoma is characterized by profound therapeutic resistance and marked heterogeneity, both at the tumour cell-intrinsic and microenvironmental levels. Although temozolomide remains the standard-of-care treatment, its mechanisms of action beyond direct tumour cell cytotoxicity, and their dependency on tumour immunogenicity remain not fully understood. Here, we performed a systematic comparative analysis of temozolomide responses in three syngeneic murine glioblastoma models—GL261, CT-2 A, and SB28—covering a spectrum of antigenicity and immune competence, using integrated in vitro and in vivo approaches. In vitro, temozolomide induced delayed and predominantly cytostatic effects in all models, with apoptosis occurring only after several replication cycles. CT-2 A cells displayed the greatest sensitivity to temozolomide, exhibiting robust caspase activation and the canonical hallmarks of immunogenic cell death, including calreticulin exposure and ATP release. In contrast, SB28 cells showed caspase activation and ATP release but inconsistent calreticulin exposure, suggesting only partial engagement of immunogenic cell death-associated pathways. GL261 cells failed to activate caspases or exhibit detectable immunogenic cell death-associated signals. However, these cell-intrinsic responses failed to predict therapeutic outcome in vivo. Using an immune-respectful metronomic temozolomide schedule, we observed strikingly divergent in vivo responses: GL261 tumours showed a robust survival benefit and occasional complete regressions, CT-2 A tumours displayed only transient growth delay, whereas SB28 tumours failed to show a survival benefit. Analysis of the tumour microenvironment revealed distinct immune profiles associated with treatment outcome. In GL261 tumours, TMZ treatment was associated with increased markers consistent with M1-like macrophage polarization and enhanced T-cell infiltration, whereas CT-2 A and SB28 tumours displayed immune profiles suggestive of a predominance of M2-like immunosuppressive features. Additionally, PD-L1 expression was increased in TMZ-treated GL261 tumours, consistent with a potential adaptive immune response. Together, these findings suggest that temozolomide efficacy in the investigated immunocompetent glioblastoma models is more closely associated with tumour antigenicity and microenvironmental context, than with intrinsic tumour cell sensitivity alone, underscoring the critical importance of model selection in translational neuropathological research.