Glioblastoma cells that evade chemoradiotherapy-induced cell death exhibit a bifurcated glycolytic program

Abstract Glioblastoma (GBM), the most common malignant brain tumor in adults, remains a highly lethal and incurable cancer, with a 5-year survival rate below 10%. Standard-of-care involves surgical resection followed by concurrent temozolomide chemotherapy and radiation treatment. While these interventions can effectively shrink tumors, they fail to eradicate all malignant cells. Small populations of GBM cells invariably survive and seed recurrent disease, leading to near-universal relapse and the formation of fatal recurrent tumors, typically within 1–2 years of treatment. Here, we investigated the metabolic features that define these surviving cell populations using ten patient-derived GBM models and matched orthotopic xenograft models exposed to a clinically relevant chemoradiotherapy regimen. By sampling living cells at defined treatment intervals and integrating 13 C-glucose tracing, quantitative untargeted metabolomics, and nCounter metabolic gene expression profiling, we reconstructed the temporal evolution of glucose metabolism from therapy-naïve to post-treatment states. Across all models, GBM cells that evaded therapy-induced death exhibited a conserved and coordinated reorganization of glycolytic flux. These cells showed enhanced glucose uptake and elevated abundance of upper glycolytic enzymes such as HK1, while lower glycolytic enzymes, including ALDOA, GAPDH, ENO1, and LDHA, were suppressed, resulting in reduced lactate output. This bifurcation of glycolytic metabolism redirected carbon flux toward the pentose phosphate pathway and nucleotide biosynthesis, as well as mitochondrial metabolism, supported by the increased abundance of tricarboxylic acid cycle enzymes. Notably, these adaptations were conserved in recurrent patient-derived orthotopic xenograft tumors in vivo. Together, these findings reveal a fundamental and conserved metabolic state that defines GBM cells surviving chemoradiotherapy. This study deciphers a core metabolic architecture that enables tumor cell survival, persistence, and recurrence following therapy by shifting glycolytic flux away from lactate production to balance biosynthetic demands with mitochondrial metabolism.