Early metabolic reprogramming and carbonic anhydrase IX-mediated extracellular acidification drive radiotherapy-induced glioblastoma cell dedifferentiation

Mitochondrial metabolism is enhanced in GSC compared to their differentiated progeny. IR-induced dedifferentiation in GBM cells occurs via an early metabolic shift to OXPHOS. IR-induced metabolic shift links CA9 induction, extracellular acidification, and stemness. Glioblastomas (GBM) are brain tumors with the worst prognosis despite treatment with surgery and radio/chemotherapy, emphasizing the need for new therapies and improved treatment efficacy. Previously, we showed that clinically relevant ionizing radiation (IR) doses enhance GBM cell dedifferentiation into a stem-like phenotype, increasing stemness markers, self-renewal, and tumorigenic abilities. This work focuses on identifying early mechanisms driving this plasticity, particularly metabolic adaptations, as tumor metabolism may support therapy resistance and recurrence. In this study, primary cell lines were established from GBM biopsies of several patients and cultured either in restrictive medium to form neurospheres (NS) enriched in GBM stem-like cells (GSC) or in normal medium to obtain their differentiated progenies. These differentiated GBM cells were then subjected to a short-term dedifferentiation protocol after IR (24 to 72 h) to characterize their metabolic and phenotypic properties. We found that early stem marker increases after IR exposure coincide with higher oxygen consumption rate (OCR), mitochondrial ATP production, and extracellular acidification rate (ECAR). However, lactate production remained unchanged, and glucose uptake showed only a transient, nonspecific increase. This shift to oxidative mitochondrial metabolism, coupled with extracellular acidosis, promotes a stem-like phenotype and is associated with Carbonic Anhydrase IX (CA9) overexpression, an extracellular membrane protein producing H + to buffer intracellular pH. CA9 downregulation via ShRNA reduces extracellular acidification and blocks early IR-induced dedifferentiation, such as neurosphere (NS) formation and stem marker overexpression. Similarly, increased OCR and ECAR, often linked to CA9 overexpression, are observed in patient-derived GSC compared to their differentiated progenies. These findings highlight CA9 as a potential target to block IR-induced early dedifferentiation process as well as the specific metabolic state of GSC. Our study strengthens the therapeutic potential of CA9 inhibition to limit GBM-associated acidic extracellular environment and enhance radiotherapy efficiency by limiting tumor cell plasticity.