Plasma extracellular vesicles from recurrent GBMs carrying LDHA to activate glioblastoma stemness by enhancing glycolysis

<b>Rationale:</b> Glioblastoma multiforme (GBM) is the most aggressive primary malignant brain tumor in adults, characterized by high invasiveness and poor prognosis. Glioma stem cells (GSCs) drive GBM treatment resistance and recurrence, however, the molecular mechanisms activating intracranial GSCs remain unclear. Extracellular vesicles (EVs) are crucial signaling mediators in regulating cell metabolism and can cross the blood-brain barrier (BBB). This study aimed to elucidate how EV cargo contributes to the intracranial GSC state and validate a non-invasive diagnostic strategy for GBM relapse. <b>Methods:</b> We isolated plasma extracellular vesicles (pl-EVs) from three groups: recurrent GBM patients post-resection, non-recurrent GBM patients post-resection, and healthy individuals. Newly diagnosed GBM patients served as an additional control. EVs were characterized and co-cultured with primary GBM cell lines to assess their effect on tumor stemness. EV cargo was analyzed using proteomics to investigate specific EV subpopulations contributing to GBM relapse. Based on these findings, we generated engineered LDHA-enriched EVs (LDHA-EVs) and co-cultured them with patient-derived organoids (PDOs). Metabolomics was performed to elucidate the underlying signal transduction pathways. <b>Results:</b> Our study demonstrated that pl-EVs from recurrent GBM patients enhanced aerobic glycolysis and stemness in GBM cells. Proteomic analysis revealed that plasma EVs from recurrent GBMs encapsulated considerable amounts of the enzyme lactate dehydrogenase A (LDHA). Mechanistically, LDHA-loaded EVs promoted glycolysis, induced cAMP/ATP cycling, and accelerated lactate production, thereby maintained the GSC phenotype. Concurrently, post-surgical therapy-induced stress-modulated hypoxia in residual tumors, promoted LDHA-enriched EV release. Clinically, high levels of circulating LDHA-positive EVs correlated with increased glycolysis, poor therapeutic response, and shorter survival in recurrent GBM patients. <b>Conclusion:</b> Our study highlights LDHA-loaded EVs as key mediators promoting GSC properties and metabolic reprogramming in GBM. These findings provide insights into recurrence mechanisms and suggest potential liquid biopsy approaches for monitoring and preventing GBM relapse.