Influence of the Hypoxia-Activated Prodrug Evofosfamide (TH-302) on Glycolytic Metabolism of Canine Glioma: A Potential Improvement in Cancer Metabolism

Simple Summary: This study investigated the anti-glycolytic effects of evofosfamide (EVO) on three canine glioma (GL)-derived cell lines with activated hypoxia-inducible factor 1 alpha (HIF-1 alpha). Our clinical data showed that glycolytic activity was correlated with poorer outcomes in dogs with spontaneous GL. Our in vitro studies showed that EVO inhibited glycolytic metabolism by targeting HIF-1 alpha-positive cells under hypoxic culture conditions, resulting in the suppression of cellular ATP production. Our in vivo studies showed that EVO significantly decreased tumor development compared to controls or temozolo-mid in orthotopic murine GL models. A metabolic analysis demonstrated that EVO suppressed glycolytic activity by eliminating HIF-1 alpha-positive cells. Our findings suggest that EVO may improve cancer metabolism and restore the microenvironment for both canine and human GL. The transcription factor hypoxia-inducible factor 1 alpha (HIF-1 alpha) drives metabolic reprogramming in gliomas (GLs) under hypoxic conditions, promoting glycolysis for tumor development. Evofosfamide (EVO) releases a DNA-alkylating agent within hypoxic regions, indicating that it may serve as a hypoxia-targeted therapy. The aim of this study was to investigate the glycolytic metabolism and antitumor effects of EVO in a canine GL model. Our clinical data showed that overall survival was significantly decreased in GL dog patients with higher HIF-1 alpha expression compared to that of those with lower HIF-1 alpha expression, and there was a positive correlation between HIF-1 alpha and pyruvate dehydrogenase kinase 1 (PDK1) expression, suggesting that glycolytic activity under hypoxia conditions may contribute to poor outcomes in canine GL. Our glycolysis assay tests showed that the glycolytic ATP level was higher than the mitochondrial ATP level in three types of canine GL cell lines by activating the HIF-1 signal pathway under hypoxia conditions, resulting in an overall increase in total cellular ATP production. However, treatment with EVO inhibited the glycolytic ATP level in the GL cell lines under hypoxia conditions by targeting HIF-1 alpha-positive cells, leading to decrease in total cellular ATP production. Our in vivo tests showed that EVO significantly reduced tumor development compared to controls and temozolomide in murine GL models. A metabolic analysis demonstrated that EVO effectively suppressed glycolytic metabolism by eliminating HIF-1 alpha-positive cells, suggesting that it may restore metabolism in canine GLs. The evidence presented here supports the favorable preclinical evaluation of EVO as a potential improvement in cancer metabolism.