METB-01
METABOLIC REPROGRAMMING DURING RESISTANCE TO ANTI-ANGIOGENIC THERAPY

Clinical trials revealed limited response duration of glioblastomas to VEGF-neutralizing antibody bevacizumab. Thriving in the harsh microenvironment created by anti-angiogenic therapy requires that tumors metabolically adapt to decreased nutrients. We investigated the hypothesis that metabolic reprogramming occurs during the evolution of anti-angiogenic therapy resistance. Bevacizumab treatment lowered glucose levels 3-fold in U87 xenografts (P < 0.05). We found 50% more glucose consumption in freshly isolated cells from bevacizumab-resistant U87-Bev^R versus responsive U87-Bev^S xenografts (P < 0.05). Characterizing expression of glycolytic enzymes revealed upregulation of the glucose transporter isoform GLUT3 in U87-Bev^R relative to U87-Bev^s xenografts (P < 0.001). Using a Seahorse extracellular flux analyzer to measure glycolysis and oxidative phosphorylation, we found that U87-Bev^R cells exhibited (1) greater proton leak, (2) less ATP turnover, (3) greater non-mitochondrial respiration with unchanged maximal respiration, and (4) greater extracellular acidification than U87-Bev^S cells, suggesting impaired mitochondria and increased glycolysis. Colorimetric assays and 13-C NMR showed increased pyruvate production and pyruvate to lactate conversion in U87-Bev^R versus U87-Bev^S cells (P < 0.05). U87-Bev^R cells produced more ATP than U87-Bev^S cells in glucose and hypoxia (P < 0.001). Mitochondria from U87-Bev^R cells in conditions selecting for oxidative phosphorylation produced less ATP than U87-Bev^S cells (P < 0.01). Mitochondrial membrane potential was higher in U87-Bev^S than U87-Bev^R cells, with hypoxia worsening each (P<0.05), and unchanged numbers of mitochondria (P>0.05). HSP60 was the only assessed mitochondrial enzyme with less expression in U87-Bev^R versus U87-Bev^S xenografts (P < 0.05). Oil Red O and thin layer chromatography revealed increased lipid (P < 0.05) and free fatty acids (P < 0.05) in U87-Bev^R versus U87-Bev^S xenografts, with unchanged fatty acid synthase expression (P > 0.05). We found bevacizumab resistance to be associated with greater GLUT3-mediated uptake of the reduced glucose levels, impaired mitochondrial health, and greater lipid production. Treatments targeting these potential points of weakness in resistant tumors should be developed and considered for use alongside anti-angiogenic therapy in glioblastoma.