Site-directed biopsies of patient glioblastomas were performed with informed consent and IRB approval (number 11-06160). Author contributions MKA conceptualized the study. GLUT3 in tumor cells recapitulated bevacizumab-resistant cell features: survival and proliferation in low glucose, increased glycolysis, impaired oxidative phosphorylation, and rapid in vivo proliferation CCG 50014 only slowed by bevacizumab CCG 50014 to that of untreated bevacizumab-responsive tumors. Targeting GLUT3 or the increased glycolysis reliance in resistant tumors could unlock the potential of antiangiogenic treatments. Introduction Antiangiogenic therapies such as the VEGF-neutralizing antibody bevacizumab hold promise for the treatment of malignancies such as glioblastoma, a devastating brain cancer for which effective treatments are gravely needed. However, while the initial responses to antiangiogenic therapy are often significant, these agents have limited durations of response (1). Half of glioblastomas treated with bevacizumab acquire therapeutic resistance after an initial response (2). Acquired resistance to antiangiogenic therapy creates tumors we have found to have a poor prognosis (3, 4), owing to these resistant tumors exhibiting both increased invasiveness and increased proliferation (3), making it a significant problem and preventing these treatments from fulfilling their therapeutic promise. We (3, 5) and others (6) have found that increased hypoxia is a defining feature of tumors resistant to antiangiogenic therapy. We have previously reported hypoxia-induced autophagy as a mechanism by which tumors resistant to CCG 50014 antiangiogenic therapy survive the hypoxia induced by these treatments (5). However, the ability of these resistant tumors to thrive in the harsh microenvironment created by antiangiogenic therapy requires that they not only survive hypoxia but also metabolically adapt to CCG 50014 the decreased availability of glucose (7) that has been described after antiangiogenic therapy in order to achieve the increased proliferation we have described in these resistant tumors (3). As such, we investigated the hypothesis that a metabolic reprogramming towards more efficient glucose uptake and a more glycolytic phenotype with lesser mitochondrial capacity for oxidative phosphorylation occurs during the evolution of resistance to antiangiogenic therapy. In seeking to identify specific mediators of these changes, we found upregulation of GLUT3 to occur in 2 xenograft models of bevacizumab resistance and in patient specimens from bevacizumab-resistant glioblastomas, with GLUT3 promoting metabolic features of bevacizumab resistance in a targetable manner. Results Cells from a bevacizumab-resistant xenograft model shift towards glycolysis compared with cells from a paired isogenic responsive xenograft model. Using a colorimetric assay, we confirmed that 4 weeks of bevacizumab treatment of subcutaneous U87 glioma cell lineCderived xenografts lowered glucose levels in the tumor tissue more than 2-fold compared with IgG-treated xenografts (= 0.03; Figure 1A). We then investigated whether bevacizumab resistance was associated with a compensatory increase in the efficiency of glucose uptake to adapt to this treatment-induced low glucose microenvironment. To do so, we analyzed U87-BevS and U87-BevR, isogenic xenograft models of bevacizumab sensitivity versus resistance that we developed by serial multigenerational treatment of U87 xenografts with either bevacizumab or a control IgG antibody (8, 9). We found 50% more glucose uptake in freshly isolated cultured cells from bevacizumab-resistant U87-BevR versus sensitive U87-BevS xenografts, both under normoxic (= 0.03) and hypoxic (= 0.03) conditions (Figure 1B). To determine whether this adaptive increase in glucose uptake was associated with changes Rabbit Polyclonal to OR1A1 in glycolysis, we used a Seahorse extracellular flux analyzer to dynamically assess extracellular acidification as a measure of glycolysis in freshly isolated cells from U87-BevR versus U87-BevS xenografts. U87-BevR cells exhibited the same baseline extracellular acidification as U87-BevS cells (= 0.1C0.2). However, once stressed by inhibitors of mitochondrial oxidative phosphorylation, U87-BevR cells exhibited greater extracellular acidification than U87-BevS, consistent with greater stress-associated glycolysis (range: 0.001 to = 0.03; Figure 1C). Similarly, a colorimetric assay to measure glycolysis by detecting pyruvate production revealed no differences in pyruvate production between U87-BevR cells versus U87-BevS cells in normoxia (= 0.2), but increased pyruvate production in U87-BevR cells versus U87-BevS cells under the stress of hypoxia (= 0.008) (Figure 1D). Additional evidence supporting increased glycolysis in cells from bevacizumab-resistant xenografts came from 13C NMR.