Anti-VEGF Induced Reduction in Microvessel Density Does Not Correlate with Anti-Tumor Repsonse in Lung Cancer Xenografts

Vascular endothelial growth factor-A (VEGF) is a primary stimulant of angiogenesis in pathological conditions including tumor progression. Strategies to block VEGF activity prevent or slow tumor growth in preclinical settings; however, clinical studies with bevacizumab, a monoclonal antibody (mAb) specific for VEGF have resulted in only modest benefit to a subset of patients with lung cancer. Previous studies in our laboratory defined the therapeutic efficacy of bevacizumab and an alternative anti-VEGF mAb (r84) in 12 non-small cell lung cancer (NSCLC) xenografts. Three NSCLC xenografts (Calu-6, A549 and Calu-3) showed intrinsic resistance to bevacizumab therapy. In the present study we evaluated whether microvessel density (MVD) could be used to 1) demonstrate if the anti-VEGF mAbs were effective at reducing VEGF-driven angiogenesis and 2) if MVD changes induced by bevacizumab or r84 correlated with overall therapeutic efficacy as determined by tumor size after chronic therapy. 3-5 tumors from animals bearing NSCLC xenografts treated with a control mAb (XTLl, bevacizumab or r84 were evaluated by immunohistochemistry for endothelial cells as a measure of microvessel density. Two independent endothelial cell markers were used, endomucin and CD31. In 11 of the 12 xenografts treatment with bevaclzumab or r84 significantly reduced MVD compared to XTL treatment, suggesting that bevacizumab and r84 do reduce VEGF-driven angiogenesis. However, the reduction in MVD induced by anti-VEGF therapy did not correlate with overall tumor response to therapy. These results strongly implicate resistance to anti-VEGF therapy is not mediated by activation af alternative angiogenic programs to compensate for VEGF blockade. Further the results suggest that tumor cell adaptation to therapy-induced hypoxia underlies poor therapeutic response to anti-VEGF strategies. Microarray of gene expression analysis of control treated tumors revealed several genes associated with metabolism, proliferation, and metastasis were significantly increased in tumors that displayed intrinsic resistant to bevacizumab. We conclude that response of tumor cells to therapy-induced hypoxia is a critical feature that drives the overall efficacy of anti-VEGF strategies.