Importance of VEGF for breast cancer angiogenesis in vivo implications from intravital microscopy of combination treatments with an anti-VEGF neutralizing monoclonal antibody and doxorubicin

In the present study, we evaluated the effects of a neutralizing anti-Vascu lar Endothelial Growth Factor (VEGF) mAb, A4.6.1(200 mu g twice weekly, i.p .), on angiogenesis and growth of tumor spheroids of human breast cancer ce ll lines (MCF-7, ZR-75 and, SK-BR-3) in nude mice. Furthermore, we investig ated if in the presence of effective VEGF blockade, a conventional chemothe rapeutic drug (doxorubicin, (5 mg/kg, weekly) could be effective, and if so would there be an additive effect of the combination regimen. Tumor Sphero ids were implanted in dorsal skinfold chambers in nude mice. Tumor cells we re pre-labeled with a fluorescent vital dye (CMTMR), which allowed the esti mation of growth of implanted tumor spheroids. FITC (fluorescein isothiocya nate)-Dextran was used to evaluate formation of neo-vasculature at the tumo r site. In control animals all three cell-lines produced extensive neovascu lature and there was significant tumor growth throughout the observation pe riod. Treatment with the anti-VEGF mAb caused significant suppression of an giogenic activity for all cell lines, stressing the critical role VEGF play s in breast tumor angiogenesis. Doxorubicin alone reduced the growth rate o f MCF-7 cells, but did not significantly affect angiogenesis. Doxorubicin i n combination with A4.6.1 resulted in significant tumors regression. Histol ogy indicated that some chambers lacked viable tumor cells at the end of th e two week observation period, lending strong support that neutralization o f VEGF in combination with conventional cytotoxic agents could be a new inn ovative treatment regimen for metastatic breast cancer.