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-Vascular Endothelial Growth Factor (VEGF) mAb, A4.6.1(200 micrograms twice weekly, i.p.), on angiogenesis and growth of tumor spheroids of human breast cancer cell lines (MCF-7, ZR-75 and, SK-BR-3) in nude mice. Furthermore, we investigated if in the presence of effective VEGF blockade, a conventional chemotherapeutic drug (doxorubicin, (5 mg/kg, weekly) could be effective, and if so would there be an additive effect of the combination regimen. Tumor Spheroids were implanted in dorsal skinfold chambers in nude mice. Tumor cells were pre-labeled with a fluorescent vital dye (CMTMR), which allowed the estimation of growth of implanted tumor spheroids. FITC (fluorescein isothiocyanate)-Dextran was used to evaluate formation of neo-vasculature at the tumor site. In control animals all three cell-lines produced extensive neovasculature and there was significant tumor growth throughout the observation period. Treatment with the anti-VEGF mAb caused significant suppression of angiogenic activity for all cell lines, stressing the critical role VEGF plays in breast tumor angiogenesis. Doxorubicin alone reduced the growth rate of MCF-7 cells, but did not significantly affect angiogenesis. Doxorubicin in combination with A4.6.1 resulted in significant tumors regression. Histology indicated that some chambers lacked viable tumor cells at the end of the two week observation period, lending strong support that neutralization of VEGF in combination with conventional cytotoxic agents could be a new innovative treatment regimen for metastatic breast cancer.