We isolated three dimensional cell clusters from fresh human solid tumors and also isolated human neoplastic and normal lymphatic cells. Cells were cultured for 96 hours with and without bevacizumab and other agents. At concentrations of bevacizumab which completely removed VEGF from the culture medium, dead microvascular cells were detected through Fast Green/H&E staining as previously described. These peculiar staining characteristics suggested the involvement of calcium, and this was confirmed through staining with Alizarin red S. Using Alizarin staining as a marker for endothelial cell death permitted the use of public domain image analysis software which resulted in a sensitive and specific system for identifying active pharmaceuticals which target the tumor microvasculature at the same time direct antitumor cell effects are determined. Our results suggest an important role for calcium in endothelial cell death mediated by bevacizumab and other agents and further suggest that agents promoting calcium influx may potentiate the activity of antiangiogenic agents
