Dasatinib inhibits VEGF₁₆₅ and PDGF-BB induced proliferation, angiogenesis, and fibronectin-mediated adhesion of MM patient ECs. (A) Proliferation of HUVECs and ECs from a representative patient with MM at diagnosis (MM-PC, D), relapse (MM-PC, R), or leukemic progression (L) treated with the indicated doses (nM) of dasatinib for 48 hours (10⁴ cells/well). Data were expressed as percentage of growth inhibition. Error bars represent SD of triplicate experiments. (B) MMECs (with or without 10 ng/mL VEGF₁₆₅ or PDGF-BB) were premixed with 50 nM of dasatinib and seeded on a Matrigel surface for 16 hours (5 × 10³/well). Capillary formation was assessed by an inverted light microscope at ×4 and ×10. Photographs are representative of 3 independent experiments. (C) MMECs were serum-starved (SFM) for 16 hours and then incubated with VEGF₁₆₅ and PDGF-BB in the presence of DMSO, as a solvent control, or the indicated concentrations of dasatinib for 48 hours. Cell- cycle distribution was assessed by propidium iodide staining and FACS analysis. (D) MM patient-derived ECs (MMECs) in serum-free medium or treated with the indicated doses of dasatinib were plated (5 × 10³/well) in fibronectin- or BSA-coated 96-well plates in triplicate for 30 minutes. Cells were then fixed with 2.5% glutaraldehyde in PBS and quantified by a colorimetric assay (Cristal violet staining). Data are means (± SD) of 1 of 4 experiments. (E) Growth factor-deprived MMECs were pretreated with or without dasatinib for 30 minutes, plated on a fibronectin-coated polycarbonate membrane in a Boyden chamber, and exposed to 10 ng/mL VEGF₁₆₅ or PDGF-BB for 6 hours. Data are mean (± SD) of migrated cells in 5 fields (original magnification ×400). (F) Serum-starved MMECs (−) pretreated with the indicated doses of dasatinib (10-50 nM) for 30 minutes were stimulated with 10 ng/mL VEGF₁₆₅ or PDGF-BB for 15 minutes. Whole cell lysates were prepared and probed with the indicated antibodies.