Figure 1. VCAM-1 inhibition facilitates melanoma cell spreading on endothelial cells.

(a) Human endothelial cells were cultured without (top panel) or in the presence of TNFα (25 ng/ml, bottom panel) to stimulate expression of adhesion molecules. Expression of VCAM-1 was visualized by immunofluorescence. (b) Human endothelial cells stimulated with TNFα and expressing VCAM-1 were treated with an isotype-matched control antibody (left photomicrographs) or with a function-blocking antibody directed against VCAM-1 (right photomicrographs). Human melanoma cells (A375) fluorescently labeled with PKH26 were allowed to adhere for 40 min to these endothelial cell cultures. Images depict low (top panels) and high magnification (bottom panels), respectively. In the upper two photomicrographs, filled arrows indicate examples of round, non-spread melanoma cells, and open arrows indicate examples of spread melanoma cells. (c) The total numbers of melanoma cells adhered to TNF-stimulated endothelial cell cultures were quantitated (left graph). In addition, the numbers of non-spread (round) and spread melanoma cells, respectively, were quantitated separately both on endothelial cells treated with isotype-matched and VCAM-directed antibodies (right graph). The values shown represent averages from 12 random microscopic fields for each condition. The experiment was repeated twice. **indicates p<0.01, ***indicates p<0.001.