Transendothelial migration of A375 melanoma cells.

(A) Migration and TEM assays. Labeled A375 melanoma cells were allowed to migrate for 16 hours in transwell inserts coated with collagen, fibronectin, uncoated polycarbonate, or an EC monolayer–coated membrane, as indicated. Bars represent the mean ± SD of the percentage of migrated A375 in relation to the total number of cells added to migrate, from 2 parallel experiments in duplicate of 12 independent experiments performed (n = 24). *Statistically significant at P < .001 compared with the other conditions. (B) A375 cells transmigrated across EC monolayers at lateral junctions. BCECF-labeled A375 cells were added to confluent HUVECs grown on collagen gels. After 2 hours (i, ii) and 16 hours (iii, iv), the cocultures were fixed and stained with silver nitrate as described. Green conventional fluorescence microscopy (i, iii) and corresponding light microscopy (ii, iv) micrographs are shown. An actively migrating melanoma cell is indicated by arrows in panel Bii. Interestingly, in the fluorescence image of transmigrated melanoma in panel Biii, silver staining is also visible of the intercellular junctions of the endothelial monolayer above because of its opacity, confirming restoration of the integrity of the EC monolayer after TC passage. (C) Confocal microscopy analysis of the distribution of VE-cadherin during melanoma cell transmigration. BCECF-labeled A375 cells were added to confluent HUVECs on dehydrated collagen gels and allowed to migrate for 2 hours. After fixation, coculture was stained for VE-cadherin. Serial horizontal (xy) sections distanced 1 μm apart, and a vertical (xz) section (lower panel) are shown.Z = 0 corresponds to the level of EC-EC junctions.