Cell shape changes and cytoskeleton reorganization during transendothelial migration of human melanoma cells

An in vitro system has been established to study the migration of human melanoma cells through a monolayer of endothelial cells. Endothelial cells were cultured to confluence on Matrigel before the seeding of melanoma cells. Laser scanning confocal microscopy showed that, prior to migration, melanoma cells appeared round and showed cortical F-actin staining. The initial stage of transmigration was characterized by numerous membrane blebs protruding from basolateral surfaces of the melanoma cells, and contact regions showed an abundance of filaments arising in the underlying endothelial cells. Later, pseudopods from the melanoma cells inserted into contact regions between endothelial cells. Eventually, the melanoma cells intercalated with the endothelial cells. At this stage, many endothelial filament bundles terminated at contacts between the endothelial cells and the transmigrating melanoma cell, suggesting active interactions between the two cell types. Upon contact with the Matrigel, melanoma cells began to spread beneath the endothelium, displaying a fibroblastic morphology with prominent stress fibers. To reestablish the monolayer, adjacent endothelial cells extended processes over the melanoma cell. Tumor necrosis factor α did not affect the transmigration of melanoma cells from cell lines isolated from several stages of metastasis. However, tumor necrosis factor did promote the transmigration of melanoma cells derived from a non-metastatic lesion. These results thus define cell attachment and cell penetration of the monolayer as two distinct steps in transmigration and suggest that tumor necrosis factor may enhance the metastatic potential of tumor cells.