Tumor cell diapedesis through human microvascular endothelial cell (HMVEC) monolayers. HBL100 cells were pre-labeled with dioctadecyl-3, 3,3', 3'-tetramethylindocarbocyanine percholate (DiI; red), co-cultured with HMVEC monolayers for various time periods, fixed with paraformaldehyde and labeled for F-actin (green). Optical sections obtained by laser scanning confocal microscopy at the focal levels indicated in (a,e,i) identified three major stages of diapedesis: round on top of (a-d), migrating through (e-h), or located underneath (i-l) the endothelium. (b) Cells round on top had filopodial extensions present on the apical surface of a cell (arrows). (g) Thick bundles of F-actin were present at the interface between endothelial cells and the migrating tumor cell (arrow). (h) Migrating tumor cells with portions spread underneath the endothelium (arrows) and (i) those that completed diapedesis often contained prominent stress fibers underneath the endothelium (arrows). No major morphological differences between wild-type and connexin43 (Cx43) expressing HBL100 cells were observed. Bar = 10 μm.