Surface-bound ephrin-A5 triggers cytoskeletal reorganization, causing repulsion or adhesion of EphA3-positive tumor cells. (A) EphA3-positive LK63 human pre-B leukemia cells and LiBr melanoma cells were cultured on FN- or ephrin-A5-Fc–coated surfaces (ephrin-A5). For confocal microscopy, the cytoskeleton of fixed cells was stained with rhodamine-phalloidin. Scale bar represents 10 μm. (B) Dose-dependent adhesion of LK63 and de-adhesion of LiBr cells. LK63 leukemia and LiBr melanoma cells were seeded into wells of protein A–coated 96-well plates coupled with ephrin-A5-Fc at indicated densities. Soluble, monomeric ephrin-A5 was added as inhibitor (+inh) to parallel cultures at 100-fold molar excess. Adherent cells were quantitated by XTT assay (A₄₉₂ absorbance). Cell attachment is expressed as a percentage (mean, SE from 3 independent assays) relative to maximal adherence; ♦ represents LK63 cells; ▵, LiBr cells; ■, LK63 cells with ephrin inhibition (+inh); and ○, LiBr cells with ephrin inhibition. (C) Adhesion of LK63 cells onto ephrin-A5-Fc–coated coverslips was documented by live-cell imaging, starting immediately after first contact of the cell with the tissue culture surface. Cells were imaged every minute and representative micrographs are shown. Corresponding videos for images in the left and right columns, Video S1 and Video S2, respectively, are available as data supplements. (D) LK63 cells on ephrin-A5-Fc (ephrin)– or fibronectin (FN)–coated coverslips (“Methods”) were fixed and stained with Alexa⁴⁸⁸-phalloidin and anti-EphA3/Alexa⁵⁹⁴ secondary antibodies for confocal microscopy. Some cells were treated with 5 μM cytochalasin D or solvent (DMSO), 30 minutes before and during plating as indicated. Micrographs of typical anti-EphA3 (red), actin (green) fluorescence images, and merged images are shown. (E) The number/cell (■) and length () of filamentous protrusions of LK63 cells, treated with cytochalasin D (cytochal D) or solvent (DMSO control), was quantitated in 10 confocal microscopic field (100× lens) using IMARIS Filament Tracer software. Mean and SE estimates from n = 27 (DMSO) and n = 48 (control) cells are shown. Statistical analysis suggests significant differences (P < .001) in number and length of filamentous protrusions between treated and untreated cells.