Reconstituting MT1-MMP–dependent EphA2 processing by ectopic expression induces repulsion and single-cell invasion of MDA-MB-453 cells. (A and B) The cells transiently coexpressing GFP and EphA2, EphA2-D/I, or EphA2-G/R with or without MT1-MMP were followed by time-lapse microscopy for 4 h; GFP- and EphA2-coexpressing cells were tracked. Pie charts show the percentage of non-detaching and detaching cells after collision (A; mean ± SEM). Representative time-lapse microscopy images visualizing track plots (shown in red); relative cell moving distances are indicated in each micrograph (B; mean ± SEM; five time-lapse analyses per cell line). Broken lines in EphA2-D/I– and MT1-MMP–coexpressing cells define the position of the tracked cells at the 0 time point. P-values were determined with a Mann–Whitney U test. Bar, 300 µm. (C) The cells were cultured within 3D collagen for 6 d. Representative confocal micrographs show EphA2 and MT1-MMP in representative cell colonies. Bar, 50 µm. (D) Quantitative assessment of single-cell invasion (error bars indicate mean ± SEM; three collagen preparations per cell).