EphA2 cleavage promotes single cell phenotype of MDA-MB-231 cells in vivo. Control, EphA2, or EphA2-D/I stably expressing MDA-MB-231 cells were orthotopically injected into the mammary fat pad of SCID mice. Tumor growth was followed for 4 wk. Tumor sizes were not significantly different (mock, 567 ± 96.25 mm³; EphA2, 320 ± 145.75 mm³; EphA2-D/I, 601 ± 81.43 mm³). (A and B) EphA2 and MT1-MMP immunohistochemistry of paraffin sections of the primary xenograft tumors. Corresponding areas (A, boxed regions) and the magnified insets (B) are marked with single and double asterisks. Arrowheads indicate invasive front cells. (C and D) Immunohistochemistry of representative metastatic tumor cell colonies in lymph nodes. The broken lines demarcate the border between tumor cells and host tissue. Arrowheads indicate singly invading cells with high MT1-MMP and intracellular EphA2. Boxes (D) indicate the areas magnified in the insets (C). Bars: (A–C) 50 µm; (D) 200 µm. (E) Quantitative assessment of intercellular spaces within tumor cell colonies in mouse primary tumors and lymph nodes (error bars indicate mean ± SEM). The measured example areas are shown in Fig. S3 G. Relative intercellular space of the mock tumors is set to one. *, P < 0.01, **, P < 0.05; Mann–Whitney U test.