Fig. 6

Podocalyxin-mediated collective invasion is actomyosin contractility dependent. a MCF-7-control and MCF-7-podo cell aggregates overlaid with collagen were fixed and immunostained for exogenous mouse podocalyxin (red) and either the actin cytoskeletal linker ezrin (green, upper panel) or the apical membrane marker Muc1 (green, lower panel). Single x/y-axis confocal images show that MCF-7-control cells localize ezrin to basolateral surfaces and small membranous pockets within the spheroid interior. Apical Muc1 localized to small, closed internal domains in the MCF-control cell aggregates. In contrast, MCF-7-podo cells predominantly localized ezrin and Muc1 to expanded microluminal surfaces within the elongated invasive aggregates. Scale = 20 μm. b MCF-7-control and MCF-7-podo cell aggregates were fixed and stained for exogenous mouse podocalyxin (red) and f-actin (green). Single x/y-axis confocal micrographs (top panel) and 3-D projected images (bottom panel) show an enrichment of f-actin along the expanded microlumenal surfaces within the MCF-7-podo cell aggregates where it colocalized with podocalyxin. Also, the outer cell surfaces of the podocalyxin aggregates exhibited prominent f-actin-rich, but podocalyxin-spare, membrane projections that probed the surrounding ECM (arrows). Scale bar = 40 μm. c, d MCF-7 cell aggregates were maintained in the presence of either DMSO (vehicle control) or the myosin II inhibitor blebbistatin (10 μM) for 4 days. Phase contrast images show that blebbistatin significantly blocked MCF-7-podo cells from collectively invading the matrix by elongation c. This is quantified in d. ***p <0.001, *p <0.05, Student’s t test; scale bar = 100 μm. DMSO dimethylsulfoxide