Figure 3.

Formation of BM traversing actin protrusions and the impact of PI3-kinase inhibition on cell invasion. MCF10A acini were fixed and IF-stained. (A) 3D reconstructed confocal image stack demonstrating BM (collagen type IV, red) penetrating actin-based protrusions (F-actin, white) formed in physiological conditions within EHS matrix (fully matured hd-BM acinus after 31 days). (B) 3D cross-section through an hd-BM acinus with BM (red)-piercing F actin-rich protrusions (white) for proinvasive conditions (+EGF, 24 h on glass) including length measurement (yellow). (C) 2D cross-section of a marimastat treated ld-BM acinus (+EGF, 24 h on glass), indicating the formation of actin-rich protrusions (white arrow heads) piercing collagen type IV pores of the BM. (D) Rendered 3D image stack shows the acini–BM–substrate interface before BM transmigration started (hd-BM, 48 h on glass). Protrusive microspikes (F-actin, green) are visible in between the porous BM scaffold (collagen type IV, blue) reaching the underlying substrate. (E) Detailed view on a preinvasive hd-BM acinus adhered to a 3 kPa substrate demonstrates the colocalization of F-actin spots (green) and vinculin patches (red) within collagen pores (blue) at the BM–substrate interface. (F) The BM–substrate interface of an hd-BM acinus at the BM transmigration onset timepoint shows F-actin bundles with vinculin patches adjacent to the substrate plane (rigid glass). (G) The cumulative onset of invasion in ld-BM breast acini on stiff (12 kPa) substrate (+EGF) pretreated with the specific PI3-K inhibitor Wortmannin (ΔPI3-K). (H) Scatter plot showing the shift of the individual invasion onset timepoints of treated and untreated control groups. Bars: mean with 95% CI. n: analysed samples of at least three independent experiments. Grey plotted areas: complete invasion block for the first 17 h in wortmannin treated acini. Scale bars: 10 µm; n.s.: p ≥ 0.05; ****: p < 0.0001).