Figure 4. Co-culture experiments.

Panel (A) Schematic representation of the adopted synchronous seeding procedure, in which MCF10A and MDA-MB-231/GFP were seeded at the same time. Panels (B, C) Representative fluorescence confocal images of the S-size cages after the procedure schematized in panel (A) and after sample fixation and cell staining with Propidium Iodide. Panel B was acquired at green wavelengths, thus detecting signal only from MDA-MB-231/GFP, while panel C was acquired at red wavelengths, thus detecting signal from both cell lines. Panel (D) is the overlap between panels C and D. Panel (E) Percentage of invaded cages after 96 h of co-seeded MCF10A and MDA-MB-231/GFP at 10:1 concentration with the protocol displayed in panel A. Data are evaluated on three independent experiments and on a total of n = 15 S-cages. Error bar represents standard deviation. Panel (F) Average Number of MDA-MB-231/GFP inside S-sized cages after 96 h in co- and mono-cultures. Histogram shows the average number of cells invading a single cage in mono-culture (n = 10, red) and co-culture (n = 8, green) experiments. Error bars are standard deviation. Panel (G) Schematic representation of the adopted asynchronous seeding procedure, in which MDA-MB-231/GFP were seeded after 6 days of MCF10A culture, once confluency was obtained. Panels (H, I) Representative fluorescence confocal images of the S-size cages after the procedure schematized in panel G and after sample fixation and staining with Propidium. Panel H was acquired at green wavelengths, thus detecting signal only from MDA-MB-231/GFP, while panel G was acquired at red wavelengths, thus detecting signal from both cell lines. Panel (L) is the overlap between panels H and I. Scalebars in panels B-D, H-L represent 50 μm.