Fig. 1: C2C12 myotubes create a more restrictive environment for EO771 breast cancer cell outgrowth than MLg cells.

A Schematic representation of co-cultures. C2C12 and MLg cells were submitted to similar culture conditions for 6 days. At day 0 (D0), when C2C12 myotubes and MLg cells were fully confluent, EO771 cancer cells were seeded onto the cells. B Representative phase-contrast microscopy images of C2C12 and MLg stained with May-Grünwald and Giemsa at D0. Scale bar = 200 µm. C Number of EO771 cells per mm² attached onto C2C12 myotubes (C2C12 + EO771) and MLg cells (MLg + EO771) 3 h post-seeding at D0. D Outgrowth of EO771 cells (100 cells per well in 96-well plate) co-cultured with C2C12 myotubes (C2C12 + EO771) or MLg cells (MLg + EO771) for 2 days. EO771 area represents the percentage of fluorescent cells normalized to D0 (n = 21–24 from three independent experiments). Representative fluorescence microscopy images of EO771 cell outgrowth on C2C12 or MLg at day 2 (D2). Scale bar = 200 µm. E Outgrowth of EO771 cells in co-cultures with C2C12 myotubes or MLg cells when seeded at 500, 1000, 4000, and 8000 cells per well in a 96-well plate at day 0. Green fluorescence of EO771 was measured over 2 days without normalization (n = 24 from two independent experiments). F Representative fluorescence microscopy images of EO771 cell outgrowth (4000 cells per well in 96-well plate) on C2C12 or MLg. Scale bar = 200 µm. Data are mean ± SEM. Normality was tested with the Shapiro–Wilk test. Comparison between groups was done using the Mann–Whitney test (comparing two groups with one variable) or two-way ANOVA adjusted for multiple testing with the Šidák method (comparing two groups with two variables). ****P < 0.0001.