The mutual effect of MDA -MB -231 and MDCK cells in a 3D co -culture. (A) Co-culture of MDA-MB-231 and MDCK cells (5:1 ratio), cultured for 8 days and stained for GFP showing the MDA-MB-231 cells (green), f-actin (red) labeling the MDCK cells, and nuclei (blue). The mass of MDCK cells without any MDA-MB-231 cells attached (arrowheads) maintains the typical honey-comb pattern of cytoskeletal organization, whereas the MDCK cell masses that are in contact with MDA-MB-231 cells show disorganised f-actin arrangement. (B) A similar culture to A, stained for GFP showing the MDA-MB-231 cells (green), β-catenin (red) highlighting the MDCK cells, and nuclei (blue). The masses of MDCK cells without any MDA-MB-231 cells attached (arrows) maintain the typical honey-comb pattern of cytoskeletal organisation suggesting localisation of β-catenin to the cell membrane, whereas the large MDCK mass with MDA-MB-231 cells attached show β-catenin in the cytoplasm as well as on the cell membrane. (C) Snapshots of a time-lapse movie (Additional files 8: Movie 8) showing co-culture of MDA-MB-231 (green) and MDCK (red) cells (5:1 ratio). Throughout the first 25 hours, the MDA-MB-231 cells interact solely with one of the MDCK masses and keep the second MDCK mass intact (arrow). Once a large number of MDA-MB-231 cells fully surround the first mass, they move onto the second MDCK mass (33 h 40 m time frame). (D) A snapshot showing co-culture of MDA-MB-231 (green) and MDCK (red) cells (5:1 ratio), cultured in 3D for 11 days. The MDCK cells that are surrounded by MDA-MB-231 cells (arrow) display a debris-like structure. (E) A snapshot of Additional file 9: Movie 9 showing MDA-MB-231 and MDCK cells co-cultured in 3D. The MDCK cells (arrow) are surrounded and engulfed by the MDA-MB-231 cells. Some red particles of MDCK debris are seen in the cytoplasm of the MDA-MB-231 cells. (F) Snapshots of MDA-MB-231 (green) and MDCK (red) cells (20:1 ratio) co-cultured in 3D for 11 days. The population is now dominated by MDA-MB-231 cells. Only a few pieces of red debris are visible. (G) Co-culture of MDA-MB-231 and MDCK cells (1:20 ratio) for 8 days, stained for MDA-MB-231 (green) and for smooth muscle actin (red) mainly labelling MDCK cells. The MDCK cells maintain spherical structures while the MDA-MB-231 cells distribute around them. (H) A similar culture to (G), stained for MDA-MB-231 (green) and for β-catenin (red) mainly labeling MDCK cells. MDA-MB-231 cells do not appear to affect β-catenin expression in the MDCK cells. (I) Snapshots of a time-lapse movie (Additional file 11: Movies 11) showing a mix of MDA-MB-231 (green) and MDCK (red) cells at a ratio of 5:1 cultured for 11 days in 3D. In this field, there are only a few MDA-MB-231 cells attached to the surface of the MDCK sphere. In the first 11 hours, the MDA-MB-231 cells move while stuck to the surface of the sphere, and then suddenly appear to explode at the 11:00 hour time frame. Scale bars; 50 μm. The indicated time; hour:min.