Dynamic interaction of MDA -MB- 231 and MDCK cells in 3D culture. (A-C) 3D co-culture of; MDA-MB-231 and MDCK (A), MDA-MB-231 and MCF10A (B), MCF7 and MCF10A (C) cells at the ratio of 1:20 for 10 days. The cells were fixed and stained for GFP detecting either MDA-MB-231 or MCF7 cancer cells (green), f-actin (red) highlighting the non-cancerous epithelial cells, and nuclei (blue). In (A), most of the MDA-MB-231 cells are on the surface of a large MDCK mass, which does not show a typical cyst-like structure. In B and C, a few cancer cells (green) are integrated in the epithelial mass. (D-G) Snapshots of time-lapse movies (Additional file 4: Movies 4, Additional file 5: Movies 5, Additional file 6: Movies 6, Additional file 7: Movies 7) showing co-culture of MDA-MB-231 (green) and MDCK (red) cells. In (D) (see also Additional file 4: Movie 4), a protrusion of MDA-MB-231 cells (arrow) extends toward the MDCK cells. In (E) (see also Additional file 5: Movie 5), the MDCK cells remain static, at a distance from the MDA-MB-231 cells for the first nine hours and are then suddenly attracted towards the stream of MDA-MB-231 cells. Once the MDCK and MDA-MB-231 cells are in contact, the MDA-MB-231 cells rapidly surround the MDCK cells. In (F) (see also Additional file 6: Movie 6), several groups of MDCK cells are already in a large mass of MDA-MB-231 cells, however, at the periphery of the MDA-MB-231 mass, a few groups of MDCK cells are seen without MDA-MB-231 contact (0:00 hour time frame; arrows). These MDCK cells become surrounded by the MDA-MB-231 cells by the end of the film. (G) (see also Additional files 7: Movie 7) shows an example where there is a relatively small number of MDA-MB-231 cells present near the MDCK cyst. In the first 20 hours of filming, the MDA-MB-231 cells repeatedly attach to and detach from the MDCK sphere. Once several MDA-MB-231 cells begin to gather, they firmly attach to the MDCK sphere. The indicated time; hour:min. Scale bars; 50 μm.