Fig. 4

a Schematic showing the steps of sequential culture experiment; b–d Scanning electron microscope (SEM) micrographs of sequential culture of MCF-7 cells at days (23 + 5), (23 + 10), and (23 + 15; white circles/ellipses represent tumoroids); e–g Sequential culture of MM 231 cells at days (23 + 5), (23 + 10), and (23 + 15). (Black circles/ellipses represent disorganized clusters) (X + Y days: MSCs were cultured on polycaprolactone (PCL)/in situ HAPclay scaffolds for X days, then cancer cells were seeded and culture was continued for Y more days); h–l Representative immunofluorescence microscope images of MM 231 and MCF-7 cells cultured in 2D and 3D sequential culture after immunostaining for nuclei, h E-cadherin, i vimentin, j, k vascular endothelial growth factor (VEGF), and l) cytokeratin 18. Scale bar: 50 μm. Abbreviations: Human breast cancer cell (HBCC); human breast cancer cell (HBCC) lines MDA-MB-231 (MM 231). Reprinted with permission from [117]