Additional file 5: of A novel spheroid-based co-culture model mimics loss of keratinocyte differentiation, melanoma cell invasion, and drug-induced selection of ABCB5-expressing cells

Figure S5. Accumulation of external melanoma cells in tri-cultures is an active separation process. Tri-culture spheroids were generated by 3D cultivation of fibroblasts for 3 days, followed by simultaneous addition of keratinocytes and melanoma cells. HaCaT and SK-MEL-28 cells were pre-labeled with CellTrackerRed CMPTX and CellTrackerGreen CMFDA dyes, respectively. After another one (‘day 4’, upper row) or 2 days (‘day 5’, lower panels), tri-culture spheroids were cryosectioned into 10-μm thick slices and stained with Dapi. Representative confocal images are shown. While most melanoma cells were embedded in the keratinocyte ring on day four, they segregated from keratinocytes on day five and either accumulated in the periphery of the culture (‘external’ melanoma cells) or within the fibroblast core (‘internal’ melanoma cells). The fibroblast core is located in the center of the tri-culture and identified as Dapi-positive plus CellTracker-negative. Scale bars: 100 μm. (JPG 467 kb)