Fig. 4

Loss of external melanoma cells upon docetaxel treatment hampers mechanistic explanation of drug effects. Tri-culture spheroids were generated by 3D cultivation of fibroblasts for three days, followed by simultaneous addition of keratinocytes and melanoma cells. HaCaT and SK-MEL-28 cells were labeled with CellTrackerRed CMPTX and CellTrackerGreen CMFDA dyes, respectively. After another two days, tri-culture spheroids were treated with 0.01 ‰ of DMSO as control or 100 nM docetaxel for 15, 24, 48, and 72 h. Spheroids were cryosectioned into 10-μm thick slices and stained for CAS3. a and b Representative confocal images of control (a) and docetaxel-treated cultures (b). In overlay images, CAS3 immunostaining signals, SK-MEL-28, HaCaT, and nuclei are depicted in red, green, yellow, and blue, respectively. Scale bars: 100 μm. (C) Quantification of CAS3-positive cells. Graph displays the amounts of CAS3-positive cells as mean ± SEM (n ≥ 3 independent experiments; * P < 0.05) in percent of the whole cell count per slice. For each experiment and time point, ≥ 3 spheroids were analyzed