Figure 2.

Cancer cells-stroma interaction. (A) Cancer cell spheroids were added on top of stromal cells (BJ or MSC) monolayer and live and dead assay was performed after 48 h. (B) Cancer cell derived spheroids were added on 2D grown stromal cells (BJ or MSC) and live and dead assay was performed. Calcein (green)-alive cells; PI (red)-dead cells. The yellow color is the effect of the green/red overlapping. Both RH30 and HT29 cells stained negative for PI suggesting no occurrence of cell death. Bright yellow fluorescence for BJ-HT29 and MSC-RH30 images was due to image background signal in red. Scale bar = 100 µm. (C) LDH assay. BJ and MSC were cultured with spheroids for 48 h and LDH was measured. Absorbance fold change was calculated relatively to the control (BJ or MSC alone. Set value: 1). Both Kruskal-Wallis and Mann-Whitney tests were used to confirm the lack of toxicity. (D) GFP⁺ RH30 spheroids disperse their cells when in contact with BJ. MYOD positive staining demonstrate how cells migrate from the spheroid. (E) Supernatants of cancer cell spheroids were added on top of stromal cells (BJ or MSC) monolayer and live and dead assay was performed after 48 h. (F) Live and dead assay [calcein—green; and propidium iodide (PI) in red] in BJ and MSC cultured with the different spheroid supernatants and without supernatants. No death events (red signal) were found. (G) LDH assay. BJ and MSC were cultured with different supernatants for 48 h and LHD was measured. Absence of supernatant toxicity was confirmed by both Kruskal-Wallis and Mann-Whitney statistical tests. (H) Phase contrast microscopy show the morphology of the BJ and MSC after supernatant conditioning. n.s., not significant.