Differential Regulation of Methylation-Regulating Enzymes by Senescent Stromal Cells Drives Colorectal Cancer Cell Response to DNA-Demethylating Epi-Drugs

Figure 4

Fibroblasts and conditioned medium-induced effects on HCT116 susceptibility to DAC in spheroid cultures and expression of EMT markers. (a) Images of high stromal cell coculture spheroids showing the presence of BJ cells in the spheroid interior. The z-plane heights are indicated on the top of images. HCT116 are nonfluorescent and the exterior of the spheroid is stained blue with Hoechst. 20x objective; scale bar: 50 μm. (b) Increase in EGFP intensity in coculture spheroids of HCT116 and BJ or sBJ cells compared to HCT116 monoculture spheroids. Data are mean ± SEM, spheroids per group from 3 independent experiments, , , Kruskal-Wallis test with Dunnet’s multiple comparisons test. (c) Representative images showing the effect of DAC on HCT116 monoculture spheroids and coculture spheroids of HCT116 and BJ fibroblasts. 20x objective; scale bar: 10 μm. (d) The effect of conditioned medium from normal and sBJ cultures on DAC-induced demethylation of HCT116 monoculture spheroids. Data are mean ± SEM, spheroids per group from 3 independent experiments, one-sample t-test. (e) Increase in the size of untreated coculture spheroids of HCT116 and BJ or sBJ compared to HCT116 monoculture spheroids (left) and no effect of DAC treatment on coculture spheroid size (right). Data are mean ± SEM, spheroids per group from 3 independent experiments, , , Kruskal-Wallis test with Dunnet’s multiple comparison test. (f) Representative Western blots showing the induction of vimentin expression in HCT116 sorted from high and low stromal cell coculture spheroids of HCT116 and sBJ.

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