FIGURE 6.

Cancer-associated fibroblasts increased self-renewal and invasion of CSCs in a TGFβ-dependent manner via direct contact and paracrine effects. (A) Representative images of GFP⁺ A223 CSCs and RFP⁺ CAFs in spheroid co-culture. (B) Quantification of sphere number in each of the indicated A223 CSC treatment [vehicle (DMSO) or TGFβ inhibitor (“TGFβi”)] ± fibroblast co-culture conditions. (C,D) Representative images and quantification of A223 CSC spheres cultured with the indicated conditioned media (CM) and vehicle (DMSO) or TGFβi. (E) Transwell invasion assay: A223 CSCs were seeded in the top Matrigel-coated chamber, and CAFs or NAFs were cultured in the bottom well. After 48 h, invaded CSCs attached to the underside of the upper chamber were quantified. (F) TGFβ1 concentration in each of the indicated CM was determined by ELISA. Either three or four technical replicates were conducted for each cell type. (G) The CM of CAFs or unconditioned control media was applied to recipient A223 cells in a 24 h sphere initiation assay. RNA harvested from A223 recipient cells was evaluated by RT-qPCR for the expression of TGFβ-target genes Spp1, Junb, and Vegfa normalized to the expression of Gapdh. ^nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.