Fig. 3: Prrx1 orchestrates myofibroblast-like functions in CAFs across diverse cancer types, enhancing EMT and stemness of tumor cells.

A In public scRNA-seq data, human CAFs were virtually sorted into high (+)- and low (−)-PRRX1 CAFs. Both high- and low-Prrx1 CAFs were compared using module score analysis. Ex vivo fibroblast cultures of either Prrx1 WT or Prrx1 KO for each type of mouse fibroblast were established. Then, the two groups were compared for each type of fibroblast using GSVA analysis. Heatmap shows the module score from scRNA-seq of human CAFs and GSVA score from bulk RNA-seq of mouse fibroblast models. B After establishing PRRX1 promoter-driven model of SCAF36, apoptosis analysis was performed at each group (PRRX1 low and PRRX1 high). Experiments were conducted in triplicate. Bar plot showing percentage of cell apoptosis(%). Data are presented as the mean ± SEM; n = 3 independent experiments (two tailed t test: *p < 0.0001). C Gel contraction assay, depending on the expression of Prrx1 in fibroblasts. Representative images are shown. Data are presented as the mean ± SEM; n = 3 independent experiments (two tailed t test: *p < 0.005, **p < 0.0001). D Establishment of hanging drop direct 3D spheroid coculture system. Breast cancer cells 168FARN-Luc-GFP were cocultured with either MMTV-CAFs sgNS or sgPrrx1, and lung cancer cells LLC1-Luc-GFP were also cocultured with either wound-healing fibroblasts sgNS or sgPrrx1, using 3D spheroid systems. E H&E and immunohistochemical staining of representative spheroids in each group (DAPI: nuclei, GFP: Luc-GFP-tagged cancer cells). Bar plot showing quantification of spheroid size. Data are presented as the mean ± SEM; n = 7 independent experiments (two tailed t test: *p < 0.001). F GFP⁺ cancer cells were isolated from spheroids using FACS, and RNA-seq of these cancer cells was performed. G 168FARN-Luc-GFP⁺ cancer cells were isolated from spheroids using FACS, and then invasion, migration, and sphere forming assays were performed on isolated cancer cells. Data are presented as the mean ± SEM; n = 4 independent experiments (two tailed t test: *p < 0.005, **p < 0.0001). Source data and exact p values are provided as a Source Data file.