Figure 5.

Single-cell RNA sequencing of co-cultured EAC cell lines and CAFs treated with PDE5i

(A) Cluster analysis of MFD-1 and primary CAFs based on differentially expressed genes from single-cell RNA sequencing analysis (n = 1,122 cells). First, cells were clustered as either cancer cells (MFD-1, blue) or fibroblasts (red).

(B) Further cluster analysis showed CAFs treated with PDE5 inhibitor vardenafil (CAFp) clustered separately from vehicle-treated CAFs (CAFv). PDE5i-treated co-cultured CAFs clustered with all other CAF cultures, whereas vehicle-treated CAFs co-cultured with MFD-1 show a phenotypic shift and clustered separately.

(C) Violin plots showing typical gene expression profiles for fibroblasts (THY1, DCN, ACTA2) or epithelial cells (KRT8, KRT18, EPCAM) show good separation between the clusters in A.

(D) Heatmap shows the top differentially expressed genes between treatment groups. CAFs and MFD-1 cells have the most distinct gene expression profiles, and although PDE5i treatment and co-culture both affect gene expression of both cell types, CAFs were most affected under both conditions.