Inhibition of CAF-associated activity in vitro by PDE5i
(A) Fibroblast contraction analyzed by NOFs treated with TGF-β1 ± 50 μM vardenafil embedded in collagen-1 gel. TGF-β1 treatment induced α-SMA expression and gel contraction in normal fibroblasts, and co-treatment with vardenafil inhibited the upregulation of α-SMA and reduced gel contraction by 50%.
(B) CAF contraction was analyzed by collagen-1 gel contraction ± 50 μM vardenafil. Α-SMA expression and collagen-1 gel contraction is 2-fold greater in untreated CAFs compared with vardenafil- treated CAFs.
(C and D) Cancer cell invasion was analyzed by transwell assays using conditioned medium from fibroblasts as the chemoattractant. (C) Normal fibroblasts ± TGF-β1 ± vardenafil and TGF-β1-treated NOFs promoted invasion of FLO-1 cells with a 5-fold induction; this induction was abrogated by vardenafil treatment. (D) CAFs treated with vardenafil reduced invasion of FLO-1 cells by 60%.
(E) Organotypic co-culture of FLO-1 cells and CAFs also showed inhibition of invasion when treated with vardenafil. One-way ANOVA, p values: ∗p < 0.05, ∗∗p < 0.01. Results are representative of three independent experiments. See also Figure S1.