Pirfenidone Reduces Epithelial–Mesenchymal Transition and Spheroid Formation in Breast Carcinoma through Targeting Cancer-Associated Fibroblasts (CAFs)

SIMPLE SUMMARY: Cancer-associated fibroblasts (CAFs) stimulate phenotypic transformation and acquisition of stemness in carcinoma cells. Targeting CAF-derived cytokines may suppress initiation of these events. This study aimed to show the inhibitory effects of pirfenidone on phenotypic transformation and stemness of cancer cells. To this end, we leverage the use of a 3D microfluidic device to analyze carcinoma progression phenotypes. We found that pirfenidone decreased tumor spheroid formation and epithelial–mesenchymal transition (EMT) the inhibition of cytokine production by CAFs. In the microfluidic model, we demonstrate that pirfenidone significantly inhibits the migration of carcinoma cells and CAFs. This study highlights the potential application of pirfenidone in suppressing invasion and potentially metastasis in breast cancer which can be further investigated in vivo. ABSTRACT: The aim of this study was to assess the effects of pirfenidone (PFD) on promoting epithelial–mesenchymal-transition (EMT) and stemness features in breast carcinoma cells through targeting cancer-associated-fibroblasts (CAFs). Using The Cancer Genome Atlas (TCGA) database, we analyzed the association between stromal index, EMT, and stemness-related genes across 1084 breast cancer patients, identifying positive correlation between YAP1, EMT, and stemness genes in samples with a high-stromal index. We monitored carcinoma cell invasion and spheroid formation co-cultured with CAFs in a 3D microfluidic device, followed by exposing carcinoma cells, spheroids, and CAFs with PFD. We depicted a positive association between the high-stromal index and the expression of EMT and stemness genes. High YAP1 expression in samples correlated with more advanced EMT status and stromal index. Additionally, we found that CAFs promoted spheroid formation and induced the expression of YAP1, VIM, and CD44 in spheroids. Treatment with PFD reduced carcinoma cell migration and decreased the expression of these genes at the protein level. The cytokine profiling showed significant depletion of various EMT- and stemness-regulated cytokines, particularly IL8, CCL17, and TNF-beta. These data highlight the potential application of PFD on inhibiting EMT and stemness in carcinoma cells through the targeting of critical cytokines.