EMT induction in normal breast epithelial cells by COX2-expressing fibroblasts.

BACKGROUND: The tumor microenvironment (TME) plays a pivotal role in cancer progression, with cancer-associated fibroblasts (CAFs) significantly influencing tumor behavior. Especially, elevated COX2 expressing fibroblasts within the TME, notably in collagen-dense tumors like breast cancer, has been recently emphasized in the literature. However, the specific effect of COX2-expressing CAFs (COX2(+) CAFs) on neighboring cells and their consequent role in cancer progression is not fully elucidated. METHODS: We induced COX2(+) fibroblasts by forcing the fibroblasts forming aggregates to undergo Nemosis as a proxy for COX2(+) CAFs. This approach enabled us to simulate the paracrine interactions between COX2(+) CAFs and normal breast epithelial cells via conditioned media from COX2(+) fibroblasts. We developed an innovative in vitro platform that combines cell mechanics-based analysis and biomolecular assays to study the interactions between COX2(+) fibroblasts and normal breast epithelial cells. By focusing on the mechanical characteristics of the cells and the epithelial-mesenchymal transition (EMT) marker expressions, we aimed to elucidate the paracrine mechanisms through which COX2(+) CAFs influence the tumor microenvironment. RESULTS: Our in vitro findings demonstrate that COX2(+) fibroblasts, through conditioned media, induce significant alterations in the mechanical behavior of normal breast epithelial cells, as evidenced by monolayer expansion measurements using traction force microscopy (TFM). This transition was further corroborated by single-cell morphology and motility analyses, as well as increased expression of mesenchymal markers, including SNAI1 at the mRNA level and vimentin at the protein level. EP4 inhibition partially reversed these changes, preserving cell-cell interactions, limiting monolayer expansion, and reducing mesenchymal-like features, suggesting that PGE2-EP4 signaling plays a key role in mediating the paracrine effects of COX2(+) fibroblasts. Together, our findings support a model in which PGE2-EP4 signaling contributes to EMT induction, potentially involving SNAI1 regulation, with implications for targeting stromal-epithelial interactions in breast cancer. CONCLUSION: This study advances our understanding of the potential mechanisms by which COX2(+) CAFs influence tumor progression within the breast tumor microenvironment (TME) through controlled in vitro investigations. By integrating cell mechanics-based analysis, biomolecular assays, and innovative in vitro cell-based modeling of COX2(+) CAFs, we have delineated the contributory role of these cells in a controlled setting. These insights lay a groundwork for future studies that could explore the implications of these findings in vivo, potentially guiding targeted therapeutic strategies.