Amphiregulin Upregulated in Esophageal Squamous Cell Carcinoma Cells upon Direct Contact with Cancer-Associated Fibroblasts. Amphiregulin Upregulated in Esophageal Squamous Cell Carcinoma Cells upon Direct Contact with Cancer-Associated Fibroblasts

Cancer-associated fibroblasts (CAFs) are a pivotal component of the tumor microenvironment, significantly contributing to the progression of esophageal squamous cell carcinoma (ESCC). Direct co-culture of human bone marrow-derived mesenchymal stem cells (MSCs), one of the origins of CAFs, with ESCC cell line, led to increased amphiregulin (AREG) expression and secretion in the ESCC cells. Consequently, the expression and phosphorylation of the AREG receptor EGFR were upregulated in co-cultured ESCC cells. Moreover, AREG treatment enhanced ESCC cell survival and migration through the EGFR-Erk/p38 signaling pathway. Immunohistochemical analysis of AREG using human ESCC tissues showed a positive correlation between the intensity of AREG expression in tumor invasive front and the expression level of the CAF marker FAP. Furthermore, bioinformatics database analysis confirmed a significant upregulation of AREG expression in ESCC tissues compared to normal tissues. These findings suggest that AREG is involved in CAF-mediated ESCC progression and could be a novel therapeutic target for ESCC. Overall design: MSCs and ESCC cells were seeded in the same dish and co-cultured for 4 days. Monocultures of both cell types served as controls. Following co-culture or monoculture, cells were washed with phosphate-buffered saline (FUJIFILM Wako Pure Chemical Corporation) and detached themselves from the dish using trypsin (FUJIFILM Wako Pure Chemical Corporation) treatment. The harvested cells were then mixed with anti-CD326 (epithelial cell adhesion molecule; EpCAM) microbeads (130-061-101; Miltenyi Biotec) and subjected to magnetic separation using the autoMACS Pro Separator (Miltenyi Biotec) to isolate high-purity tumor cells. cDNA microarray analysis was performed on monocultured and co-cultured TE-9 cells.