Cancer-associated fibroblasts (CAFs) recently demonstrated tumor-promoting roles in various cancer types, yet their implication in endometrial cancer (EC) has not been fully explored. We isolated fibroblasts (CAFs) and its epithelial counterpart from human EC tissues using CD90- and CD326-antibodies conjugated magnetic beads, respectively. We collected CAFs secretion by concentrating spent supernatants from cells growing in media containing 2% fetal bovine serum. Both human EC cell lines and primary EC epithelial cells showed increased cell proliferation in a dose-dependent manner, following treatment with CAFs secretion. This was in contrast to the growth inhibitory effects shown with secretions from normal endometrial fibroblasts. EC cells also demonstrated increased cell motility and invasiveness in response to CAFs secretion. Using cytokine array and ELISA, we further identified several cytokines which were overexpressed in CAFs compared to normal endometrial fibroblasts: macrophage chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), growth regulated oncogene (GRO), regulated on activation, normal T cell expressed and secreted (RANTES) and vascular endothelial growth factor (VEGF). These cytokines may activate MAPK/Erk and/or PI3K/Akt pathways to promote EC cell proliferation, as shown by immunoblotting and ELISA. Indeed, CAFs-mediated EC proliferation was suppressed independently by specific inhibitors for PI3K/Akt (LY294002) and MAPK/Erk (U0126). To determine if CAFs promote EC tumor proliferation in vivo, we inoculated fluorescently labeled-epithelial cell and CAFs subcutaneously at the flank of nude mice in the ratio of 1:2 and 1:4. Tumor sizes were measured twice a week using calipers and monitored weekly using in vivo imaging. Inoculation of CAFs and normal fibroblasts alone did not induce any tumor growth; however, co-injection of EC with CAFs (1:4) showed approximately 3 times higher growth rate when compared to EC alone. More interestingly, there was no sign of tumor growth in mice inoculated with combination of EC and normal fibroblasts (1:4) at the end of experiment. Taken together, our data suggests that CAFs exert tumor-promoting effects both in vitro and in vivo partly via specific cytokines-mediated activation of MAPK/Erk and PI3K/Akt pathways. Delineating the roles of CAFs in EC tumor microenvironment may provide potential therapeutic targets for the treatment of EC.

Citation Format: Ivy Chung, Kavita S. Subramaniam, Seng Tian Tham, Zahurin Mohamed, Noor Azmi Mat Adenan, Yin Ling Woo. Cancer-associated fibroblasts promote endometrial cancer cell proliferation in vitro and in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1078. doi:10.1158/1538-7445.AM2014-1078