Peritoneal mesothelial cell secretory products increase invasion of endometrial stromal cells in an in vitro model of the early endometriotic lesion

Abstract

OBJECTIVE: We previously demonstrated that co-culture with peritoneal mesothelial cells (PMCs) increases invasion of endometrial stromal cells (ESCs) through Matrigel (MTGL), a basement membrane (BM) matrix. Also, conditioned media (CM) from cultured PMCs alters ESC expression of genes involved in invasion and metastasis. The purpose of the present study was to evaluate the effect of CM on ESC invasion through MTGL. DESIGN: In vitro study. MATERIALS AND METHODS: LP9 PMCs were grown to subconfluence and CM was collected. YHES immortalized ESCs were fluorescence labeled and cultured over MTGL-coated porous membranes. Invasion through the membranes was measured as previously described (1). Initially, the rate of invasion in the presence of CM was compared with control media (CTRL) containing 10% charcoal stripped, heat inactivated serum. Subsequently, the invasion of YHES cells in the presence of CM was compared to the rate of invasion in the presence of CM subjected to charcoal filtration as well as CM subjected to heat inactivation. We also evaluated the effect of CTRL supplementation with fibronectin (FN, 4-70 mcg/ml) and hepatocyte growth factor (HGF, 0.5 ng/ml), factors reported to be produced by cultured PMCs and implicated in cell invasion. RESULTS: Compared with CTRL, CM increased the rate of YHES ESCs invasion by greater than 20 fold (p<.02). Charcoal stripping and heat inactivation of the CM did not appreciably diminish the increased rate of invasion of YHES cells. Supplementation of CTRL with FN or HGF did not increase invasion compared with CTRL alone. Enzyme linked immunoabsorbant assay (ELISA) demonstrated an increased FN concentration in CM vs CTRL (3.9 mcg/ml vs 0.7 mcg/ml, respectively). ELISA demonstrated that HGF concentrations were similar in CM and CTRL (25.9 pg/ml and 31.6 pg/ml, respectively). CONCLUSIONS: While many investigators have concluded that PMCs act as a barrier to peritoneal invasion, the present study shows that PMC secretory products increase the rate of ESC invasion through BM proteins. Factors leading to this increased rate of invasion are not steroids, and are not heat labile. FN and HGF, previously reported to increase the rate of ovarian cancer cell invasion through MTGL (2,3), had minimal effects on ESC invasion. Studies are ongoing to elucidate which PMC product(s) lead to increased ESC invasion. 1. Nair AS et al. Fertil Steril, in press 2. Shibata K et al. Cancer Res 1997; 57:5416 3. Yoshida S et al. J Clin Endocrinol Metab 2004;89:823.