Selective Inhibition of Prostaglandin E2 Receptors EP2 and EP4 Inhibits Growth of Experimental Endometriosis in Nude Mice Evaluated by Real-time Noninvasive Imaging and Fluorescence Microscopy.

Endometriosis is a highly prevalent estrogen-dependent gynecological inflammatory disease of reproductive-age women characterized by the presence of functional endometrial cells outside the uterus commonly leading to inflammation, pain and infertility. Current anti-estrogen therapies can only be prescribed for a short time and result in undesirable side effects. Therefore, identification of novel signaling pathways that inhibit growth and function of endometriotic cells are needed not only to understand the pathogenesis of endometriosis but also to develop molecular-targeted therapies. The objectives of this study were to (1) develop a new in vivo model for real-time imaging and fluorescence guided assessment of endometriosis and (2) determine the effects of inhibition of PGE2 receptors EP2 and EP4 on the growth of endometriosis. Cell lines derived from endometriosis, epithelial cells (12Z) and stromal cells (22B) were transduced with green fluorescence protein (GFP) or red fluorescence protein (RFP) by lentiviral vectors, respectively. These stable 12Z-GFP and/or 22B-RFP cells were xenografted into the peritoneal cavity of bilaterally overectomized and estroginized nude mice on Day 1. Beginning on Day 14, mice were treated with different doses of EP2 and EP4 inhibitors for 2 weeks. The growth of 12Z-GFP and 22B-RFP cells were measured real-time using a NightOWL in vivo imager from Days 1-28 at weekly intervals. On day 28, the mice were euthanized. Xenograft of mixed populations of 12Z-GFP and 22B-RFP cells into the peritoneal cavity of nude mice established peritoneal endometriosis. A linear increase of 12Z-GFP or 22B-RFP cells was detected with the in vivo imager. Using a fluorescence stereo microscope, ~18-20 lesions per mice were detectable whereas gross examination or stereo microscopy revealed ~5-7 lesions per mice in the peritoneal cavity. Whole animal imaging and fluorescence stereo microscopy analyses indicated that selective inhibition of EP2 and EP4 decreased growth of endometriosis in a dose-dependent manner. Immunohistochemistry analysis indicated that inhibition of EP2 and EP4 decreased expression of p-ERK1/2 and p-AKT proteins in 12ZGFP and 22B-RFP cells. Histomorphology analysis indicated that xenograft of 12Z-GFP or 22B-RFP cells alone did not form endometrial glands. In contrast, combination of 12Z-GFP and 22B-RFP cells resulted in the formation of endometrial glands at ectopic sites. Results of the present study indicate that xenograft of 12Z-GFP and 22B-RFP cells into nude mice constitute a valuable model for functional genomics and experimental therapeutics to understand molecular pathogenesis of endometriosis. Selective inhibition of EP2 and EP4 receptors inhibited growth of endometriosis by suppressing ERK1/2 and AKT pathways and thus could emerge as potential nonsteroidal therapy for endometriosis. Supported by NIH/NICHD grants 1R21HD065138-01A1 and 1R21HD066248-01A1 to JAA.