Invasive cell behavior is a component of normal (blastocyst implantation, extravasation) and pathogenic processes (metastasis, endometriosis), and is initiated by heterotypic contacts between cells. There is strong evidence, particularly in metastatic cancer, that gap junction (GJ) mediated intercellular coupling (GJIC) between the invading cells and target tissue plays a critical role. Yet gap junctions have not been studied in the most common invasive pathogenesis endometriosis. Endometriosis is thought to arise through retrograde menstruation of endometrial tissue to the peritoneum (occurring in most women), but there is active debate as to whether the disease is due to changes in the endometrium (the seed) or a permissive peritoneal environment (the soil). Project 1 demonstrated a striking downregulation of GJ genes in stromal cells (ESCs), and an inverse upregulation in epithelial cells (EECs) that follows the progression of the disease, and could serve as a diagnostic. Here we show that ESCs from endometriosis patients are unique in showing a large induction of GJIC upon interaction with peritoneal mesothelial cells (PMCs). This heterotypic GJIC was critical for the ESC invasiveness by inducing a disruption of the barrier function of the mesothelium. Conversely, PMCs also selectively affect ESCs from endometriosis patients by triggering Cx43 assembly into GJs, and by inducing a Mesenchymal to Epithelial Transition (MET). These results lend strong support the seed hypothesis. The current proposal builds on these results within the collaborative environment of a P01 that will allow a comprehensive analysis of how intercellular interactions in endometriosis lead to lesion formation. This will include understanding: (a) How GJ, and related adhesive genes, are regulated in endometriosis (Project 1); (b) how ESCs and PMCs initially recognize and stably adhere to one another (Project 3), leading to GJ formation, and; (c) how these GJs induce both modification of the PMC barrier function and promote ESC invasive behavior (Project 2). The goals of Project 2 will be achieved through three aims: Aim 1: Establish what functions of Cx43 are important for initiating invasiveness (i.e. GJIC, hemichannels, protein scaffolds) (1.1) and identify the molecular changes induced in PMCs that disrupt barrier function (1.2). Aim 2: Explore the converse effects of PMCs on endometriosis-derived ESCs by assessing if HA-CD44 interactions (Proj. 3), or other signals, induce heterotypic GJ formation (2.1), exploring the dependence of molecular changes induced in ESCs by PMCs (e.g. MET) on heterotypic GJIC (2.2), and whether PMCs derived from the peritoneum of control or endometriosis patients (the soil) differ in the effects they elicit (2.3). Aim 3: Validate the in vitro models for relevance to endometriotic lesion formation in a syngeneic mouse transplant model, exploring the roles of Cx43 in both endometrium (3.1) and peritoneal me...