Simple SummaryOne major challenge to understanding and targeting deadly epithelial ovarian cancer is the heterogeneous nature of the disease, including intrinsic physical and genetic differences in cells and differences in the spatial microenvironmental cues in the tumor. In this study, we show that microenvironmental cues that mimic tumor promoting aspects of the in vivo tumor niche can alter the pattern of clustered epithelial cancer cells and induce the formation of invasive subpopulations. We identified the invasive subpopulations based on analysis of single cell physical characteristics and underlying structural protein organization. Our analysis highlights that peripheral cancer cells in a cluster display increased invasiveness in response to the external cues.AbstractMetastatic progression of epithelial ovarian cancer (EOC) involves the partial epithelial-to-mesenchymal transition (EMT) of cancer cells in the primary tumor and dissemination into peritoneal fluid. In part to the high degree of heterogeneity in EOC cells, the identification of EMT in highly epithelial cells in response to differences in matrix mechanics, growth factor signaling, and tissue hypoxia is very difficult. We analyzed different degrees of EMT by tracking changes in cell and nuclear morphology, along with the organization of cytoskeletal proteins. In our analysis, we see a small percentage of individual cells that show dramatic response to TGF-β1 and hypoxia treatment. We demonstrate that EOC cells are spatially aware of their surroundings, with a subpopulation of EOC cells at the periphery of a cell cluster in 2D environments exhibited a greater degree of EMT. These peripheral cancer cells underwent partial EMT, displaying a hybrid of mesenchymal and epithelial characteristics, which often included less cortical actin and more perinuclear cytokeratin expression. Collectively, these data show that tumor-promoting microenvironment conditions can mediate invasive cell behavior in a spatially regulated context in a small subpopulation of highly epithelial clustered cancer cells that maintain epithelial characteristics while also acquiring some mesenchymal traits through partial EMT.