Abstract Due to the lack of early symptoms and valid screening methods, most patients with the most prevalent histotype of ovarian cancer, high-grade serous ovarian cancer (HGSOC), are diagnosed at late stage, at which point metastasis has already occurred. The purpose of this study was to characterize the migratory, invasive, and adhesive abilities of HGSOC cells, representing different stages of disease evolution, in both two-dimensional (2D) and three-dimensional (3D) organotypic assays. Five HGSOC cell lines, collected at different stages of disease progression were used throughout this study; PEO1, PEO4, and PEO6 cells were obtained from a first patient, whereas PEO14 and PEO23 cells were isolated from a second patient. Both wound healing (WH) and Boyden chamber (BC) assays were performed to study the migratory capacity of each cell line, while invasion was assessed by adding a layer of extracellular matrix to the BC assays. Finally, 2D adhesion assays were performed on fibronectin coated plates. On the other hand, 3D organotypic models composed of a mixture of collagen I and fibroblasts, topped with a monolayer of mesothelial cells, were constructed to further study the metastatic potential of all five HGSOC cell lines; HGSOC were plated on top of the mesothelial cells, and their adhesive behavior and capacity to displace mesothelial cells was followed by fluorescence microscopy. In 2D studies, the migratory and invasive capacities of the HGSOC cells decreased along disease evolution in both cell line series. Moreover, a distinct migration pattern was observed; cell lines representing early-stage disease had a tendency to migrate as individual cells, while cells representing late stage disease migrated in clusters. Also in 2D conditions, adhesion rates were found to be similar between all cell lines. Noteworthy, these results obtained in 2D conditions were somewhat contradicted by the results obtained in the 3D organotypic models, as both late-state disease PEO6 and PEO23 cells adhered more rapidly to the mesothelial cell monolayer than their early-stage counterparts. Furthermore, cell lines previously found to be tumorigenic in vivo such as PEO4, PEO6, and PEO14, demonstrated to trigger higher rates of mesothelial cell displacement in the 3D organotypic model system. This mesothelial cell displacement was also observed after exposure of mesothelial cells to conditioned media derived from PEO4, PEO6, or PEO14 cultures. In conclusion, 2D in vitro assays demonstrated lower migration and invasion capacities at late- rather than early-stage disease, while the opposite was observed in the organotypic models. Mesothelial cell displacement was found to be associated with tumorigenicity in vivo and to be independent of physical interactions between cancer cells and mesothelial cells. Citation Format: Sabrina J. Ritch, Alicia A. Goyeneche, Abu S. Noman, Carlos M. Telleria. Discrepancies in the metastatic potential of high-grade serous ovarian cancer cells representing disease progression when comparing two-dimensional assays versus three-dimensional organotypic culture model systems [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3456.
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