Background Previous work in our laboratory demonstrated that antiprogestin mifepristone impairs the growth and adhesion of highly metastatic cancer cells, and causes changes in their cellular morphology. In this study, we further assess the anti-metastatic properties of mifepristone, by studying whether cytostatic doses of the drug can inhibit the migration and invasion of various cancer cell lines using a double fluorescence cytochemical labeling approach. Methods Cell lines representing cancers of the ovary (SKOV-3), breast (MDA-MB-231), glia (U87MG), or prostate (LNCaP) were treated with cytostatic concentrations of mifepristone. Wound healing and Boyden chamber assays were utilized to study cellular migration. To study cellular invasion, the Boyden chamber assay was prepared by adding a layer of extracellular matrix over the polycarbonate membrane. We enhanced the assays with the addition of double fluorescence cytochemical staining for fibrillar actin (F-actin) and DNA to observe the patterns of cytoskeletal distribution and nuclear positioning while cells migrate and invade. Results When exposed to cytostatic concentrations of mifepristone, all cancer cells lines demonstrated a decrease in both migration and invasion capacities measured using standard approaches. Double fluorescence cytochemical labeling validated that mifepristone-treated cancer cells exhibit reduced migration and invasion, and allowed to unveil a distinct migration pattern among the different cell lines, different arrays of nuclear localization during migration, and apparent redistribution of F-actin to the nucleus. Conclusion This study reports that antiprogestin mifepristone inhibits migration and invasion of highly metastatic cancer cell lines, and that double fluorescence cytochemical labeling increases the value of well-known approaches to study cell movement. Electronic supplementary material The online version of this article (10.1186/s12885-019-5587-3) contains supplementary material, which is available to authorized users.

Epithelial ovarian cancer (EOC) is considered the deadliest gynecological disease and is normally diagnosed at late stages, at which point metastasis has already occurred. Throughout disease progression, EOC will encounter various ecosystems and the communication between cancer cells and these microenvironments will promote the survival and dissemination of EOC. The primary tumor is thought to develop within the ovaries or the fallopian tubes, both of which provide a microenvironment with high risk of causing DNA damage and enhanced proliferation. EOC disseminates by direct extension from the primary tumors, as single cells or multicellular aggregates. Under the influence of cellular and non-cellular factors, EOC spheroids use the natural flow of peritoneal fluid to reach distant organs within the peritoneal cavity. These cells can then implant and seed distant organs or tissues, which develop rapidly into secondary tumor nodules. The peritoneal tissue and the omentum are two common sites of EOC metastasis, providing a microenvironment that supports EOC invasion and survival. Current treatment for EOC involves debulking surgery followed by platinum-taxane combination chemotherapy; however, most patients will relapse with a chemoresistant disease with tumors developed within the peritoneum. Therefore, understanding the role of the unique microenvironments that promote EOC transcoelomic dissemination is important in improving patient outcomes from this disease. In this review article, we address the process of ovarian cancer cellular fate at the site of its origin in the secretory cells of the fallopian tube or in the ovarian surface epithelial cells, their detachment process, how the cells survive in the peritoneal fluid avoiding cell death triggers, and how cancer- associated cells help them in the process. Finally, we report the mechanisms used by the ovarian cancer cells to adhere and migrate through the mesothelial monolayer lining the peritoneum. We also discuss the involvement of the transcoelomic ecosystem on the development of chemoresistance of EOC.

Background Simplistic two-dimensional (2D) in vitro assays have long been the standard for studying the metastatic abilities of cancer cells. However, tri-dimensional (3D) organotypic models provide a more complex environment, closer to that seen in patients, and thereby provide a more accurate representation of their true capabilities. Our laboratory has previously shown that the antiprogestin and antiglucocorticoid mifepristone can reduce the growth, adhesion, migration, and invasion of various aggressive cancer cells assessed using 2D assays. In this study, we characterize the metastatic capabilities of high-grade serous ovarian cancer cells generated along disease progression, in both 2D and 3D assays, and the ability of cytostatic doses of mifepristone to inhibit them. Methods High-grade serous ovarian cancer cells collected from two separate patients at different stages of their disease were used throughout the study. The 2D wound healing and Boyden chamber assays were used to study migration, while a layer of extracellular matrix was added to the Boyden chamber to study invasion. A 3D organotypic model, composed of fibroblasts embedded in collagen I and topped with a monolayer of mesothelial cells was used to further study cancer cell adhesion and mesothelial displacement. All assays were studied in cells, which were originally harvested from two patients at different stages of disease progression, in the absence or presence of cytostatic doses of mifepristone. Results 2D in vitro assays demonstrated that the migration and invasive rates of the cells isolated from both patients decreased along disease progression. Conversely, in both patients, cells representing late-stage disease demonstrated a higher adhesion capacity to the 3D organotypic model than those representing an early-stage disease. This adhesive behavior is associated with the in vivo tumor capacity of the cells. Regardless of these differences in adhesive, migratory, and invasive behavior among the experimental protocols used, cytostatic doses of mifepristone were able to inhibit the adhesion, migration, and invasion rates of all cells studied, regardless of their basal capabilities over simplistic or organotypic metastatic in vitro model systems. Finally, we demonstrate that when cells acquire the capacity to grow spontaneously as spheroids, they do attach to a 3D organotypic model system when pre-incubated with conditioned media. Of relevance, mifepristone was able to cause dissociation of these multicellular structures. Conclusion Differences in cellular behaviours were observed between 2 and 3D assays when studying the metastatic capabilities of high-grade serous ovarian cancer cells representing disease progression. Mifepristone inhibited these metastatic capabilities in all assays studied.

High-grade serous ovarian cancer (HGSOC) is a lethal disease often diagnosed in grade III International Federation of Gynecology and Obstetrics (FIGO) upon colonization of the peritoneal cavity, or in grade IV FIGO when it colonizes the pleural cavity. When in the pleural cavity, the disease often manifests with accumulation of pleural effusions containing multicellular structures (MCS). In this work, we studied whether MCS isolated from a pleural effusion of a patient that did not respond to chemotherapy are sufficient to cause peritoneal carcinomatosis in immunosuppressed mice when implanted orthotopically. Nude mice were injected intraperitoneally with MCS isolated from the pleural effusions of a heavily treated patient. We determined the time taken for the disease to develop, the tissues targeted, presence or absence of ascites, the macroscopic and microscopic disease appearance, and the expression of standard biomarkers of HGSOC. We contrasted the histopathology of the tumors recreated in mice against that of the original tumor taken during diagnostic surgery. MCS from pleural effusions were sufficient to recreate disease in the mice, which reached their humane-endpoint seven months following MCS injection (n=3). The animals developed abundant cellular peritoneal ascites. In addition, macro and microscopic discrete disease was observed in the fat surrounding the ovaries, the omental-pancreatic area, the peritoneal wall, a lymph node, the diaphragm, and the lung. The histopathology of the disease showed infiltrating papillary architecture, areas with cells arranged in a solid pattern, and tumor nests with slit-like lumens that stained positive for mutant p53, p16, and WT1. Our results provide proof of principle that MCS isolated from pleural effusions of a HGSOC patient are sufficient to recreate peritoneal carcinomatosis in nude mice, depicting most morphological features found at postoperative diagnosis. Citation Format: Alicia A. Goyeneche, Ashwyna Sunassee, Sabrina J. Ritch, Michael Koch, Maria Bell, Zu-hua Gao, Carlos M. Telleria. Multicellular structures of serous ovarian cancer nature isolated from pleural effusions are sufficient to recreate peritoneal carcinomatosis in immunosuppressed mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3001.

BackgroundReductionist two-dimensional (2D) in vitro assays have long been the standard for studying the metastatic abilities of cancer cells. However, tri-dimensional (3D) organotypic models provide a more complex environment, closer to that seen in patients, and thereby provide a more accurate representation of their true capabilities. Our laboratory has previously shown that the antiprogestin and antiglucocorticoid mifepristone can reduce the growth, adhesion, migration, and invasion of various aggressive cancer cells assessed using 2D assays. In this study, we characterize the metastatic capabilities of high-grade serous ovarian cancer cells generated along disease progression, in both 2D and 3D assays, and the ability of cytostatic doses of mifepristone to inhibit them.MethodsHigh-grade serous ovarian cancer cells collected from two separate patients at different stages of their disease were used throughout the study. The 2D wound healing and Boyden chamber assays were used to study migration, while a layer of extracellular matrix was added to the Boyden chamber to study invasion. A 3D organotypic model, composed of fibroblasts embedded in collagen I and topped with a monolayer of mesothelial cells was used to further study cancer cell adhesion and mesothelial displacement. All assays were studied in cells representing different stages of disease progression in the absence or presence of cytostatic doses of mifepristone.Results2D in vitro assays demonstrated that the migration and invasive rates of the cells isolated from both patients decreased along disease progression. Conversely, in both patients, cells representing late-stage disease demonstrated a higher adhesion capacity to the 3D organotypic model than those representing an early-stage disease. This adhesive behavior is associated with the in vivo tumor capacity of the cells. Regardless of these differences in adhesive, migratory, and invasive behavior among the experimental protocols used, cytostatic doses of mifepristone were able to inhibit the adhesion, migration, and invasion rates of all cells studied, regardless of their basal capabilities over reductionist or organotypic metastatic in vitro model systems. Finally, we demonstrate that when cells acquire the capacity to grow spontaneously as spheroids, they do attach to a 3D organotypic model system when pre-incubated with conditioned media. Of relevance, mifepristone was able to cause dissociation or “cleavage” of these multicellular structures.ConclusionDifferences in cellular behaviours were observed between reductionist 2D and 3D assays when studying the metastatic capabilities of high-grade serous ovarian cancer cells representing disease progression. Mifepristone inhibited these metastatic capabilities in all assays studied.

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.

Epithelial ovarian cancer (EOC) represents 90% of all ovarian tumors and is considered to be the deadliest gynecological disease. High grade serous ovarian cancer (HGSOC) is the most aggressive form of EOC and represents 70% of all deaths from the disease. Due to a lack of early symptoms and valid screening methods, HGSOC is often diagnosed at late stages, when metastasis has already occurred. HGSOC disseminates from the primary tumor as single cells or clusters that typically adheres to the peritoneal organs and wall. The purpose of this study was to characterize the migratory, invasive, and adhesive abilities of HGSOC cells representing different stages of disease evolution. Both wound healing (WH) and Boyden chamber (BC) assays were performed to study the migratory capacity of five HGSOC cell lines; PEO1, PEO4 and PEO6, from a first patient, and PEO14 and PEO23, from a second patient. The cell lines were generated along different stages of the disease (e.g. at presentation, recurrence, and before death), thus representing disease evolution. To study the invasive capacity of each cell line, a layer of extracellular matrix was added to the BC assays. Adhesion assays were performed on fibronectin coated plates. It was found that the migratory and invasive capacities of the HGSOC cell lines decreased along disease evolution in both cellular series. PEO1 and PEO14, two chemo-sensitive cell lines, were found to have the highest migratory and invasive capacities, while their chemoresistant counterparts, PEO4, PEO6 and PEO23, showed a decrease in migration and invasion. Moreover, a distinct migration pattern was found to be conserved in both WH and BC assays. Cell lines representing an early stage disease had a tendency to migrate as individual cells, while cells representing a late stage disease were observed migrating in clusters. Adhesion rates were found to be similar between all cells lines. The results suggest that HGSOC disease progression seems to be associated with reduced migratory and invasive capacities, contradicting the idea that advanced stage EOC cancers have higher metastatic potential. Citation Format: Sabrina J. Ritch, Alicia A. Goyeneche, Abu S. Noman, Carlos M. Telleria. The metastatic potential of high-grade serous ovarian cancer cells along disease progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2897.