Yuliya Klymenko

Complex Determinants of Epithelial: Mesenchymal Phenotypic Plasticity in Ovarian Cancer

Unlike most epithelial malignancies which metastasize hematogenously, metastasis of epithelial ovarian cancer (EOC) occurs primarily via transcoelomic dissemination, characterized by exfoliation of cells from the primary tumor, avoidance of detachment-induced cell death (anoikis), movement throughout the peritoneal cavity as individual cells and multi-cellular aggregates (MCAs), adhesion to and disruption of the mesothelial lining of the peritoneum, and submesothelial matrix anchoring and proliferation to generate widely disseminated metastases. This exceptional microenvironment is highly permissive for phenotypic plasticity, enabling mesenchymal-to-epithelial (MET) and epithelial-to-mesenchymal (EMT) transitions. In this review, we summarize current knowledge on EOC heterogeneity in an EMT context, outline major regulators of EMT in ovarian cancer, address controversies in EMT and EOC chemoresistance, and highlight computational modeling approaches toward understanding EMT/MET in EOC.

Heterogeneous Cadherin Expression and Multicellular Aggregate Dynamics in Ovarian Cancer Dissemination

Epithelial ovarian carcinoma spreads via shedding of cells and multicellular aggregates (MCAs) from the primary tumor into peritoneal cavity, with subsequent intraperitoneal tumor cell:mesothelial cell adhesion as a key early event in metastatic seeding. Evaluation of human tumor extracts and tissues confirms that well-differentiated ovarian tumors express abundant E-cadherin (Ecad), whereas advanced lesions exhibit upregulated N-cadherin (Ncad). Two expression patterns are observed: “mixed cadherin,” in which distinct cells within the same tumor express either E- or Ncad, and “hybrid cadherin,” wherein single tumor cell(s) simultaneously expresses both cadherins. We demonstrate striking cadherin-dependent differences in cell-cell interactions, MCA formation, and aggregate ultrastructure. Mesenchymal-type Ncad+ cells formed stable, highly cohesive solid spheroids, whereas Ecad+ epithelial-type cells generated loosely adhesive cell clusters covered by uniform microvilli. Generation of “mixed cadherin” MCAs using fluorescently tagged cell populations revealed preferential sorting into cadherin-dependent clusters, whereas mixing of cell lines with common cadherin profiles generated homogeneous aggregates. Recapitulation of the “hybrid cadherin” Ecad+/Ncad+ phenotype, via insertion of the CDH2 gene into Ecad+ cells, resulted in the ability to form heterogeneous clusters with Ncad+ cells, significantly enhanced adhesion to organotypic mesomimetic cultures and peritoneal explants, and increased both migration and matrix invasion. Alternatively, insertion of CDH1 gene into Ncad+ cells greatly reduced cell-to-collagen, cell-to-mesothelium, and cell-to-peritoneum adhesion. Acquisition of the hybrid cadherin phenotype resulted in altered MCA surface morphology with increased surface projections and increased cell proliferation. Overall, these findings support the hypothesis that MCA cadherin composition impacts intraperitoneal cell and MCA dynamics and thereby affects ultimate metastatic success.

Aging Increases Susceptibility to Ovarian Cancer Metastasis in Murine Allograft Models and Alters Immune Composition of Peritoneal Adipose Tissue

Ovarian cancer, the most deadly gynecological malignancy in U.S. women, metastasizes uniquely, spreading through the peritoneal cavity and often generating widespread metastatic sites before diagnosis. The vast majority of ovarian cancer cases occur in women over 40 and the median age at diagnosis is 63. Additionally, elderly women receive poorer prognoses when diagnosed with ovarian cancer. Despite age being a significant risk factor for the development of this cancer, there are little published data which address the impact of aging on ovarian cancer metastasis. Here we report that the aged host is more susceptible to metastatic success using two murine syngeneic allograft models of ovarian cancer metastasis. This age-related increase in metastatic tumor burden corresponds with an increase in tumor infiltrating lymphocytes (TILs) in tumor-bearing mice and alteration of B cell-related pathways in gonadal adipose tissue. Based on this work, further studies elucidating the status of B cell TILs in mouse models of metastasis and human tumors in the context of aging are warranted.

Multiparity activates interferon pathways in peritoneal adipose tissue and decreases susceptibility to ovarian cancer metastasis in a murine allograft model

Ovarian cancer is the fifth leading cause of cancer deaths in U.S. women and the deadliest gynecologic malignancy. This lethality is largely due to the fact that most cases are diagnosed at metastatic stages of the disease when the prognosis is poor. Epidemiologic studies consistently demonstrate that parous women have a reduced risk of developing ovarian cancer, with a greater number of births affording greater protection; however little is known about the impact of parity on ovarian cancer metastasis. Here we report that multiparous mice are less susceptible to ovarian cancer metastasis in an age-matched syngeneic murine allograft model. Interferon pathways were found to be upregulated in healthy adipose tissue of multiparous mice, suggesting a possible mechanism for the multiparous-related protective effect against metastasis. This protective effect was found to be lost with age. Based on this work, future studies exploring therapeutic strategies which harness the multiparity-associated protective effect demonstrated here are warranted.

Abstract B6: Cadherin switching, multicellular aggregate dynamics and metastatic success

Epithelial Ovarian Carcinoma (EOC) is the fifth leading cause of womens cancer-related death and deadliest of all gynecological cancers with 22,240 new and 14,230 fatal cases predicted in 2013 ( American Cancer Society Estimates, 2013 ), primarily due to detection at late metastatic, prognostically poor stages of the disease. As opposed to other malignancies, EOC metastasis occurs by shedding of cancer cells from the primary tumor directly into the peritoneal cavity where they form multicellular aggregates (MCAs). These metastatic units along with single cells travel with the peritoneal fluid flow, adhere to peritoneum, migrate through mesothelial cell layer into submesothelial matrix wherein they subsequently proliferate into secondary tumor masses. The contribution of MCA dynamics to metastatic success is largely unknown. A feature that distinguishes EOC from most other carcinomas is an increase of E-cadherin (Ecad) expression at early stages of metastasis, frequently together with conserved expression of N-cadherin (Ncad). Our human tumor tissue array dual label immunofluorescence microscopy data confirm simultaneous expression of both cadherins in the majority of ovarian cancer tissues. Two patterns of expression are observed: “mixed cadherin” in which distinct cells within the same tumor express E- or N-Cad and “hybrid cadherin” wherein single tumor cells simultaneously express both cadherins. The mechanisms regulating initial cell detachment, MCA generation, survival in ascitic fluid and secondary anchorage and the role of cadherin dynamics in these steps remain poorly understood. Our research has revealed striking cadherin-dependent differences in cell-cell interactions, MCA formation, aggregate surface morphology and inner ultrastructure. In particular, mesenchymal-type DOV13 and SKOV3 (Ncad+) cells formed stable, highly cohesive smooth solid spheroids, while epithelial-type OvCa433 and OvCa429 (Ecad+) cells generated less adhesive cell clusters, loosely conglomerated and covered by uniform microvilli. To recapitulate the “hybrid cadherin” phenotype observed in human tumors, OvCa433 cells (Ecad) were transfected with the CDH2 (Ncad) gene to generate hybrid cadherin cells, leading to relevant changes in MCA surface morphology with increased superficial lamellipodia and filopodia, resembling the intermediate phenotype of true hybrid OvCa432 and OVCAR3 (Ecad+/Ncad+) aggregates. Alternatively, generation of “mixed cadherin” MCAs using fluorescently tagged cell populations revealed a sorting rather than mixing phenotype, with cells sorting into individual MCAs rather than generating heterogeneous Ecad/Ncad clusters. However, cell lines with common cadherin profiles [OvCa433/OvCa429 (Ecad/Ecad); DOV13/SKOV3 (Ncad/Ncad)] readily formed homogeneous aggregates. Interestingly, hybrid OvCa433 cells (Ecad/Ncad) formed heterogeneous MCAs with DOV13 (Ncad). Our data support the hypothesis that cadherin switching may regulate MCA dynamics and thereby affect metastatic unit cohesivity, survival, and ultimate metastatic success. Citation Format: Yuliya Klymenko, M. Sharon Stack. Cadherin switching, multicellular aggregate dynamics and metastatic success. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B6.

Methods for the visualization and analysis of extracellular matrix protein structure and degradation

This chapter highlights methods for visualization and analysis of extracellular matrix (ECM) proteins, with particular emphasis on collagen type I, the most abundant protein in mammals. Protocols described range from advanced imaging of complex in vivo matrices to simple biochemical analysis of individual ECM proteins. The first section of this chapter describes common methods to image ECM components and includes protocols for second harmonic generation, scanning electron microscopy, and several histological methods of ECM localization and degradation analysis, including immunohistochemistry, Trichrome staining, and in situ zymography. The second section of this chapter details both a common transwell invasion assay and a novel live imaging method to investigate cellular behavior with respect to collagen and other ECM proteins of interest. The final section consists of common electrophoresis-based biochemical methods that are used in analysis of ECM proteins. Use of the methods described herein will enable researchers to gain a greater understanding of the role of ECM structure and degradation in development and matrix-related diseases such as cancer and connective tissue disorders.

Modeling the effect of ascites-induced compression on ovarian cancer multicellular aggregates

ABSTRACT Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy. EOC dissemination is predominantly via direct extension of cells and multicellular aggregates (MCAs) into the peritoneal cavity, which adhere to and induce retraction of peritoneal mesothelium and proliferate in the submesothelial matrix to generate metastatic lesions. Metastasis is facilitated by the accumulation of malignant ascites (500 ml to >2 l), resulting in physical discomfort and abdominal distension, and leading to poor prognosis. Although intraperitoneal fluid pressure is normally subatmospheric, an average intraperitoneal pressure of 30 cmH2O (22.1 mmHg) has been reported in women with EOC. In this study, to enable experimental evaluation of the impact of high intraperitoneal pressure on EOC progression, two new in vitro model systems were developed. Initial experiments evaluated EOC MCAs in pressure vessels connected to an Instron to apply short-term compressive force. A Flexcell Compression Plus system was then used to enable longer-term compression of MCAs in custom-designed hydrogel carriers. Results show changes in the expression of genes related to epithelial-mesenchymal transition as well as altered dispersal of compressed MCAs on collagen gels. These new model systems have utility for future analyses of compression-induced mechanotransduction and the resulting impact on cellular responses related to intraperitoneal metastatic dissemination. This article has an associated First Person interview with the first authors of the paper. Summary: Development of new model systems with which to evaluate cellular responses to compression enables experimental modeling of the intraperitoneal force environment of ovarian cancer patients with tense ascites.

Epigenetic Crosstalk between the Tumor Microenvironment and Ovarian Cancer Cells: A Therapeutic Road Less Traveled

Metastatic dissemination of epithelial ovarian cancer (EOC) predominantly occurs through direct cell shedding from the primary tumor into the intra-abdominal cavity that is filled with malignant ascitic effusions. Facilitated by the fluid flow, cells distribute throughout the cavity, broadly seed and invade through peritoneal lining, and resume secondary tumor growth in abdominal and pelvic organs. At all steps of this unique metastatic process, cancer cells exist within a multidimensional tumor microenvironment consisting of intraperitoneally residing cancer-reprogramed fibroblasts, adipose, immune, mesenchymal stem, mesothelial, and vascular cells that exert miscellaneous bioactive molecules into malignant ascites and contribute to EOC progression and metastasis via distinct molecular mechanisms and epigenetic dysregulation. This review outlines basic epigenetic mechanisms, including DNA methylation, histone modifications, chromatin remodeling, and non-coding RNA regulators, and summarizes current knowledge on reciprocal interactions between each participant of the EOC cellular milieu and tumor cells in the context of aberrant epigenetic crosstalk. Promising research directions and potential therapeutic strategies that may encompass epigenetic tailoring as a component of complex EOC treatment are discussed.

Chemical Analysis of Morphological Changes in Lysophosphatidic Acid-Treated Ovarian Cancer Cells

Ovarian cancer (OvCa) cells are reported to undergo biochemical changes at the cell surface in response to treatment with lysophosphatidic acid (LPA). Here we use scanning electron microscopy (SEM) and multiplex coherent anti-Stokes Raman scattering (CARS) imaging via supercontinuum excitation to probe morphological changes that result from LPA treatment. SEM images show distinct shedding of microvilli-like features upon treatment with LPA. Analysis of multiplex CARS images can distinguish between molecular components, such as lipids and proteins. Our results indicate that OvCa429 and SKOV3ip epithelial ovarian cancer cells undergo similar morphological and chemical responses to treatment with LPA. The microvilli-like structures on the surface of multicellular aggregates (MCAs) are removed by treatment with LPA. The CARS analysis shows a distinct decrease in protein and increase in lipid composition on the surface of LPA-treated cells. Importantly, the CARS signals from cellular sheddings from MCAs with LPA treatment are consistent with cleavage of proteins originally present. Mass spectrometry on the cellular sheddings show that a large number of proteins, both membrane and intracellular, are present. An increased number of peptides are detected for the mesenchymal cell line relative to the epithelial cell indicating a differential response to LPA treatment with cancer progression.

Abstract TMEM-025: IMPACT OF MESOTHELIN EXPRESSION ON THE METASTATIC SUCCESS OF OVARIAN CANCER

Ovarian cancer is the most lethal gynecological cancer in U.S. women. Poor 5-year survival rates ( 90%. Metastasizing tumor cells grow rapidly and aggressively attach to the mesothelium of all organs within the peritoneal cavity, including the parietal peritoneum and the omentum, producing secondary lesions. Mesothelin (MSLN), a 40kDa glycoprotein that is over expressed in many cancers including ovarian and mesotheliomas is suggested to play a role in cell survival, proliferation, tumor progression and adherence. However, the biological function of mesothelin is not fully understood as MSLN knockout mice do not present with an abnormal phenotype. Conversely, MSLN has been shown to bind to the ovarian cancer antigen, CA-125, and thought to play a role in the peritoneal diffusion of ovarian tumor cells. Taking into consideration the potential importance of MSLN/CA-125 binding in ovarian tumor metastasis within the peritoneum, MSLN wild type (WT) and knockout (KO) mice were used to explore the role of mesothelin on the susceptibility of ovarian tumor cells to adhere to the mesothelium of the organs in the peritoneal cavity. An ex vivo peritoneal assay, using CA-125 positive human ovarian tumor cells OVCAR8-GFP and peritoneal explants from MSLN WT and KO mice demonstrated a decrease in OVCAR8-GFP cell adhesion to peritoneal tissues from MSLN KO mice compared to MSLN WT mice. Furthermore, allograft tumor studies using MSLN WT and KO mice injected intraperitoneally with fluorescently-tagged syngeneic murine ovarian cancer cells (ID8-RFP) was performed. Disease progression was evaluated post injection by fluorescent in vivo imaging prior to end point dissection (~8 weeks). Abdominal organs were dissected, imaged ex vivo and organ-specific tumor burden was quantified by tumor area. Tumor burden was significantly decreased in the liver and omentum of MSLN KO mice compared to MSLN WT mice. Together, the results demonstrate a loss of mesothelial cell-ovarian tumor cell adhesion in the omentum and peritoneum of mice that do not express MSLN. Citation Format: Tyvette Hilliard, Kyle Iwamoto, Elizabeth Loughran, Marwa Asem, Yueying Liu, Jing Yang, Laura Tarwater, Yuliya Klymenko, Jeff Johnson, Zonggao Shi, and M. Sharon Stack. IMPACT OF MESOTHELIN EXPRESSION ON THE METASTATIC SUCCESS OF OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-025.