Lysophosphatidic acid modulates ovarian cancer multicellular aggregate assembly and metastatic dissemination.
ABSTRACT: Epithelial ovarian cancer (EOC) metastasis occurs by exfoliation of cells and multicellular aggregates (MCAs) from the tumor into the peritoneal cavity, adhesion to and retraction of peritoneal mesothelial cells and subsequent anchoring. Elevated levels of lysophosphatidic acid (LPA) have been linked to aberrant cell proliferation, oncogenesis, and metastasis. LPA disrupts junctional integrity and epithelial cohesion in vitro however, the fate of free-floating cells/MCAs and the response of host peritoneal tissues to LPA remain unclear. EOC MCAs displayed significant LPA-induced changes in surface ultrastructure with the loss of cell surface protrusions and poor aggregation, resulting in increased dissemination of small clusters compared to untreated control MCAs. LPA also diminished the adhesive capacity of EOC single cells and MCAs to murine peritoneal explants and impaired MCA survival and mesothelial clearance competence. Peritoneal tissues from healthy mice injected with LPA exhibited enhanced mesothelial surface microvilli. Ultrastructural alterations were associated with restricted peritoneal susceptibility to metastatic colonization by single cells as well as epithelial-type MCAs. The functional consequence is an LPA-induced dissemination of small mesenchymal-type clusters, promoting a miliary mode of peritoneal seeding that complicates surgical removal and is associated with worse prognosis.
Project description:During epithelial ovarian cancer (EOC) progression, intraperitoneally disseminating tumor cells and multicellular aggregates (MCAs) present in ascites fluid adhere to the peritoneum and induce retraction of the peritoneal mesothelial monolayer prior to invasion of the collagen-rich submesothelial matrix and proliferation into macro-metastases. Clinical studies have shown heterogeneity among EOC metastatic units with respect to cadherin expression profiles and invasive behavior; however, the impact of distinct cadherin profiles on peritoneal anchoring of metastatic lesions remains poorly understood. In the current study, we demonstrate that metastasis-associated behaviors of ovarian cancer cells and MCAs are influenced by cellular cadherin composition. Our results show that mesenchymal N-cadherin-expressing (Ncad+) cells and MCAs invade much more efficiently than E-cadherin-expressing (Ecad+) cells. Ncad+ MCAs exhibit rapid lateral dispersal prior to penetration of three-dimensional collagen matrices. When seeded as individual cells, lateral migration and cell-cell junction formation precede matrix invasion. Neutralizing the Ncad extracellular domain with the monoclonal antibody GC-4 suppresses lateral dispersal and cell penetration of collagen gels. In contrast, use of a broad-spectrum matrix metalloproteinase (MMP) inhibitor (GM6001) to block endogenous membrane type 1 matrix metalloproteinase (MT1-MMP) activity does not fully inhibit cell invasion. Using intact tissue explants, Ncad+ MCAs were also shown to efficiently rupture peritoneal mesothelial cells, exposing the submesothelial collagen matrix. Acquisition of Ncad by Ecad+ cells increased mesothelial clearance activity but was not sufficient to induce matrix invasion. Furthermore, co-culture of Ncad+ with Ecad+ cells did not promote a 'leader-follower' mode of collective cell invasion, demonstrating that matrix remodeling and creation of invasive micro-tracks are not sufficient for cell penetration of collagen matrices in the absence of Ncad. Collectively, our data emphasize the role of Ncad in intraperitoneal seeding of EOC and provide the rationale for future studies targeting Ncad in preclinical models of EOC metastasis.
Project description:High tumor kallikrein-related-peptidase 4 (KLK4) levels are associated with a poor outcome for women with serous epithelial ovarian cancer (EOC), for which peritoneal dissemination and chemoresistance are key events. To determine the role of KLK4 in these events, we examined KLK4-transfected SKOV-3 and endogenous KLK4 expressing OVCA432 cells in 3-dimensional (3D) suspension culture to mimic the ascites microenvironment. KLK4-SKOV-3 cells formed multicellular aggregates (MCAs) as seen in ascites, as did SKOV-3 cells treated with active KLK4. MCA formation was reduced by treatment with a KLK4 blocking antibody or the selective active site KLK4 sunflower trypsin inhibitor (SFTI-FCQR). KLK4-MCAs formed larger cancer cell foci in mesothelial cell monolayers than those formed by vector and native SKOV-3 cells, suggesting KLK4-MCAs are highly invasive in the peritoneal microenvironment. A high level of KLK4 is expressed by ascitic EOC cells compared to matched primary tumor cells, further supporting its role in the ascitic microenvironment. Interestingly, KLK4 transfected SKOV-3 cells expressed high levels of the KLK4 substrate, urokinase plasminogen activator (uPA), particularly in 3D-suspension, and high levels of both KLK4 and uPA were observed in patient cells taken from ascites. Importantly, the KLK4-MCAs were paclitaxel resistant which was reversed by SFTI-FCQR and to a lesser degree by the general serine protease inhibitor, Aprotinin, suggesting that in addition to uPA, other as yet unidentified substrates of KLK4 must be involved. Nonetheless, these data suggest that KLK4 inhibition, in conjunction with paclitaxel, may improve the outcome for women with serous epithelial ovarian cancer and high KLK4 levels in their tumors.
Project description: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.
Project description:UNLABELLED:More than 60% of patients who are diagnosed with epithelial ovarian cancer (EOC) present with extensive peritoneal carcinomatosis. EOC cells typically disseminate by shedding into the peritoneal fluid in which they survive as multicellular aggregates and then implant onto peritoneal surfaces. However, the mechanism that facilitates aggregation and implantation of EOC cells is poorly understood. The cell adhesion molecule P-cadherin has been reported to be induced during early progression of EOC and to promote tumor cell migration. In this study, P-cadherin not only promoted migration of EOC cells, but also facilitated the assembly of floating EOC cells into multicellular aggregates and inhibited anoikis in vitro. Furthermore, inhibiting P-cadherin by short hairpin RNAs (shRNA) or a neutralizing antibody prevented EOC cells from attaching to peritoneal mesothelial cells in vitro. In mouse intraperitoneal xenograft models of EOC, inhibition of P-cadherin decreased the aggregation and survival of floating tumor cells in ascites and reduced the number of tumor implants on peritoneal surfaces. These findings indicate that P-cadherin promotes intraperitoneal dissemination of EOC by facilitating tumor cell aggregation and tumor-peritoneum interactions in addition to promoting tumor cell migration. IMPLICATIONS:Inhibiting P-cadherin blocks multiple key steps of EOC progression and has therapeutic potential.
Project description:The interplay between peritoneal mesothelial cells and ovarian cancer cells is critical for the initiation and peritoneal dissemination of, and ascites formation in, ovarian cancer. The production of lysophosphatidic acid (LPA) by both peritoneal mesothelial cells and ovarian cancer cells has been shown to promote metastatic phenotype in ovarian cancer. Herein, we report that exogenous addition or ectopic overexpression of the matricellular protein SPARC (secreted protein acidic and rich in cysteine) significantly attenuated LPA-induced proliferation, chemotaxis, and invasion in both highly metastatic SKOV3 and less metastatic OVCAR3 ovarian cancer cell lines. SPARC appears to modulate these functions, at least in part, through the regulation of LPA receptor levels and the attenuation of extracellular signal-regulated kinase (ERK) 1/2 and protein kinase B/AKT signaling. Moreover, our results show that SPARC not only significantly inhibited both basal and LPA-induced interleukin (IL) 6 production in both cell lines but also attenuated IL-6-induced mitogenic, chemotactic, and proinvasive effects, in part, through significant suppression of ERK1/2 and, to a lesser extent, of signal transducers and activators of transcription 3 signaling pathways. Our results strongly suggest that SPARC exerts a dual inhibitory effect on LPA-induced mesothelial-ovarian cancer cell crosstalk through the regulation of both LPA-induced IL-6 production and function. Taken together, our findings underscore the use of SPARC as a potential therapeutic candidate in peritoneal ovarian carcinomatosis.
Project description:Ascitic multicellular aggregates (MCAs) promote peritoneal metastasis of ovarian cancer. The aim of the present study was to elucidate the role of cancer?associated fibroblasts (CAFs) in MCA formation and metastasis in patients with high?grade serous ovarian cancer (HGSOC). Immunohistochemistry was used to identify the cell phenotypes and the presence of CAFs in ascitic MCAs. The role of CAFs in tumor?cell MCA formation was assessed by co?culture in suspension. Primary ascitic tumor cells and omental CAFs were used to generate ex vivo MCAs in hanging drops, and the invasiveness of MCAs was evaluated by mesothelial clearance and adhesion assays in vitro and in vivo. MCAs containing CAFs and tumor cells were identified in the ascitic fluid. CAFs facilitated tumor cell aggregation and compaction to form MCAs, and enhanced the mesothelial clearance and adhesion abilities of tumor?cell MCAs. These findings suggest that ascitic CAFs promote peritoneal metastasis by forming heterotypic aggregates with tumor cells, and that they may serve as potential targets for the treatment of HGSOC.
Project description: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.
Project description: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.
Project description:The cause of death among the majority of epithelial ovarian cancer (EOC) patients involves passive dissemination of cancer cells within the peritoneal cavity and subsequent implantation of cancer spheroids into adjacent organs. Thus, it is important to identify the factors that mediate EOC metastasis and implantation, including clearance of the mesothelium. Sushi domain containing 2 (SUSD2) encodes a type I transmembrane protein containing several functional domains inherent to adhesion molecules. Immunohistochemical analysis determined the presence of SUSD2 in several subtypes of EOC, with the strongest staining observed in high-grade serous ovarian carcinomas (HGSOCs). A high-density, clinically annotated HGSOC tissue microarray was stained with an anti-SUSD2 antibody. Patients with tumors that had a low percentage of SUSD2 staining cells had a shorter median survival (31.7 months) compared with patients who had tumors with extensive SUSD2 staining (49.1 months; P-value=0.0083). To investigate the role of SUSD2 in HGSOCs, stable OVCAR3, OVSAHO and KURAMOCHI cell lines were established with knockdown (KD) or non-targeting (NT) of SUSD2. Boyden chamber and wound-healing assays demonstrated that OVCAR3, OVSAHO and KURAMOCHI SUSD2-KD cells migrated at significantly higher rates compared with their SUSD2 NT counterpart cell lines. Quantitative reverse transcription-PCR and western immunoblot analysis indicated an inverse relationship between SUSD2 and well-characterized mesenchymal proteins, including Twist-1, Zeb-1, N-cadherin, STEAP1, AHNAK, Snail-1, COL5A2 and Snail-3 in OVCAR3, OVSAHO and KURAMOCHI cell line models. In addition, OVCAR3 and KURAMOCHI SUSD2-KD spheroids displayed increased mesothelial clearance ability compared with cells that express endogenous levels of SUSD2. These data suggest that SUSD2 has a role in the inhibition of mesothelial clearance, which is required for metastasis. Altogether, our findings indicate that SUSD2 impedes migration, epithelial-to-mesenchymal transitional and mesothelial clearance of HGSOC cells, consistent with prolonged survival of patients with SUSD2-expressing tumors.
Project description:The transcriptomes of three immortalized ovarian surface epithelial cell lines (iOSE, PMID: 17266044) and primary OSE cells (Innoprot, Derio, Spain) and four immortalized fallopian tube secretory epithelial (iFTE) cell lines (PMID: 21502498, 22936217) were compared. RNA-sequencing was done from rRNA depleted total RNA (Ribo-Zero rRNA Removal Kit) to approx. 20 million 50 bp paired end reads per sample. A discriminative gene expression signature comprised of 211 genes was developed and used to classify isolated and EpCAM enriched primary ovarian cancer cells (PMID: 25991672). Impact of this signature on overall survival was assessed from several publicly available ovarian cancer gene expression data sets. Background: High grade serous ovarian cancer (HGSOC) is characterized by extensive local, i.e. peritoneal, tumor spread, manifested in two different clinical presentations, miliary (many millet sized peritoneal implants) and non-miliary (few large exophytically growing peritoneal nodes), and an overall unfavorable outcome. HGSOC is thought to arise from fallopian tube secretory epithelial cells, via so called serous tubal intraepithelial carcinomas (STICs) but an ovarian origin was never ruled out for at least some cases. Comparative transcriptome analyses of isolated tumor cells from fresh HGSOC tissues and (immortalized) ovarian surface epithelial and fallopian tube secretory epithelial cell lines revealed a close relation between putative origin and tumor spread characteristic, i.e. miliary from tubes and non-miliary from ovaries.