The Transcoelomic Ecosystem and Epithelial Ovarian Cancer Dissemination.

Ovarian cancer is considered the deadliest gynecological disease and the fourth leading cause of cancer death in women worldwide. Epithelial ovarian cancer (EOC) is the most common form of ovarian cancer and is classified into different histotypes. The various subtypes of EOC can be divided into Type 1 and Type 2 disease. Type 1 EOCs typically grow slowly, are diagnosed at early-stage disease (stage I or II) and tend to be TP53 wild type. Type 1 EOCs include mucinous, endometrioid, clear cell, and low-grade serous. Type 2 EOC tend to be quite aggressive, often diagnosed at late-stage disease (stage III or IV) and are typically P53 mutant. High-grade serous ovarian cancer (HGSOC), a type II EOC, is the most aggressive form of the disease and accounts for 70% of all deaths from EOC. HGSOC is most often detected at late stages of the disease when the cancer has already metastasized to distant sites within the peritoneal cavity. Initial treatment with a combination of platinum-based chemotherapy shows promising results in most patients, with 70% of patients responding. However, 95% of patients will eventually relapse with a more severe and chemoresistant disease ( 1 ). HGSOC metastasizes from the site of origin by direct extension from the tumor, either as single cells or as multicellular structures (MCS). These MCS use the natural rotational flow of peritoneal fluid to travel through the peritoneal cavity and reach distant sites where they establish themselves and form metastatic lesions ( 2 ). The peritoneal cavity is a closed off compartment in which the abdominal and reproductive organs reside. Organs within this cavity are covered in a protective layer of peritoneal tissue and are suspended in the peritoneal fluid. The peritoneal tissue is composed of an underlying stroma of fibroblasts embedded and dispersed within an extracellular matrix (ECM), primarily composed of collagen I, collagen IV, and fibronectin, and topped with a monolayer of mesothelial cells. The mesothelial cells play a protective role by providing a non-adhesive barrier allowing for the free movement of internal organs. This non-adhesive quality of the mesothelial cell layer is the result of the glycolax. Made up of glycoproteins, the glycolax is produced by the mesothelial cells and functions mainly to maintain hydration and reduce frictional forces against peritoneal organs ( 3 ). Under normal conditions, 5-20 mL of peritoneal fluid can be found within the peritoneal cavity. This fluid is mostly composed of ovarian exudate, plasma transudate, retrograde menstruation, and macrophage and mesothelial cell secretions. The peritoneal fluid contains a multitude of immune cells as well as floating mesothelial cells and functions to reduce friction between peritoneal organs, fluid exchange with plasma, and exchange of immune cells. The peritoneal fluid is constantly circulating within the peritoneal cavity in a circular motion, with gravity aiding with downward flow while pressure from respiratory movements lead to upward flow of the fluid. Under pathological conditions, it is common to observe an increase in the peritoneal fluid, often caused by a disruption in the balance between secretion and drainage. This accumulation is called ascites fluid and plays an important role in the dissemination of HGSOC ( 4 ). Throughout the process of dissemination, HGSOC cells encounter varying microenvironments that influence their progression. This review covers how HGSOC cells are influenced by the microenvironments at their site of origin in the secretory cells of the fallopian tube or the ovarian surface epithelium, their survival through the peritoneal fluid, and the adhesion and infiltration into the peritoneum. We also review the mechanisms within this transcoelomic ecosystem that lead to HGSOC chemoresistance.

When ovarian cancer cells detach from their site of origin, either in the ovary or the fallopian tubes, they tend to form MCSs or spheroids within the peritoneal cavity. Spheroids have many advantages over cancer cells travelling individually, including increased survival and ability to adhere and invade at a distant site. The number of spheroids found in ascites increases with advanced disease and has been associated with chemoresistance in patients ( 85 ). Spheroids detach from the primary tumor as clusters of cells, rarely mixing amongst each other once in the peritoneal fluid ( 86 ). Vitronectin and αv integrins have been observed between ovarian cancer cells in spheroids, and spheroids were unable to form when exposed to blocking antibodies against both adhesion proteins ( 87 ). Nectin-4, a cell adhesion molecule, has also been shown to play an important role in spheroid formation. HGSOC cells expressing Nectin-4 form compact spheroids in culture and this effect is inhibited when Nectin-4 is knocked down ( 88 ). The composition of ovarian cancer spheroids is still being debated. In many cases, the presence of cancer-associated fibroblasts (CAFs) and TAMs within the spheroids has been reported. One study showed that when ovarian cancer cells were injected into mice, spheroids formed with TAMs located at their center. These TAMs secreted epidermal growth factor (EGF), which increased surface adhesion molecules on both TAMs and ovarian cancer cells, further promoting the adhesion between the two cell types. Elevated EGF levels also increased levels of VEGF and its receptor on ovarian cancer cells, further promoting ovarian cancer cell proliferation and migration ( 89 ). A separate study found that spheroids collected from patient samples were PAX8 positive, a marker specific for EOC. In this case, TAMs were mainly located outside the ovarian cancer spheroids. Ovarian cancer cells making up the spheroids were found to have a mixture of epithelial and mesenchymal phenotypes, with mesenchymal cells located more in the center of the clusters ( 90 ). There is also evidence that in certain cases, CAFs can be found within the core of HGSOC spheroids. CAFs have been shown to enhance the invasion capacity of HGSOC cells by forming spheroids with cancer cells through integrin α5 and EGF secretion. CAFs lead these spheroids to the peritoneum and help with their survival ( 91 ). Epithelial-to-mesenchymal transition (EMT) is the process leading to a phenotypic change from an epithelial-like cell to a more mesenchymal-like cell. EMT is characterized by the loss of tight junctions between cells, a decrease in E-cadherin (E-Cad), an increase in N-cadherin (N-Cad) expression, and a more migratory and invasive behavior. Ovarian cancer cells seem to undergo EMT when forming spheroids and reverse this process when adhering to a distant site. HGSOC spheroids have reduced E-cad expression compared to the levels found in a primary tumor. This decrease in E-Cad in the spheroids is associated with an increase in mesenchymal markers, such as N-Cad, Snail, Twist1, Twist2, and Zeb2. EMT seems to be initiated by TGF-ß signaling in the peritoneal fluid ( 92 , 93 ). However, recent evidence has shown that the cadherin profile of ovarian cancer spheroids may not be as straightforward as previously believed. Many studies have demonstrated that spheroids may contain a mix of E-Cad+ and N-Cad+ cells, as well as hybrid cells which are positive for both E-Cad and N-Cad. Differences in the cadherin profile changes the compactness and the aggressiveness of the spheroid, with E-Cad+ dominant spheroids being less compact and aggressive than N-Cad+ dominant spheroids ( 94 , 95 ). The presence of mixed cadherin and hybrid spheroids suggests differing roles between the various cells within the spheroid. It has been suggested that the N-Cad+ cells provide a metastatic advantage at secondary sites while the E-Cad+ cells provide an advantage during the free-floating stage by promoting resistance to anoikis and chemotherapy ( 96 – 99 ). Anoikis is a form of apoptotic cell death initiated by the loss of cell adhesion. In order to survive transport by the peritoneal fluid, ovarian cancer spheroids must develop resistance to anoikis. The exact mechanism behind anoikis resistance is still unclear; however, the activation of integrin-dependent pathways seems to be the mechanism behind this resistance to cell death. α5ß3-integrin, a cell adhesion molecule, activates pro-survival pathways and the anti-apoptotic factor Bcl-2. When cells adhere to ECM, α5ß3 integrin activates FAK/PI3K/Akt signaling, which, in turn, promotes the transcription of Bcl-2. Many studies have suggested that this pathway plays an important role in the development of anoikis resistance in ovarian cancer spheroids ( 100 , 101 ). Protein kinase C (PKC) can also be activated by α5ß3-integrin leading to the activation of the ERK pathway. ERK1/2 phosphorylation has been found to promote ovarian cancer spheroid survival and play a role in the resistance to anoikis ( 102 ). A loss of adhesion to ECM can lead to an increase in ROS activation within ovarian cancer cells, which can trigger anoikis. Rho-specific guanine nucleotide exchange factor (Rgnef) is activated downstream of integrin pro-survival pathways and has been shown to reduce ROS activation through NF-κB activation ( 103 ).

The transcoelomic ecosystem in EOC plays a crucial role in the progression of the disease. The pro-inflammatory environment in both the OSE and the fallopian tubes provides opportunities for the accumulation of DNA damage, potentially leading to the formation of precursor lesions with increased proliferative capacities. The lack of anatomical barriers between the ovaries, fallopian tubes, and the fluid-filled peritoneal cavity allows for primary EOC tumors to influence the microenvironment within the peritoneal fluid and facilitate their dissemination and survival. EOC cells form spheroids that provide them with the ability to avoid immune detection and to resist to anoikis, while secreting factors that activate the tumor-supporting functions of surrounding macrophages. Cell-cell communication between EOC cells and cells within the peritoneal ecosystem further promotes the survival and dissemination of EOC, in particular to the peritoneal tissue and the omentum. Fibroblasts, mesothelial cells and adipocytes play crucial roles in inducing adhesion and invasion at distant metastatic sites. Finally, the cellular players involved in EOC dissemination all play a further role in the development of chemoresistance, a common occurrence in ovarian cancer. The understanding of the transcoelomic ecosystem in EOC is important in creating new therapeutic targets and furthering the development of novel treatment options for this devastating disease.

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The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The current treatment for HGSOC involves debulking surgery to remove any macroscopic masses, followed by platinum-based combination chemotherapy. The platinum-based agents approved in the clinic, cisplatin, carboplatin, and oxaliplatin, induce toxicity in ovarian cancer cells by generating DNA crosslinking, leading to DNA damage and triggering apoptosis. This treatment has remained unchanged since the approval of cisplatin in the 1980s followed by carboplatin in 1989, and oxaliplatin in 2000, even though the development of chemoresistance disease occurs in approximately 95% of patients. The 5-year survival rate after treatment has remained at less than 40% for the past three decades, creating a desperate need for new therapeutic options for this disease ( 1 ). There are three mechanisms known to promote chemoresistance in HGSOC: the reduction of intracellular influx of platinum, the control of DNA repair pathways, and the blocking of DNA damage-induced cell death ( 183 ). The complex microenvironment plays a large role in the progression of HGSOC as well as the development of chemoresistance through each one of these mechanisms. The microenvironment in ascites fluid contains immune cells, reprogrammed to have tumor-promoting functions. HGSOC cells treated with cisplatin have been shown to increase the polarization of macrophages towards a TAM phenotype by releasing IL-6 and PGE2 inflammatory cytokines. These TAMs showed increased expression of STAT3, known to promote tumor survival, and a decrease in STAT6 and STAT1, which are immune potentiators ( 184 ). A microenvironment composed of TAMs can further contribute to a chemoresistant phenotype in ovarian cancer. TAMs have been shown to release exosomes containing miR-223 that promotes drug resistance in ovarian cancer cells by blocking PTEN expression, a protein known to block cell survival pathways. This reduction allows for the increased expression of the PI3K/AKT cell survival pathway ( 185 ). CAFs play important roles in tumor progression as well as immune regulation and a multitude of evidence points to a potential role in the development of chemoresistance in HGSOC. CAFs reduce the uptake of cisplatin by HGSOC cells by increasing intracellular glutathione (GSH) levels. CAFs release high levels of GSH, which is then taken up by HGSOC cells. GSH can bind internalized cisplatin and the GSH-cisplatin complex effluxes from the cell. CD8+ T cells were found to counteract this effect by promoting GSH degradation through IFN-γ release ( 186 ). CAFs were also found to induce EMT, through the activation of the JAK/STAT3 pathway, in ovarian cancer cells by secreting IL-6 the JAK/STAT3 pathway leads to a decrease in platinum-induced apoptosis due to a decrease in the pro-apoptotic protein Bax and caspase-3 ( 187 , 188 ). Interestingly, ECM stiffness also plays a role in platinum resistance. HGSOC cells were found to be more resistant to chemotherapy when adhered to stiff substrate compared to a soft substrate. Specifically, substrates composed of collagen-6 and fibronectin were found to induce chemoresistance caused by an increase in integrin activation ( 189 ). HGSOC cells influence the pro-tumor functions of mesothelial cells through the release of TGF-ß. Osteopontin (OPN) is a glycoprotein released by mesothelial cells under the influence of TGF-ß and potentiates chemoresistance in ovarian cancer cells. It signals through the binding of CD44 and integrins on cancer cells and activates PI3k/AKT tumor survival pathway and the expression of ATP-binding cassette (ABC) transporters involved in drug efflux, a similar mechanism to the upregulation of copper transporters ATP7A and ATP7B that function to efflux platinum from the cell leading to chemoresistance ( 190 , 191 ). Another study showed that the upregulation of fibronectin on the surface of mesothelial cells, induced by TGF-ß, caused chemoresistance in ovarian cancer cells via the PI3K/AKT pathway ( 119 ). This study suggests a similar mechanism in which the binding of cell surface adhesion molecules on ovarian cancer cells activates a pro-survival signaling pathway. Adipocytes secrete arachidonic acid (AA), a fatty acid that induces chemoresistance in HGSOC cells by activating the PI3K/AKT survival pathway, thereby blocking apoptosis ( 192 ). Adipocytes release exosomes containing miRNAs, which can induce platinum resistance in ovarian cancer cells. miR21, found in exosomes released by both adipocytes and CAFs, downregulate APAF1, a protein found to be involved in chemoresistance and reduced apoptosis ( 193 ) Finally, HGSOC cells become chemoresistant when placed under the fluidic stress within the ascitic fluid. Ovarian cancer cells treated with cisplatin and paclitaxel under a microfluidic model had a higher viability than those treated under static conditions. Chemoresistance induced by fluid stress has been associated with activation of PI3K/Akt pathway and EGFR ( 194 , 195 ).

This research was funded through the OvCAN initiative of Ovarian Cancer Canada.

SR drafted the manuscript and compiled the figures. CT supervised the graduate student and revised and approved the final version of the manuscript.