Peritoneal metastasis of high-grade serous ovarian cancer (HGSOC) occurs when tumor cells suspended in ascites adhere to mesothelial cells. Despite the strong relationship between metastatic burden and prognosis in HGSOC, there are currently no therapies specifically targeting the metastatic process. We utilized a coculture model and multivariate analysis to examine how interactions between tumor cells, mesothelial cells, and alternatively-activated macrophages (AAM) influence the adhesion of tumor cells to mesothelial cells. We found that AAM-secreted MIP-1β activates CCR5/PI3K signaling in mesothelial cells, resulting in expression of P-selectin on the mesothelial cell surface. Tumor cells attached to this P-selectin through CD24, resulting in increased tumor cell adhesion in static conditions and rolling underflow. C57/BL6 mice treated with MIP-1β exhibited increased P-selectin expression on mesothelial cells lining peritoneal tissues, which enhanced CaOV3 adhesion and ID8 adhesion Analysis of samples from patients with HGSOC confirmed increased MIP-1β and P-selectin, suggesting that this novel multicellular mechanism could be targeted to slow or stop metastasis in HGSOC by repurposing anti-CCR5 and P-selectin therapies developed for other indications. This study reports novel insights on the peritoneal dissemination occurring during progression of ovarian cancer and has potential for therapeutic intervention. http://cancerres.aacrjournals.org/content/canres/78/13/3560/F1.large.jpg .

<div>Abstract<p>Peritoneal metastasis of high-grade serous ovarian cancer (HGSOC) occurs when tumor cells suspended in ascites adhere to mesothelial cells. Despite the strong relationship between metastatic burden and prognosis in HGSOC, there are currently no therapies specifically targeting the metastatic process. We utilized a coculture model and multivariate analysis to examine how interactions between tumor cells, mesothelial cells, and alternatively-activated macrophages (AAM) influence the adhesion of tumor cells to mesothelial cells. We found that AAM-secreted MIP-1β activates CCR5/PI3K signaling in mesothelial cells, resulting in expression of P-selectin on the mesothelial cell surface. Tumor cells attached to this <i>de novo</i> P-selectin through CD24, resulting in increased tumor cell adhesion in static conditions and rolling underflow. C57/BL6 mice treated with MIP-1β exhibited increased P-selectin expression on mesothelial cells lining peritoneal tissues, which enhanced CaOV3 adhesion <i>ex vivo</i> and ID8 adhesion <i>in vivo</i>. Analysis of samples from patients with HGSOC confirmed increased MIP-1β and P-selectin, suggesting that this novel multicellular mechanism could be targeted to slow or stop metastasis in HGSOC by repurposing anti-CCR5 and P-selectin therapies developed for other indications.</p><p><b>Significance:</b> This study reports novel insights on the peritoneal dissemination occurring during progression of ovarian cancer and has potential for therapeutic intervention.</p><p><b>Graphical Abstract:</b> <a href="http://cancerres.aacrjournals.org/content/canres/78/13/3560/F1.large.jpg" target="_blank">http://cancerres.aacrjournals.org/content/canres/78/13/3560/F1.large.jpg</a>. <i>Cancer Res; 78(13); 3560–73. ©2018 AACR</i>.</p></div>

<p>Supplemental File contains 8 Figures: SF1 shows a schematic of the co-culture device, SF2 shows additional details of PLSR model and validation of MIP-1β, SF3 shows validation of MIP-1β impact on SELP/P-selectin in mesothelial cell lines, SF4 shows CD24 expression in HGSOC lines, SF5 shows additional CD24 data for HGSOC lines and validation of CD24 siRNA knockdown, SF6 shows additional IHC of in vivo tissues for P-selectin, SF7 shows the impact of ascites on additional HGSOC lines, and SF8 shows additional immunofluorescent images of P-selectin in HGSOC patients. There are also 4 Tables: ST1 lists genes differentially expressed in LP-9 with AAM co-culture, ST2 lists soluble factors in Bioplex arrays that were not detected, ST3 provides CCR1 and CCR5 qRT-PCR results, and ST4 details the prognostic impact of CD24 in HGSOC patients.</p>

High-grade serous ovarian cancer (HGSOC) is the most lethal gynecologic cancer, and patients with HGSOC are often subject to extensive metastasis. The primary mode of metastasis in HGSOC is transcoelomic, where tumor cells detach and float in the peritoneal fluid before reattaching to the mesothelial-lined peritoneum. Levels of alternatively activated macrophages (AAMs) in ascites in the peritoneal cavity are correlated with disease stage, but it is unclear if AAMs play a role in transcoelomic spread. We hypothesized that AAM-secreted ligands enhance adhesion of ovarian cancer to mesothelial cells, which would support tumor progression. To address this hypothesis, we adapted a novel coculture device from our lab to create an in vitro model of the metastatic microenvironment and partial-least squares regression (PLSR) modeling to identify AAM-secreted ligands that enhanced adhesion. Methods: The device consists of two parallel culture surfaces (a well within a 24-well tissue culture plate and a glass coverslip) that are separated by a PDMS ring such that only paracrine interactions can occur between cells cultured on the surfaces. LP-9 or LP-3 mesothelial cells were seeded to confluency onto adsorbed collagen I within the PDMS ring and primary monocytes isolated from whole blood were differentiated to the M2 phenotype on the coverslip. Mesothelial cells were then cultured for 24 hours with a primary AAM coverslip, mimicking paracrine signaling between macrophages and the peritoneal mesothelium. As a control, mesothelial cells were cultured with a cell-free coverslip for 24 hours. Cell-Tracker Green-labeled ovarian cancer cells (OVCAR5, CaOV3, or OV-90) were seeded on top of the mesothelial cells and allowed to adhere for 3 hours. Nonadherent cells were washed off and the adherent cells were quantified. Media were collected from the devices and screened for 36 cytokines and matrix metalloproteinases using Bioplex assays. The data were analyzed using PLSR to identify secreted factors that correlated to the observed changes in cell adhesion. To validate the model, adhesion experiments were repeated using blocking antibodies and recombinant proteins. To examine the presence of these factors in patients, ascites fluid from patients with benign conditions or HGSOC were obtained under protocols approved by the Institutional Review Board at UW-Madison. Results and Conclusions: Coculture of primary AAMs with mesothelial cells increased ovarian cancer cell adhesion by at least 40% in all three cell lines. Bioplex analysis revealed that cytokines such as MIP-1β, MMP-7, and IL-13 were present in the media of AAM cultures. A PLSR model constructed with this data indicated a strong correlation between the ligands measured in the cultures and cell adhesion (R2Y= 0.95, Q2Y= 0.84). MIP-1β correlated strongly with adhesion and contributed highly to the model’s predictive capacity. To validate this model-predicted relationship, we experimentally tested the impact of inhibiting MIP-1β and found that while the baseline adhesion was unaffected, the ability of AAMs to enhance adhesion was lost with all three tumor cell lines. Similarly, treatment of mesothelial cells with MIP-1β alone was sufficient to increase tumor cell adhesion. Additionally, analysis of benign and HGSOC ascites showed elevated levels of MIP-1β in HGSOC patients. Overall, our findings suggest a role of AAMs in the multicellular process of transcoelomic spread of HGSOC, and identify MIP-1β as a potential target in the peritoneal cavity. Ongoing efforts are decoding the mechanisms by which MIP-1β increases adhesion between mesothelial and tumor cells. Citation Format: Molly J. Carroll, Kaitlin C. Fogg, Harin A. Patel, Anne-Sophie Mancha, Manish S. Patankar, Pamela K. Kreeger. Computational methods and novel in vitro model elucidate a therapeutic target against ovarian cancer metastasis. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A21.

<p>Supplemental File contains 8 Figures: SF1 shows a schematic of the co-culture device, SF2 shows additional details of PLSR model and validation of MIP-1β, SF3 shows validation of MIP-1β impact on SELP/P-selectin in mesothelial cell lines, SF4 shows CD24 expression in HGSOC lines, SF5 shows additional CD24 data for HGSOC lines and validation of CD24 siRNA knockdown, SF6 shows additional IHC of in vivo tissues for P-selectin, SF7 shows the impact of ascites on additional HGSOC lines, and SF8 shows additional immunofluorescent images of P-selectin in HGSOC patients. There are also 4 Tables: ST1 lists genes differentially expressed in LP-9 with AAM co-culture, ST2 lists soluble factors in Bioplex arrays that were not detected, ST3 provides CCR1 and CCR5 qRT-PCR results, and ST4 details the prognostic impact of CD24 in HGSOC patients.</p>