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 .

Utilizing microfluidics to mimic the dynamic temporal changes of growth factor and cytokine concentrations in vivo has greatly increased our understanding of how signal transduction pathways are structured to encode extracellular stimuli. To date, these devices have focused on delivering pulses of varying frequency, and there are limited cell culture models for delivering slowly increasing concentrations of stimuli that cells may experience in vivo. To examine this setting, we developed and validated a microfluidic device that can deliver increasing concentrations of growth factor over periods ranging from 6 to 24 h. Using this device and a fluorescent biosensor of extracellular-regulated kinase (ERK) activity, we delivered a slowly increasing concentration of epidermal growth factor (EGF) to human mammary epithelial cells and surprisingly observed minimal ERK activation, even at concentrations that stimulate robust activity in bolus delivery. The cells remained unresponsive to subsequent challenges with EGF, and immunocytochemistry suggested that the loss of an epidermal growth factor receptor was responsible. Cells were then challenged with faster rates of change of EGF, revealing an increased ERK activity as a function of rate of change. Specifically, both the fraction of cells that responded and the length of ERK activation time increased with the rate of change. This microfluidic device fills a gap in the current repertoire of in vitro microfluidic devices and demonstrates that slower, more physiological changes in growth factor presentation can reveal new regulatory mechanisms for how signal transduction pathways encode changes in the extracellular growth factor milieu.

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<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>

<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>