Exploratory cytokine analysis of ascites from OC patients has been limited to date. Only a few studies focused on characterizing cytokines levels in OC ascites and the difference in the cytokine profile expression pattern that distinguishes between non-malignant and malignant ascites and between the various OC subtypes. Furthermore, the number of cytokines analyzed in these studies has been limited to 20 cytokines or less. One of the distinctive features of our study was the simultaneous measurement of 120 cytokines in OC ascites. The characterization of the tumor environment in which OC develops and metastasizes is essential for understanding how this environment alters the behavior of tumor cells. In the present study, the cytokine production patterns of OC ascites obtained from newly diagnosed patients were determined using a multiplex cytokine array that measures 120 different cytokines. Interestingly, our analysis has identified multiple cytokines present at high levels in most malignant ascites that, to our knowledge, have not been previously reported, including adiponectin, angiogenin, angiopoietin-2, GRO, ICAM-1, HGF, IGFBP-1, leptin, MIP-3α, NAP-2. It is also interesting to note that cytokines such as HCC-4, IL-6, IL-8, IL-10, leptin, MIP-1β, MIP-3α, PARC, PDGF, and TNF-R1 are differentially expressed in the serum of healthy women versus patients with OC [29
]. The differential production of some cytokines in ascites points toward an important role of these cytokines in the progression of OC. It is therefore not surprising to find growth factors or growth factor-related proteins such as EGF-R, Fit-3 ligand, and HGF, proinflammatory cytokines (IL-6, IL-6R and IL-10), and pro-angiogenic factors (angiogenin, angiopoietin, IL-8 and PDGF) among the cytokines present in OC ascites. For example, angiopoietin is an angiogenic factor that support angiogenesis during tumor growth [31
]. Angiogenesis in OC also plays a major role in the growth of disseminated tumors [32
]. Patients with OC have higher serum levels of angiopoietin than patients with benign or borderline tumors [33
]. The high production of angiopoietin in OC ascites is therefore consistent with its known function in angiogenesis.
Cytokine profiling of ascites could serve as a valuable tool to identify putative key soluble factors that determine the biological activity of ascites. Applying the multiplex cytokine array to a small number of malignant ascites enabled us to identify candidate cytokines which expression was subsequently validated in a larger cohort by ELISA. Using this approach, we show that IL-10 is one of the factors that promote the prosurvival activity of ascites. We demonstrate that IL-10 depletion from IL-10 containing ascites by an IL-10 blocking antibody partially inhibits the survival-promoting activity of these ascites against TRAIL, whereas the IL-10 neutralizing antibody does not affect the survival-promoting activity of an ascites that does not contain measurable levels of IL-10. Interestingly, the inhibitory effect of the IL-10 neutralizing antibody is selective for TRAIL as it does not alter the prosurvival activity of ascites against cisplatin. To our knowledge, IL-10 has not been previously associated with TRAIL resistance and further work is therefore needed to understand the mechanism by which IL-10 selectively decreases TRAIL-induced cell death but not cisplatin-induced cell death. Importantly, our data underscore the interplay that exists between different cytokines in OC ascites to regulate TRAIL-induced apopotosis. As OPG was identified as another potential candidate, we incubated different ascites with an OPG neutralizing antibody but we could not show any effect on the survival-promoting activity of ascites against TRAIL or cisplatin (data not shown). Nonetheless, these data underscore the potential of cytokine multiplex array to provide a framework for biomarker discovery in ascites.
In this study, we showed that high ascites levels of IL-10, OPG and leptin correlated with shorter PFS. Whether these cytokines represent independent prognostic factors remain to be established because multivariate analysis was not performed in this study mainly because of the small number of patients. Nonetheless, the presence of these cytokines in OC ascites and their association with patient’s outcome suggest that the tumor environment plays an active role in the progression of OC. IL-10 was detectable in about 75% of OC ascites. IL-10 is known to inhibit T helper cell proliferation, hamper dendritic cell maturation and inhibit co-stimulatory molecules [34
]. In this context, IL-10 is regarded as a potential immune suppressive component during tumor development. The high concentration of IL-10 in ascites and its association with shorter PFS might reflect its ability to allow tumor cells to escape from immune surveillance. IL-10 could also promote the prosurvival of ascites as shown it this study. OPG is a secreted member of the tumor necrosis factor (TNF) receptor superfamily that binds to the ligand for receptor activator of nuclear factor κB (RANKL) and inhibits bone resorption. OPG can also bind and inhibit the activity of TRAIL, raising the possibility that the physiologic mechanism of tumor surveillance exerted by soluble TRAIL may be abrogated in the ascites where OPG expression is high [37
]. This is supported by the observation that overexpression of OPG is associated with significantly worse overall survival and relapse-free survival in colon cancer patients [38
]. Moreover, overexpression of the OPG protein is an independent risk factor for colon cancer recurrence [38
]. Leptin is a 16KD adipokine produced predominantly by adipocytes and leptin mediated signaling pathways play an important role in cancer cell proliferation, invasion and metastasis [39
]. The role of leptin in OC development and progression has not been extensively investigated but available studies have shown that leptin stimulates OC cell growth at least in vitro
]. Further studies are needed to determine the precise role of these cytokines in malignant ascites.
In summary, this study provides the first in depth analysis of cytokine production in OC ascites from newly diagnosed patients. Comparative analysis of the cytokine profiles in OC ascites identified a subset of cytokines produced at high levels in OC ascites, which may contribute to the pathogenesis of OC. A number of these cytokines appeared to correlate with the clinical outcome and high levels in ascites were associated with shorter PFS. Among these cytokines, IL-10 was shown to promote the prosurvival activity of ascites. These data support the need to continue proteomic analysis of malignant ascites to understand the role of soluble factors. Cytokine array is a promising tool with which to characterize and prioritize key cytokines in malignant ascites.