The Research Of Potent Suppression Mechanisms Of NK Cells Response Mediated By CA125in Epithelial Ovarian Cancer

[Background]Ovarian cancer is one of the most common type of cancer in female genital system. Themorbidity ranked the third in female genital system malignancy, but the mortality wasthe first. For these patients, although the highest standard of multimodality therapy withsurgery and cytotoxic chemotherapy, long term survival remains low and the5-yearsurvival remains less than30%. Conventional surgery and radiotherapy andchemotherapy are difficult to significantly improve the long-term survival rate ofpatients, especially survival rate of advanced stage patients, it is hightime to looking fornew methods of treatment of ovarian cancer.Natural killer cells are important immunocytes in the body. In addition, they can notonly application to tumor,s adoptive immunotherapy (AIT), but also eliminatepostoperation minimal residual and reduce recurrence of the disease as an adjuvanttherapy.The tumor-associated antigen CA125was discovered in the late1980s using the thennew monoclonal technology to develop antibodies for treatment of ovarian cancer,rather than for diagnosis of the disease. Over the last three decades CA125has beenevaluated for monitoring response to treatment, detecting recurrent disease anddistinguishing malignant from benign pelvic masses, as well as for early detection.While the potential for each of these applications became apparent soon afterdevelopment of an immunoassay, understanding the structure and function of CA125 developed more gradually, the current study indicates that CA125should be considerednot only as a biomarker, but also as a target that can contribute to the pathogenesis andprogression of epithelial ovarian cancer. In this study, we investigated the role forCA125in immunomodulatory, modulating tumor growth, invasive and metastaticproperties.[Objective]To explore the potent suppression of NK cell function was induced by CA125inepithelial ovarian cancer, provid experience basis for the application of adoptive NKcell-based immunotherapy to ovarian carcinoma.[Methods]1. In vitro:1) Cell-mediated cytotoxicity assay were performed to determine the effect of CA125on the ability of human NK cells to lyse the target cell line K562and the test NKcells were exposed to different concentrations of CA125for72h.2) FCM were used to find the phenotypic alteration associated with test NK cells.3) The test NK cells were monitored for apoptosis by flow cytometry using standardannexin V binding assays.4) Cell proliferation was evaluated using the CellTiter96AQueous One SolutionCell Proliferation Assay.2. In vivo:1) SKOV3cells (1×106) were transplanted into the left shoulder department skin ofCD1nu/nu nude mouse, in100μl RPMI1640medium or PBS.2) Tumor volumes were measured twice weekly and calculated using the followingformula:(length×width2)/2. 3) When CD1nu/nu nude mouse subcutaneous tumor grow until5weeks,open theskin, stripping subcutaneous tumor.3. Analysis of clinical data:1) Flow cytometry instrument and polymerase chain reaction (PCR) detection ofactivating and inhibitory receptor profile on PB-and PFNK of EOC patients.2) Secretion of cytokines IL-8, IL-12, IL-6, IL-10and TNF-α in EOC peritoneal fluidwas assayed with CBA Kits.3) Flow cytometry instrument detection of phenotypic changes of NK cells induced bythe PF.[Results]1. In vitro:1) Cell-mediated cytotoxicity assay were performed to determine the effect of CA125on the ability of human NK cells to lyse the target cell line K562.Test NK cellswere exposed to different concentrations of CA125for72h，we found that therewas significant concentration-dependent inhibition of NK cell-mediated killing ofK562cells after a72-h preincubation.2) Further more we decided to find the phenotypic alteration associated with test NKcells. Incubation of NK cells with CA125did not alter the expression ofCD226,Nkp30and Nkp46. On the other hand, CA125decreased the expression ofCD16,CD94/NKG2A,NKG2D and Nkp44, respectively, based on differences inMFI.3) The functional capacity of CA125does not induce apoptosis of NK cells iscompromised.2. In vivo:mice injected with CA125displayed a larger tumor burden through the peritoneal cavity when compared to the control (P=0.001).3. Analysis of clinical data:1）The phenotypic differences between these samples were shown, PT PBNK andPFNK appear to express lower amounts of NK cell receptors than their HD PBNKcounterparts. In our experiments,we confirmed NK cell receptors are also detected byRT-PCR experiments using two separate primer sets on mRNA isolated from healthydonors PBMC,but receptors expression in EOC PFNK are not obvious.2）Concentrations of Th1-related cytokines(IL-8, IL-12) were quite low,and levels ofTh2-related cytokines(IL-6,IL-10,TNF-α) were particularly higher. Ascitesenvironment obviously appeared Th2/Th1drift.3）The HD derived PBMCs were incubated in PF from five EOC patients and culturedfor72hr to at least partly mimic the long-term exposure scenario that the immune cellslikely encounter in the peritoneal environment. The average expression levels for eachone of these receptors appear lower in PF counterparts than control. In this case wecould prove Phenotypic changes of NK cells are induced by the PF.[Conclusion]In conclusion, we provide the direct evidence for a functional role of CA125. Ourresults demonstrated that CA125might inhibit NK cell cytotoxicity, and thus couldchange the immune microenvironment and affect the patients’ prognosis.