Ovarian Epithelium Carcinoma Induce Abnormal Differentiation Of Dendritic Cell Precursors

IntroductionEpithelial ovarian cancer(EOC)is the fifth most common cause of cancermortality in women.Because of its early and rapid but mainly located metastasis in theperitoneal cavity even at the advanced stage,it has been proposed that there is a localimmune deficiency in tumor microenvironment.Tumor microenvironment is mainly composed of tumour cells,stromal cells,theextracellular matrix and body immune system.Studies carried out on tumormicroenvironment are focused on cytokines,dendritic cells(DC),T cells,NK cells,andso on.Researchers have found that inbalaneced expression of Thl/Th2/Th3 cytokines intumor microenvironment played an important role in changing immune response towarda Th2 cytokine profile,which prevents the anti-tumor reactivity by Th1 response.However,the source of elevated Th2 cytokines in local rnicroenvironment of carcinomapatients is complicated and heterogeneous.The direct evidence that EOC cells produce more Th2 cytokines and other immune inhibitory factors remain a subject for debate.EOC mainly arises in ovarian surface epithelium(OSE)cells.This modifiedmesothelium also has the ability to secrete bioactive inflammatory cytokines,whichplay an important role in lysis and repair of ovarian surface during ovulation.Little isknown,however,about the profiles of secretion between EOC cells and OSE cells,andthis may be an important factor in the local immune deficiency of EOC patients.As the most important immunity cells in tumour microenviroment,the deficiencyof DC function may be the main factor in tumor escaping from host immunity system.DC is the most important and potent professional antigen presentation cells(APC).Twomain subtypes are described as myeloid DC(mDC)and plasmacytoid DC(pDC).ThemDC is well characterized so as to stimulate a primary T-lymphocyte response,whilepDC was found to induce naive T cells differentiating into regulatory T cells withsuppressive activity.The defects of DC in tumor microenvironment are decreasednumber of mature mDC,accumulation of immature mDC,and increased number ofinhibitory pDC.But it is unclear that whether this is associated with carcinoma cells.Ithas been demonstrated that tumor cells can secrete cytokines to regulate thedifferentiation,maturation,and activation of DC.But it is also unclear that if thishappens in EOC patients.It is important to study the cytokines expression of EOC andto ensure that elevated Th2 cytokines derived from EOC cells have influence to DCdifferentiation and maturation.In this study we used cytokine antibody array to screen the different expressionprofiles of EOC cells and OSE cells,and then to confirm it in vivo.In order to study theeffect of supernantants of cultured EOC cells on DC precursors differentiation,maturation,and function,we also established model of DC precursors differentiation in vitro.In the last part of the study,we used antibody neutralization tests to confirm theeffect of cytokines secreted by EOC cells on DC.The subject of this study is todemonstrate the mechanism of EOC miroenvironment defect,and to offer theoryevidence to improve DC vaccine for EOC. PARTⅠDifference of cytokine expression between epithelialovarian carcinoma and normal ovarian epitheliumObjective:The aim of this study was to detect different expression of cytokines in epithelialovarian carcinoma(EOC)cells and normal ovarian surface epithelial(OSE)cells invitro and the levels of those with elevated expression in the EOC patients,and toanalyze the contribution of cytokine profiles to tumor immune deficiency.Materials and Methods:Cytokine antibody array was used to detect cytokine profiles in two cell lines ofEOC(SKOV3 and CaoV3),primarily cultured EOC and OSE cells.The levels ofleukemia inhibitory factor(LIF),interleukin-10(IL-10),IL-4,and transforming growthfactor-β1(TGF-β1)in peritoneal fluids and sera in the patients with EOC and benigngynecological tumors were detected by enzyme-linked immunosorbent assay(ELISA).Results:The levels of LIF,IL-10,and IL-4 were detected two times higher in the culturesupematants of the EOC cell lines than those in OSE cells by cytokine antibody array.Both LIF and IL-10 levels were more increased in ascites of EOC patients than in thosein benign gynecological tumor patients(P=0.02,P=0.001).The level of IL-4 was notdetectable in any samples of ascites or sera.No difference of TGF-β1 value wasdetected between patients with EOC and benign gynecological tumors.Conclusion:Epithelium ovarian carcinoma cells can produce more LIF,IL-10 and IL-4 thanOSE cells,and contribute to the elevated levels of those cytokines in EOC patients, which probably participates in the development of immune deficiency in the peritonealcavity of EOC patients. PARTⅡOvarian carcinoma cells influence differentiation ofdendritic cell precursors into two mature subtypes in vitroObjective:Decreased number and impaired function of dendritic cells(DCs)have been foundin ovarian carcinoma microenvironment.The study was designed to detect if thisphenomenon was associated with abnormal DC differentiation influenced by ovariancarcinoma cells.Materials and methods:FLT-3L and SCF were used for expanding DC precursors from CD34~+ progenitors.GM-CSF and TNF-αwere used to induce mature DCs.Supematants of cultured ovariancarcinoma cell line SKOV3 were added,in order to study their influence on thedifferentiation and maturation of Lin~-CD45RA~-DC precursors.Flow cytometry wasused to analyze cell subtypes and molecular surfacemarkers.MLR was used to exam stimulatory activity of DCs.IL-12 secretion was testedby ELISA.Results:Lin~-CD45RA~-DC precursors cultured with GM-CSF and TNF-αgeneratedHLA~-DR~+CD11C~+CD123~-myeloid DCs(mDCs)and HLA-DR~+CD11C~-CD123~+plasmacytoid DCs(pDCs)in vitro.The supematants from ovarian carcinoma cell lineSKOV3(SKOV3-supematants)increased pDCs and decreased mDCs compared withpure medium or supernatants of normal ovarian surface epithelial(OSE)cells.Therewere no significantly different expressions of HLA-DR and CD80 by DCs between withand without SKOV3-supernatants.But DCs treated with SKOV3-supernatants were shown to have impaired immune activity to stimulate proliferation of allogeneic CD3+T cells and secrete IL-12.Conclusion:Ovarian carcinoma cells influence differentiation of Lin-CD45RA-DC precursorsinto subtypes of mature DCs in vitro.This resulted in fewer mDCs,increased number ofpDCs,and impairment of mature DCs immune activity. PARTⅢAction of cytokines on DC precusor differentiationinfluenced by EOC cellsObjective:to study the effect of IL-10 and LIF on DC prcursors differentiation and if blockingof these two cytokines can reverse the differentiation influenced by EOC cells.Materials and methods:FLT-3L and SCF were used for expanding DC precursors from CD34+ progenitors.GM-CSF and TNF-αwere used to induce mature DCs.Four groups were designed.Thefirst group of DC was cultured with RPMI 1640 medium.The second group was addedby SKOV3 culture surpernatants.The third one was influenced by SKOV3 andanti-IL-10 and anti-LIF antibodies at the same time.IL-10 and LIF were added intomedium in the fourth group.Flow cytometry was used to analyze cell subtypes andmolecular surface markers.Allogeneic T-cell proliferation assay was used to examstimulatory activity of DCs.IL-12 secretion was tested by ELISA.Results:DC treated with IL-10 and LIF showed significantly decreased expression ofHLA-DR and CD80.And these two cytokines could also decrease the ratio ofmDC andincrease ratio of pDC,with impairing the DC function of stimulating proliferation ofallogeneic CD3+ T cells and secreting IL-12.While anti-IL-10 and anti-LIF antibodieswere added into SKOV3 culture condition,they also had the same effect as SKOV3 hadon DC precursor differentiation and DC function defect.Conclusion:IL-10 and LIF influenced mDC precusors differentiated into more pDC and less mDC,as well as impaired DC maturation and function.But anti-IL-10 and anti-LIFcould not reverse the influence caused by ovarian carcinoma cells on the differentiationof Lin~-CD45R~A-DC precursors into subtypes of mature DCs in vitro,which resulted infewer mDCs,increased number of pDCs,and impairment of mature DCs immuneactivity.