Ovarian Cancer Cells Induce Peripheral Mature DCs To Differentiate Into Macrophages In Vitro

Tumor can escape from immune surveillance and clearance through various pathways.Previous studies on dysfunction of anti-tumor immunity mostly focused on deficiencies of adaptive cellular immunity,including tumor associated-antigen specific T cell immunity,antigen-presenting cells,especially dendritic cells,and regulatory T cells.However,more recent researchers have found that inflammation in the tumor microenvironment has many tumor-promoting effects;it aids in the proliferation and survival of malignant cells,promotes angiogenesis and tumor metastasis,subverts adaptive immune responses and alters responses to hormones and chemotherapeutic agents.Macrophages,the scavengers of immune system,are important inflammatory cells in innate immunity and also possess the ability to present antigen in adaptive immunity. Evidences from clinical and experimental studies indicates that macrophages can produce many compounds ranging from metagenic oxygen and nitrogen radicals to angiogenic factors during an inflammatory response which can contribute to cancer initiation and promotion.Macrophages derive from circulating monocytes that are common precursors of both macrophages and peripheral myeloid dendritic cells.It is well known that dendritic cells are the most potent professional antigen presenting cells and initiators of adaptive immune response.Previous studies revealed that some tumor-derived factors or tumor-associated molecules could skew monocytes differentiation from dendritic cells to macrophages.However,it is generally recognized that this transformation function of dendritic cells disappears after they are maturated. No studies are reported on mature dendritic cells differentiating into macrophages up to date,to our knowledge.Undoubtedly,if this differentiation pathway exists,it could favor cancer development and progression greatly because of simultaneous suppression of adaptive cellular immunity and facilitation of non-specific inflammatory response in tumor microenvironment..Ovarian cancer is the leading cause of deaths in gynecological malignancies,most of the patients present advanced disease at their primary diagnosis and the rates of long-term survival among patients with advanced disease is Only about 10-30 percent. One of the most biological characteristics of ovarian carcinoma is widely intraperitoneal distribution of disease.Accumulated evidences revealed there exists peritoneal and systematic immunodeficiency for patients with ovarian cancer.Our recent study showed that that the supernatant from ovarian cancer cell line influence differentiation of Lin-CD45RA- dendritic cell precursors into 2 subtypes of mature dendritic cells in vitro with fewer myeloid dendritic cells and increased number of plasmacytoid dendritic cells favoring tumor cells,suggesting that ovarian cancer cells possess the capacity to change differentiation of dendritic cells.In current study,we first establish a method to induce mature DC from peripheral monocytes in vitro by combination culture.Second,we investigate whether ovarian cancer cells could induce mature DC to differentiate to macrophage-like cells by using ascites from ovarian cancer patients and supernatants from ovarian cancer cell culture to mimic ovarian cancer microenvironment,and further explore possible mechanisms mediating this differentiation pathway.PartⅠExpansion and maturation of DCs from human peripheral blood in vitroObjective:To isolate and purify monocytes from human peripheral blood,culture them with cocktail cytokines and induce them to differentiate into mature DC in vitro.Methods:After isolation from peripheral blood by using a high-gradient magnetic cell sorting system(MACS),CD14~+monocytes were cultured with rhGM- CSF and rhIL-4 for 6days,LPS was added for another 24 hours to induce maturation.Morphologic and functional studies were identified by invert microscope,FACS,mixed lymphocyte reaction(MLR).Results:1.Cells obtained typical DC morphologic features after culture for 7 days.2.Fresh isolated monocytes were CD14+CD1a- and expressed low level of CD83,after culture for 7 days,most cells differentiated into CD14-CD1a±dendritic cells. Conclusion:Peripheral blood monocytes can be induced to differentiate into mature DC under the culture condition of GM-CSF,IL-4 and LPS.PartⅡOvarian cancer cells induce peripheral mature DCs to differentiate into macrophagesObjective:To investigate whether ovarian cancer cells could induce peripheral mature dendritic cells to differentiate into macrophages.Methods:Mature DCs were divided into 2 culture system:1.Ascites(peritoneal fluid ) culture system,DCs were divided into 2 groups:(1)(?) control group:50% non-malignant peritoneal fluid was added,(2)ascites group:50%ovarian cancer ascites was added.2.Supernatant culture system,DCs were divided into 3 groups:(?)(1)control group:50%control supernatant was added,(2)SKOV3 supernatant group:50% supernatant from SKOV3 was added,(3)CAOV3 supernatant group:50%supernatant from CAOV3was added.After coculture for 48 hours,expression of CD14/CD1a, CD83 and phagocytosis of FITC-dextran were detected by FACS;MLR was taken to evaluate the T-cell costimulatory capacity. Results:1,After culture for 48 hours,cells cocultured with ovarian cancer ascites presented significantly increased CD14+CD1a- and decreased CD83 expressions compared with control group.Furthermore,cells cocultured with ovarian cancer ascites had poorer T-cell costimulatory properties.Lastly,the FITC-dextran analysis showed that these cells had stronger phagocytosis.2,Supernatant from both cell lines,the same as ovarian cancer ascites,induced a group of macrophage-like cells that exhibited CD14+CD1a-CD83- and possessed low T-cell costimulatory properties and stronger phagocytosis.Conclusions:1,Ovarian cancer ascites induced peripheral mature dendritic cells to differentiate into macrophage-like cells.2,Supernatant of ovarian cancer cells induce peripheral mature dendritic cells to differentiate into macrophage-like cells.PartⅢIL-10 secreted by ovarian cancer cells mediates the differentiation of peripheral mature DCs into macrophagesObjective:To investigate the effects of IL-10 and LIF secreted by ovarian cancer cells on differentiation of peripheral mature dendritic cells into macrophage -like cells. Methods:Mature dendritic cells were divided into 3 culture system:1.Cytokine culture system,falls into 3 group,(1) blank control group,(2) IL-10 group:100ng/ml IL-10 was added,(3) LIF group:100ng/ml LIF was added.2.Ascites(peritoneal fluid ) culture system,DCs falls into 4 groups:(1) control group:50%non-malignant peritoneal fluid was added,(2) ascites group:50%ovarian cancer ascites was added,(3) ascites+antiIL-10 group:50%ovarian cancer ascites and neutralizing IL-10 antibody (2ug/ml) were added,(4) ascites +anti-LIF group:50%ovarian cancer ascites and neutralizing LIF(2ug/ml) antibody were added.3.Supernatant culture system,falls into 7 groups:(1)□control group:50%control supernatant was added,(2) SKOV3 supernatant group:50%supernatant from SKOV3 was added,(3) SKOV3 supernatant +anti-IL-10 group:50%supernatant from SKOV3 and neutralizing IL-10 antibody (2ug/ml) were added;(4)SKOV3 supernatant +anti-LIF group:50%SKOV3 supernatant and neutralizing LIF antibody(2ug/ml) were added;(5)CAOV3 supernatant group:50%supernatant from CAOV3 was added,(6) CAOV3 supernatant +anti-IL-10 group:50%supernatant from CAOV3 and neutralizing IL-10 antibody(2ug/ml) were added;(7) CAOV3 supernatant +anti-LIF group:50%supernatant from CAOV3 and neutralizing LIF antibody(2ug/ml) were added.After coculture for 48 hours,expression of CD14/CDla,CD83 and phagocytosis of FITC-dextran were detected by FACS,MLR was taken to evaluate the T-cell costimulatory capacity.Results:1,In the cytokine system,IL-10 induced a group of macrophage-like cells that exhibited CD14+CD1a-CD83- and possessed low T-cell costimulatory properties and stronger phagocytosis,but LIF did not possess this capacity. 2,In the ascites(peritoneal fluid ) culture system:neutralizing IL-10 antibody blocked the differentiation of peripheral mature dendritic cells into macrophage -like cells induced by ovarian cancer ascites,but neutralizing LIF antibody did not.3,In the supernatant culture system:neutralizing IL-10 antibody blocked the differentiation of peripheral mature dendritic cells into macrophage -like cells induced by supernatants from ovarian cancer cells,but neutralizing LIF antibody did not.Conclusion:IL-10 but not LIF mediated differentiation pathway of peripheral mature DCs into macrophages-like cells.