The Study Of Endothelial-like Differentiation Tendency Of Dendritic Cells In The Supernatant Of Sw620

Background:Dendritic cells(DCs) are the best professional antigen-presenting cells(APCs) of the immune system.They play the central role in initiating,regulating and maintaining immune responses.They are important for the specific anti-tumor T-cell response.However,most studies have shown that both circulating and tumor-infiltrating DCs(TIDCs) from cancer patients appear to be phenotypically and functionally defective.The tumor tissue secretes kinds of immune factors,which can inhibit maturation of DCs.Tumors require blood supply for expansive growth.Until recently,angiogenesis, or sprouting of endothelial cells(ECs) from existing vessels,was the only accepted mechanism of tumor vascularization.Recent studies have suggested that vasculogenesis,or recruitment of endothelial progenitors(EPCs) that differentiate into endothelial cells,might contribute to the formation of tumor neovessels.The origin of this endothelium may be either bone marrow-derived EPCs or from human peripheral blood containing CD34⁺ population.Increased VEGF levels have been found in a variety tumor.Numerous studies have shown that VEGF stimulates ECs proliferation and differentiation via mitogen-activated protein kinase/extracellular signal-regulated kinase(p42/44-MAPK/ERK1/2) signaling,which can contribute to vessel formation in tumor.MAPK/ERK pathway has been shown to be one of the key signaling pathways in the differentiation of various cell types both in vivo and in vitro. Recently,a research reported a novel leukocyte subset in mouse and human ovarian carcinomas that coexpressed endothelial and dendritic cells markers.Other studies suggest the tumor-specific culture conditions induce the differentiation of tumor-invading monocytes into tumor-associated dendritic cells(TADCs).Additional incubation of tumor-associated DCs with pro-angiogenic factors,such as vascular endothelial growth factor and oncostatin M,led to transdifferentiation into endothelial-like cells.Also,they were able to form network-like structures on matrigel.Our prior studies have shown that the supernatant of SW620 can inhibit the growth of DCs and its expression of relative antigen.In additional,in the presence of the supernatant of SW620 immature DCs have the potential to differentiate into a cell type resembling endothelial-like cells(ELCs).These study indicates DCs were inhibited in anti-tumor immunity and some DCs differentiate into endothelial-like cells in tumor microenvironment.There are many reports about the use of intratumoural DCs inoculation as a means for tumor vaccination in the world,but research on endothelialisation of DCs is still limited.The effects of this phenomenon and mechanisms also need further investigation.Experiment aim:To study the degree of endothelialisation in the microenvironment created by dissoluble cytokines derived from colon carcinoma cells SW620 on the differentiations of different stages dendritic cells from human peripheral blood mononuclear cell(PBMC),and observe the precise role of MAPK/ERK signaling pathway in the process of dendritic cells differentiation into endothelial-like cells in vitro.Methods:1.The supernatant of SW620 was prepared.2.Precursors of DCs were separated from peripheral blood mononuclear cells (PBMC) divided from healthy volunteers' peripheral blood by Ficoll density gradient centrifuge.After 3 hours incubation,remove non-adherent cells.The left precursors were cultured at 3×10⁶/ml with RPMI1640 medium containing rhGM-CSF(100ng/ml), IL-4(5ng/ml).At the fifth day of culture added LPS(5ng/ml).Inaddition,the supernatant of SW620 cells were added to DCs on the 2^nd(immature DCs induced group),7^th(mature DCs induced group) days of culture in experimental group.And half changed the medium every other day and every group is cultured to continue 7 days.The DCs(DCs cultured with normo-medium) and experimental dendritic cells cultured with supernatant of SW620 were collected at 9^th,14^th for further study. Observed the appearance character of DCs with microscope during the culture process. The vWF and VE-cadherin expression on the protein level was detected by western blotting.The endothelial-specific Weibel-Palade body was observed in the cytoplasm by transmission electron microscope(TEM).Mix lymphocyte reaction(MLR) and efficiency of kill SW620 cell after loaded tumor antigen reflected the ability of DCs. Ac-LDL uptake was analyzed with a microscope.The phosphorylations of ERK1/2 was assayed by western blotting method 15,30 and 60 minutes after the treatment of supernatant of SW620.In order to elucidate whether MAPK/ERK1/2 is essential for the differentiation process,we examined the effect of PD98059,a selective inhibitor of MEK1 and 2,the upstream regulator of MAPK/ERK 1 and 2 phosphorylation,on the differentiation of iDCs to ELCs.3.Cultured masculine groups of HUVEC with trypsase and ECM.All data were expressed as mean value±S.D and analyzed by statistical software SPSS12.0.P＜0.05 was considered to be statistically significant.Statistical analysis was performed using one-way ANOVA.Results:1.The immature DCs induced group cells' growth speed was slower than the control cells',and the cell density was lower.In addition,they were able to form tube-like and cord structures after 7 days of culture in the supernatant of SW620 cell. The morphologic changes of normal control cells were coincide with the typical DCs' reported.However,there was no significant difference between mature DCs induced groups and control groups.2.Compared with the normal control groups cells,the expression of vWF and VE-cadherin in the immature DCs induced groups were increased by the tumor-conditioned medium,and there was statistics disparation between control groups and experimental groups.The endothelial-specific Weibel-Palade body was detected in the immature induced group cells' cytoplasm by TEM.The APC function of DCs is decrease than the normal control groups.Meanwhile the experimental groups had the ability to uptake DiL-Ac-LDL,which were the features of endothelial cell.Supernatant of SW620 activated the MAPK/ERK1/2 pathway in a time—dependent manner.Incubation of iDCs with MAPK/ERK1/2 phosphorylation inhibitor PD98059,blocked the sustained SW620 supernatant induced MAPK/ERK1/2 phosphorylation.Inhibition of MAPK/ERK1/2 phosphorylation by PD98059 also blocked the expression of vWF and VE-cadherin in these cells and their differentiation to ELCs.Conclusion:1.The supernatant of SW620 can inhibit the growth and function of DCs and the immature DCs induced group cells showed strong expression of classical ECs characteristics.These results suggest that in the presence of the supernatant of SW620 immature DCs have the potential to differentiate into a cell type resembling endothelial-like cells(ELCs).2.These data suggest that the supernatant of SW620 induces immature DCs differentiation into ELCs via a MAPK/ERK1/2 signaling pathway-mediated mechanism in vitro.Inhibition of MAPK/ERK1/2 phosphorylation by PD98059 also blocked their differentiation to ELCs.