Endothelial-like Differentiation And Its Mechanism Of Dendritic Cells In The Microenvironment Of Human Esophageal Squamous Cell Carcinoma

Dendritic cells(DCs) are the best professional antigen presenting cells,which are uniquely able to activate a naive T-cell response.DCs play the central role in regulating immune response.The T cell immune response activated by DCs plays a key role in anti-tumor immune responses.The defection on function and numbers of DCs in cancer patients is the main reason for the tumor immune escape.Therefore,many studies focus on enhancement of antigen presenting function of DCs and effective DCs vaccine preparation.On 2004,Coukos G reported a novel leukocyte subset that coexpressed EC and DC markers within murine and human ovarian carcinoma which secret a high level of. vascular endothelial growth factor A(VEGF-A).VEGF-A is one of the most potent direct-acting angiogenic molecule secreted by tumor cells,which is a highly effective and specific mitogen for endothelial cells(ECs),promoting tumor angiogenesis and metastasis.This study also proved that murine bone marrow-derived DCs could undergo endothelialization in vitro after incubation in high level of VEGF-A,and involved in tumor angiogenesis in vivo.Vessel formation is an important indicator of tumour in rapid growth.Classically, vascularization has been considered through angiogenesis,which involves sprouting from preexisting endothelial cells.Recent studies have suggested that vasculogenesis, which occurs more prominently during embryonic development,can also contribute to vessel formation in adults.Vasculogenesis is the development of vessels from precursor cells that migrate to the site of vascularization.There is a similar event in tumor.On 2006,Young MR reported that CD34~+ progenitor cells,which are capable of developing into dendritic cells,addition of conditioned medium of murine Lewis lung carcinoma cells redirected their differentiation into ECs.On 2007,E.Gottfried showed that tumor-associated DCs incubated additionally with pro-angiogenic factors,such as VEGF and oncostatin M,could trans-differentiate into endothelial-like cells,which may be an alternative pathway of tumour angiogenesis.Endothelial-like differentiation of DCs is a new phenomenon,and its mechanisms are unclear.Many growth factors/cytokines and vasoactive substances can led to cell differentiation through cellular signal transduction.MAPK/ERK1/2 is the common signaling pathway which leds to cell differentiation by various of extracellular signals. So,it is worthwhile to research further whether the MAPK/ERK1/2 signaling pathway mediate the endothelial-like differentiation of DCs.Esophageal cancer is one of the most frequently diagnosed cancers,and has high-incidence and high mortality rate.Esophageal squamous cell carcinoma(ESCC) is still the main type of esophageal cancer,which has strong invasive and bad prognosis. It is unclear wether ESCC and its metabolites can induce endothelial-like differentiation of DCs to led immune escape of ESCC ? In this study,Firstly,to simulate the ESCC microenvironment by EC9706 cell supernatant and ESCC tissue homogenate supernatant,and research the effect on the endothelial-like differentiation of immature DCs(iDCs) and mature DCs(mDCs).Secondly,to investigate the mechanisms of endothelial-like differentiation of iDCs in ESCC microenvironment through investigating the effect of VEGF-A on the endothelial-like differentiation of DCs,the effect of VEGF-A on EC9706 cell supernatant inducing the endothelial-like differentiation of iDCs,and the MAPK/ERK1/2/CREB signaling pathway mediating the endothelial-like differentiation of iDCs induced by EC9706 cell supernatant or by VEGF-A.This research will rich the theory of ESCC immune escape,and may bring forward to strategy of blocking endothelial-like differentiation of iDCs to make DCs could be used better as an anti-ESCC vaccine. PartⅠEffect of the ESCC microenvironment on the endothelial-like differentiation of iDCs and mDCsChapter 1 Study on cell morphology and associated antigen of EC9706 cell supernatant on the endothelial-like differentiation of iDCs and mDCsMethods1.Experimental grouping Control DCs:human peripheral blood monocytes were induced toward DCs by rhIL-4,rhGM-CSF and LPS.DCs were harvested at day 9.and day 14.Cells of iDCs induced group:40%EC9706 supernatant was added at the end of day 2(iDCs) for 7 days induction,and induced cells were harvested at day 9.Cells of mDCs induced group:40%EC9706 supernatant was added at the end of day 7(mDCs)for 7 days induction,and induced cells were harvested at day 14.Positive control human umbilical vein endothelial cells(HUVECs):HUVECs were acquired by digestion of trypsin and cultivation of endothelial cell medium(ECM).2.Observing cell morphology under the optical microscope.3.Flow-cytometry(FCM) analysis of DC surface antigen CD86,CD11c and CD1a on Cells of iDCs induced group and mDCs induced group.4.Detection of the expression of CD1a,vWF,CD144 on monocytes,cells of iDCs induced group and mDCs induced group,and HUVECs by RT-PCR.5.Detection of the expression of vWF on cells of iDCs induced group and mDCs induced group,and HUVECs by Immunofluorescence.6.Western blotting detection of the expression of vWF,CD144 on cells of iDCs induced group and mDCs induced group,and HUVECs.Results1.Morphology and the expression of associated antigen of control DCs and HUVECsOn day 7,control DCs displayed morphology feature of DCs under electronic microscope.There were many typical dendrites on cell surfaces,and plenty of mitochondria,microfilament in cytoplasm.Comparing with monocytes,the expression levels of related surface antigens were increased obviously(P＜0.01). Positive control HUVECs presented cobblestone,spindle,whirlpool-shaped and lumens structure,highly expressed vWF and CD144,and showed strong uptake of Dil-acetylated low-density lipoprotein(Dil-Ac-LDL).2.The effect of EC9706 supernatant on cell morphologyOn day 8,Control DCs became big and round,there were many typical dendrites on cell surfaces,some displayed suspension,iDCs induced group had slender branch, lumen-like structure,some displayed cord-arranged.There was no difference between mDCs induced group and control DCs.3.The expression of DC surface antigen CD86,CD11c and CD1a after induction of EC9706 supernatantFCM showed that the expression levels of CD86,CD1a and CD11c were decreased in iDCs induced group cells compared with the control DCs(P＜0.05),but the associated surface antigens expressed on cells of mDCs induced group were not different from the control DCs(P＞0.05).4.The expression of CD1a,CD144 and vWF pre-and post-induction of EC9706 supernatantRT-PCR showed that the expression of CD144 and vWF could not be detected and CD1a was very weak in monocytes.The CD1a expression decreased and the CD144 and vWF expression increased in iDCs induced group cells as compared with the control DCs.There was no CD144 or vWF expression in mDCs induced group cells and its control DCs,and only CD1a could be detected.Immunofluorescence analysis showed that the vWF expression level of iDCs induced group cells was greater than that in the control DCs(P＜0.05).However,there was no significant difference between mDCs induced group and its control DCs (P＞0.05).Western blot analysis demonstrated that CD144,vWF expression was higher in the iDCs induced group cells than its control DCs(P＜0.01).In both mDCs induced group cells and the control DCs,the CD144,vWF protein was not detected. Chapter 2 Study of EC9706 cell supernatant on the endothelial-like differentiation function of iDCs and mDCsMethods1.Detection of the tube-like structure formation of the cells of iDCs induced group and mDCs induced group on fibronectin-coated plates in vitro.2.Dil-Ac-LDL and India ink uptake assay of the PBMCs,cells of iDCs induced group and mDCs induced group.3.Transmission electron microscope(TEM) detection of the EC-specific structure of WP bodies in iDCs induced group cells and control DCs.4.Detection of T cell proliferation of iDCs induced group cells and killing effect of CTL on EC9706 cells by CCK-8 in vitro.Results1.The effect of EC9706 supernatant on EC associated functionsTube-like structure formation assay showed that the cells of iDCs induced group appeared in fusiform shape and had the tendency of vortex distribution in the first 24 h. They tended to form tube-like structure in the second 24 h which was very similar to the positive control HUVECs,However,the cells were distributed evenly in control DCs of iDCs induced group,mDCs induced group and its control DCs.Uptake assay showed that monocytes uptaked Indian ink and a few Dil-Ac-LDL. The cells of iDCs induced group did not uptake Indian ink,which excluded the possibility that monocytes and macrophages were mixed in with the induced cells.The increased uptake of Dil-Ac-LDL in cells of iDCs induced group.There was no difference between cells of mDCs induced group and its control DCs.WP body is the EC specific functional structure.TEM showed that WP bodies and plenty of mitochondria were presented in the cells of iDCs induced group,which was similar to HUVECs.No WP body was found in the control DCs.2.The effect of EC9706 supernatant on DC associated functionsComparing with control DCs,the T cell proliferation and CTL activity for killing EC9706 cells in iDCs induced group decreased obviously(P＜0.01),which showed that the microenviroment produced by EC9706 cell supernatant dramatically disabled the antigen presenting function of cells in iDCs induced group.Chapter 3 Effect of ESCC tissue homogenate supernatant on the endothelial-like differentiation of iDCsMethods1.Collection of fresh ESCC and peri-carcinoma tissues 10 cases to prepare homogenate supernatant by grinding method.2.Experimental grouping Control DCs:human peripheral blood monocytes were induced toward DCs by rhIL-4,rhGM-CSF and LPS.Carcinoma homogenate supernatant group:40%carcinoma homogenate supernatant was added at the end of day 2(iDCs) for 7 days induction,and induced cells were harvested at day 9. Peri-carcinoma homogenate supernatant group:40%peri-carcinoma homogenate supernatant was added at the end of day 2(iDCs) for 7 days induction.Positive control HUVECs.3.Western blotting detection of the expression of vWF,CD144 on cells of carcinoma homogenate supernatant group,peri- carcinoma homogenate supernatant group and control DCs.4.Detection of the expression of vWF on cells of carcinoma homogenate supernatant group,peri-carcinoma homogenate supernatant group and control DCs.5.Dil-Ac-LDL and India ink uptake assay of the ceils of carcinoma homogenate supernatant group,peri-carcinoma homogenate supernatant group and control DCs.6.Checking T cell proliferation of cells of carcinoma homogenate supernatant group, peri-carcinoma homogenate supernatant group and control DCs,and killing effect of CTL on EC9706 cells by CCK-8.Results1.The effect of ESSC carcinoma and peri-carcinoma homogenate supernatant on iDC morphologyOn day 8,cells of control DCs and peri-carcinoma homogenate supernatant group displayed round,some suspended,and there were many typical dendrites on cell surfaces.However,cells of carcinoma homogenate supernatant group displayed slender spindle.2.The expression of CD144 and vWF after the induction of carcinoma and peri-careinoma homogenate supernatantWestern blotting analysis demonstrated that the CD144,vWF expression was higher in the cells of carcinoma homogenate supernatant group than control DCs and peri-carcinoma group(P＜0.01).However,there was no difference between peri-carcinoma group and control DCs(P＞0.05).Immunofluorescence analysis showed that the vWF expression was stronger in the cells of carcinoma homogenate supernatant group than in control DCs and peri-carcinoma group(P＜0.05).There was no difference between peri-carcinoma group and control DCs(P＞0.05).3.The effect on uptake function of ECs after the induction of carcinoma and peri-carcinoma homogenate supernatantThe uptake of Dil-Ac-LDL in cells of carcinoma homogenate supernatan group was stronger than in peri-carcinoma group and control DCs.4.The effect of carcinoma and peri-carcinoma homogenate supernatant on antigen presentation of DCsComparing with control DCs and peri-carcinoma group,the T cell proliferation and CTL activity for killing EC9706 cells of carcinoma homogenate supernatant group decreased obviously(P＜0.01),there was no difference between peri-carcinoma group and control DCs(P＞0.05).PartⅡMechanisms of the ESCC microenvironment induction of the endothelial-like differentiation of iDCsChapter 1 Effect of VEGF-A on the endothelial-like differentiation of DCs Methods1.Experimental grouping Control DCs:human peripheral blood monocytes were induced toward DCs by rhIL-4,rhGM-CSF and LPS.VEGF-A iDCs induced group and mDCs induced group:20 ng/ml VEGF-A was added at the end of day 2(iDCs), and at the end of day 7(mDCs) respectively,after 7 days induction,induced cells were harvested at day 9 and day 14 respectively.Positive control HUVECs.2.FCM analysis of DC surface markers CD86,CD11c and CD1a on Cells of VEGF-A iDCs induced group and mDCs induced group3.Detection of the expression of CD1a,vWF and CD144 on cells of VEGF-A iDCs induced group and mDCs induced group by RT-PCR,Western blotting and Immunofluorescence.4.Dil-Ac-LDL and India ink uptake assay of the cells of VEGF-A iDCs induced group and mDCs induced group.5.TEM detection of the WP bodies in VEGF-A iDCs induced group cells and its controls.6.Detection of T cell proliferation of VEGF-A iDCs induced group cells and killing effect of CTL on EC9706 cells by CCK-8 in vitro.Results1.The effect of VEGF-A on cell morphologyThe cells of VEGF-A iDCs induced group grew slowly.On day 8,Control DCs became big and round,however,most cells of VEGF-A iDCs induced group had slender branch,spindle,lumen-like structure,some displayed cord-arranged.There was no difference between VEGF-A mDCs induced group and control DCs.2.The expression of DC surface antigen CD86,CD11c and CD1a after induction of VEGF-AFCM showed that the expression levels of CD86,CD1a and CD11c were decreased in VEGF-A iDCs induced group cells comparing with the control DCs (P＜0.05),but the surface antigens expressed in cells of VEGF-A mDCs induced group were not different from the control DCs(P＞0.05).3.The expression of CD1a,CD144 and vWF after induction of VEGF-ART-PCR,Western blotting and Immunofluorescence showed that The CD1a expression decreased and CD144 and vWF expression increased in VEGF-A iDCs induced group cells(P＜0.05).There was no difference between VEGF-A mDCs induced group and control DCs(P＞0.05).4.The effect of EC associated functions and antigen presentation of DCs after induction of VEGF-AUptake assay showed that the cells of VEGF-A iDCs induced group did not uptake Indian ink,but the uptake of Dil-Ac-LDL increased in cells of VEGF-A iDCs induced group.There was no difference between cells of mDCs induced group and its control DCs.TEM showed that WP bodies and plenty of mitochondria were present in the cells of VEGF-A iDCs induced group,which was similar to HUVECs.No WP body was found in the control DCs.Comparing with control DCs,the T cell proliferation and CTL activity for killing EC9706 cells of VEGF-A iDCs induced group decreased obviously(P＜0.01).Chapter 2 Effect of VEGF-A blocked on EC9706 cell supernatant inducing the endothelial-like differentiation of iDCsMethods1.Detection of the content of VEGF-A in EC9706 cell supernatant,esophageal carcinoma and peri-cancer tissues homogenate supernatant by ELISA.2.Experimental grouping Control DCs:human peripheral blood monocytes were induced toward DCs by rhIL-4,rhGM-CSF and LPS.EC9706 supernatant group: 40%EC9706 supernatant was added at the end of day 2(iDCs) for 7 days induction. Blocking VEGF-A group:40%EC9706 supernatant and VEGF-A antibody was added at the end of day 2(iDCs) for 7 days induction.Positive control HUVECs.3.After blocking VEGF-A,detecting the expression of vWF on cells of EC9706 supernatant group by Immunofluorescence.4.After blocking VEGF-A,detecting the expression of vWF on cells of EC9706 supematant group by Western blotting. 5.After blocking VEGF-A,detecting Dil-Ac-LDL uptake on cells of EC9706 supernatant group.Results1.The content of VEGF-A in the EC9706 cell supernatant and homogenate supematant of esophageal carcinoma was significantly higher than that of the peri-carcinoma homogenate supernatant(P＜0.05).2.Cell morphology:after blocking VEGF-A The cells of EC9706 supernatant group showed slender spindle,which was similar to HUVECs.Control DCs became big and round.There was almost no slender branch in cells of blocking VEGF-A group.3.The expression of CD144 and vWF after blocking VEGF-AImmunofluorescence showed that comparing with the cells of EC9706 supernatant group,vWF expression decreased in blocking VEGF-A group(P＜0.05).There was no difference between VEGF-A blocking Group and control DCs(P＞0.05).Western blotting showed that comparing with the cells of EC9706 supernatant group,CD144 and vWF expression decreased in blocking VEGF-A group(P＜0.01).4.The effect on uptake function of ECs after the induction of carcinoma and peri-carcinoma homogenate supernatantUptake assay showed that the uptake of Dil-Ac-LDL decreased in VEGF-A blocking group.There was no difference between blocking VEGF-A Group and control DCs.Chapter 3 MAPK/ERK1/2/CREB signal pathway activated by VEGF-A mediates endothelial-like differentiation of iDCs in the ESCC microenvironmentMethods1.Experimental grouping EC9706 supernatant group:40%EC9706 supernatant was added at the end of day 2(iDCs) for the induction of 0 min,15 min,30 min,60 min and 7 days respectively.VEGF-A group:20 ng/ml VEGF-A was added at the end of day 2(iDCs) for the induction of 0 min,15 min,30 min,60 min and 7 days respectively. PD98059 60min blocking group:after 50μM PD98059 1 h incubation,40%EC9706 supernatant or 20 ng/mlVEGF-A was added for 1 h induction.PD98059 7 d blocking group:EC9706 supernatant and 10μM PD98059 or VEGF-A and 10μM PD98059 was added for 7 days induction.2.Western blotting detection of the phospho-MAPK/ERK1/2 and phospho-CREB activated by EC9706 supernatant or VEGF-A.Detection of the level of p-ERK1/2 and p-CREB,and the expression of vWF and CD144 after PD98059 blocking.3.After PD98059 blocking,detection of the expression of vWF on cells of EC9706 supernatant group and VEGF-A group by Immunofluorescence.4.Immunofluorescence Double Staining(FITC,PI) detection of nuclear translocation of p-ERK1/2 actived by EC9706 supernatant or VEGF-A.5.After PD98059 blocking,detecting Dil-Ac-LDL uptake of cells of EC9706 supernatant group and VEGF-A group.6.After PD98059 blocking,detecting the level of p-ERK1/2 and p-CREB on cells of EC9706 supernatant group by Western blotting.7.EMSA detection of the CRE binding capacity of the activation of CREB in the nucleus of EC9706 supernatant group or VEGF-A group.Results1.The activation of MAPK/ERK1/2 and CREB during the endothelial-like differentiation of iDCsEC9706 supernatant or VEGF-A induced the phosphorylation of MAPK/ERK1 /2 and CREB in a time dependent manner.PD98059 is a selective inhibitor of MEK1/2. MEK1/2 is the upstream regulator of MAPK/ERK1/2 phosphorylation.With PD98059 pretreatment,the phosphorylation level of MAPK/ERK and CREB decreased obviously in EC9706 supernatant 60 min group or in VEGF-A 60 min group(P＜0.01).2.Inhibition of MAPK/ERK phosphorylation resulted in the inhibition of the endothelial-like differentiation of iDCsInhibition of MAPK/ERK phosphorylation by PD98059 was accompanied by morphological changes,a significantly decreased expression of CD144 and vWF (P＜0.01),and Dil-Ac-LDL uptake dramatically reduced in the cells of EC9706 supernatant group or VEGF-A group in the presence of PD98059.Thus,inhibition of MAPK/ERK1/2 phosphorylation by PD98059 resulted in the inhibition of the endothelial-like differentiation of iDCs which were induced by EC9706 supernatant or VEGF-A,both morphologically and functionally.3.Nuclear transiocation of phosphorylation of MAPK/ERK1/2Nuclear translocation following phosphorylation of MAPK/ERK1/2 is required to induce transcriptional changes leading to proliferation,survival and differentiation. iDCs incubated with EC9706 supernatant or VEGF-A for 7 days showed that the presence of phosphorylation of MAPK/ERK1/2(green) in the nuclei(red) of iDCs.In the presence of PD98059,however,EC9706 supernatant or VEGF-induced MAPK/ ERK1/2 phosphorylation as well as nuclear translocation were significantly inhibited on day 9.4.Effect of VEGF-A blocked on EC9706 cell supernatant inducing the endothelial-like differentiation of iDCsWestern blotting showed that comparing with EC9706 supernatant,the level of p-ERK1/2 and p-CREB decreased in VEGF-A blocking group(P＜0.01),which showed VEGF-A played a starter role during the process of MAPK/ERK1/2/CREB activated by EC9706 supernatant.5.Activation of CREB binding activity to CREEMSA showed that CREB activated by EC9706 supernatant or VEGF-A had high binding capacity of CRE DNA sequence.Promoter sequences upstream of CD144 and vWF gene contain the CRE element.Activation of CREB may bind CRE element to mediate CD144 and vWF transcription to up-regulate their expression levels.Conclusions1.EC9706 cell supernatant induces iDCs to differentiate from the DC pathway toward endothelial cells,and form endothelial-like cells.But it has no obvious influence on mDCs,which may bring forward evidence that the mDCs can be used relatively safely in an anti-ESCC vaccine.2.The ESCC tissue homogenate supernatant also induce the endothelial-like differentiation of iDCs,however,peri-carcinoma homogenate supernatant has no this effect,which shows that the endothelial-like differentiation of iDCs is induced by the ESCC microenvironment,which may be one of the mechanisms of ESCC immune escape.3.VEGF-A induces iDCs to up-regulate some EC markers and down-regulate some DC markers,simultaneously,EC morphology features and functions observed,and form endothelial-like cells.But VEGF-A doesn't induce the endothelial-like differentiation of mDCs.4.EC9706 supernatant or VEGF-A induces the phosphorylation of MAPK/ERK1/2 and CREB in a time dependent manner,and the activation persists in whole induction process.The nuclear translocation of p-ERK1/2 activates CREB,may led to CD144, vWF gene transcription.5.MAPK/ERK1/2/CREB signal pathway mediates the endothelial-like differentiation of iDCs induced by EC9706 supernatant or VEGF-A.Blocking MEK by PD98059 results in the inhibition of MAPK/ERK1/2 phosphorylation,and inhibition of the endothelial-like differentiation of imDCs induced by EC9706 supernatant or VEGF-A.6.Blocking VEGF-A in EC9706 supernatant results in the inhibition of MAPK/ERK 1/2 with CREB phosphorylation,and inhibition of the endothelial-like differentiation of iDCs,which shows that VEGF-A in EC9706 supernatant is a starter to activate MAPK/ERK 1/2/CREB signal pathway,which mediates the endothelial-like differentiation of iDCs in the ESCC microenvironment.This study provids the new strategy to inhibite the endothelial-like differentiation of iDCs.