VEGF Regulates The Migration And Differentiation Of DC In Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma(OSCC)is the most common malignant epithelial neoplasm of the head and neck.One of the main pathogenesis is that the abnormal of innate or adaptive immunity.It has been suggested that dysfunction of dendritic cell(DC)induced by the tumor is one of the critical mechanisms for the escape of immune surveillance.DC is the most potent antigen presenting cells and it can activate or suppress the nalve T cell response.Immature DC suppressed the activation of T cells and mature DC can activate the T cell response.The DC function was determined by the microenvironment in which it detained.The activation of Signal Transducer and Activator of Transcription 3(STAT3)in some kinds of tumor were common and its activation made the dysdifferentiation and resulted the ineffective immunity.Vascular Endothelial Growth Factor(VEGF)was over-expression in some kinds of cancers and the function of VEGF was to improve the angiogesis and immunosuppressive.Our previous study had confirmed that the proportion of DC decreased in OSCC patients but VEGF was elevated,and VEGF expression had a inverse correlation with the number and the portion of mature DC.We also proved VEGF inhibited the maturation of immature DC into mature DC in vivo in the OSCC microenvironment.So we hypothesized that DC is dysdifferentiation in OSCC and VEGF accumulated the pre-DC in tumor.VEGF regulated the differentiation of pre-DC into dysfunction DC by JAK/STAT3 pathway.To identify if pre-DC was dysdifferentiation in OSCC,we obtained the PBMCs of 47 OSCC patients.Then we assessed the proportion of myeloid DC(mDC),plasmid DC(pDC)and immunosuppressive myeloid derived cell(MDSC).We also assessed the proportion of CD4+ Th cell,CD8+ Ts cell and CD4+ CD25+ FoxP3+ Treg cell by flowcytometry.We found that the proportion of circulating mDC and pDC decreased in OSCC.However,the portion of MDSC increased in OSCC patients.The proportion of CD4+ T cells decreased and CD8+ T cells and Treg cells increased significantly.Furthermore,to determine if VEGF engaged in the dysdifferentiation of pre-DC in OSCC,the pre-DC derived from the healthy was cocultured with HSC-3 cell or HSC-3 added Avastin which is the antibody of VEGF.We found the proportion of MDSC elevated.To determine if VEGF recruited the pre-DC to tumor nest and promote the progression of cancer.The expression of VEGF,the infiltration number of CD 14+pre-DC and CD208+ mature DC in different parts of the samples including the normal epithelial part,the hyperplasia epithelial part and the cancer part including the cancer nest and cancer stroma were evaluated on 79 OSCC by immunohistochemistry(IHC).The expression of VEGF and the infiltration CD14+ pre-DC and CD208+ mature DC were higher in hyperplasia parts than in normal parts but they were lower than cancer parts.The expression of VEGF in cancer nest but not in cancer stroma correlated with the TNM stage.The number of CD14+ pre-DC in the cancer stroma but not in the cancer nest correlated with the WHO stage,and the number of CD208 correlated with the lymphnode mestatasis.The gene expression of VEGF,CD 14 and MCP-1 were higher in tumor than in adjacent non-neoplastic part,and VEGF correlated with MCP-1 positively.Thus,we inferred VEGF promoted the migration of pre-DC to tumor part and promoted the progression of OSCC.To determine whether VEGF promote the migration of pre-DC from peripheral tumor microenvironment to the tumor site,we mimicked the tumor microenvironment with OSCC tumor cell supernant including high concentration of secreted VEGF by tumor cells.The bone marrow derived pre-DC pretreated with low concentration of VEGF was cultured with supernant or supernant adding Avastin which is the antibody of VEGF.Then Transwell experiment was carried out and the surface markers and the endocytosis ability of DC were assessed.We demonstrated VEGF promoted the migration of pre-DC to the tumor site.And it was interesting that the ability of migration of pre-DC treated with low concentration VEGF was stronger than the pre-DC cultured in the normal status at the early stage(2,4 day).However,at the later stage(6,8 day)the ability of migration of normal pre-DC was stronger than pre-DC pretreated with low concentration of VEGF.We also demonstrated that VEGF inhibited the expressions of CD1c,CD40,CD80,CD86 and MHC-? on pre-DC.The endocytosis of immature DC treated with VEGF was stronger than normal immature DC.These results demonstrated that VEGF not only promoted the accumulation of pre-DC but also inhibited the differentiation of pre-DC.How did VEGF inhibit the differentiation of pre-DC?STAT3 was activated in some kinds of tumors no matter in the tumor cells or in the infiltrated immune cells.But was it involved in the dysdifferentiation of pre-DC?The expression of STAT3 and p-STAT3 in different parts of the samples including the normal epithelial part,the hyperplasia epithelial part and the cancer part including the cancer nest and cancer stroma were evaluated on 79 OSCC by IHC.The expression of VEGF,STAT3 and p-STAT3 were higher in hyperplasia parts than in normal parts but were lower than in cancer parts.The expression of p-STAT3 in cancer nest correlated with the WHO stage.The expression of p-STAT3 in cancer stroma was correlated with inflammation.VEGF expression in cancer nest was positively correlated with the activation of STAT3 in cancer nest and in cancer stroma.The gene expressions of VEGF and STAT3 were higher in PBMC and tumor was higher than controls.Furthermore,pre-DC was treated with supemant which mimic the OSCC tumor microenvironment then Western blot was carried.We found in OSCC microenvironment VEGF inhibited the differentiation of pre-DC by JAK/STAT3 pathway.Thus,we can infer VEGF can promote the migration of pre-DC to tumor part and promoting the progression of OSCC.In conclusion,we found that pre-DC diverge to immunosuppressive MDSC but not to functional DC in the OSCC microenvironment containing high concentration VEGF secreted by tumor cells for the first time.The expression of VEGF,STAT3,P-STAT3 and the infiltration of pre-DC engaged in the progression of OSCC.Also,for the first time we demonstrated the activation of STAT3 in the cancer stoma was correlated with the infiltration of immune cells.The secreted VEGF by tumor cells can promote the migration of pre-DC and made the dysdifferentiation of pre-DC and chronic inflammation by JAN/STAT3 pathway.