DC Tolerogenic Transformation Induced By VEGF In Oral Squamous Cell Carcinoma And Its Mechanism

[Purpose]The purpose of this study is to detect the expression of DC surface IDO and PD-L1 in tumor tissues and peripheral blood of patients with OSCC,and to analyze the prognosis of survival.The expression of VEGF in tumor tissues and peripheral blood of OSCC patients and its correlation with tol-DC were preliminary.To explore the mechanism of VEGF-induced tol-DC.[Methods]1.Immunofluorescence was used to detect the presence of IDO+CD1a+ and PD-L1+CD1a+ double positive cells in frozen sections of OSCC tissues.2.50 cases of OSCC tumor tissues were collected.The expression of tol-DC,IDO+CD1a+ and PD-L1+CD1a+ and VEGF in tumor tissues of OSCC patients were detected by immunohistochemical double staining and immunohistochemical staining.Tol-Correlation between survival prognosis and clinicopathological parameters of DC,and correlation with VEGF.3.25 patients with OSCC and 20 healthy people with peripheral blood,flow cytometry to detect the proportion of Mo-DC,pDC,and the expression of PD-L1,Elisa detection of peripheral blood serum VEGF content,and Analyze its relevance.4.PBMC was induced to DC in peripheral blood.During the differentiation process of immature DC to mature DC,exogenous VEGF factor was added to stimulate the change of phenotype and the expression level of related factors.5.Mouse bone marrow induced into DC,group:(1)iDC group;(2)mDC group;(3)VEGF+mDC group;(4)VEGF+ VEGFR1 inhibitor+ mDC group;(5)VEGF+VEGFR2 inhibition Agent + mDC group.Western blot was used to detect the expression of JAK2,p-JAK2,STAT3,p-STAT3,IDO and PD-L1.[Results]1.Immunofluorescence results demonstrated that IDO+CD1a+ and PD-L1+CDla+existed in OSCC tissues,providing a basis for further experiments.The results of immunohistochemical double staining showed that the expression level of IDO+CD1a+DC in tumor tissues of OSCC patients was significantly higher than that of normal tissues(P<0.05),which was used to analyze IDO+CD1a+,PD-L1+CD1a+ in OSCC.The significance of expression for patient prognosis,we assessed the correlation between IDO+CDla+,PD-L1+CD1a+Dand clinicopathological features.Of the 50 patients,21 had high expression of IDO+CD1a+Dand were closely associated with tumor stage(P=0.0065),lymph node metastasis(P<0.0001),recurrence(P=0.029),and WPOI(P=0.044).Correlation.Among the 50 patients,19 had high expression of PD-L1+CDla+DC,and were closely related to lymph node metastasis(P=0.0169),recurrence(P=0.0147),and degree of differentiation(P=0.0187).Our data indicate that patients with higher expression of IDO+CD1a+and PD-L1+CD1a+ have a statistically significant association with OS and DFS.COX regression analysis showed that IDO+CD1a+(OS:P=0.029;DFS:P=0.036)and PD-L1+CD1a+(OS:P=0.017;DFS:P=0.037)were independent of OS and DFS in OSCC.Risk factors can be used as an independent prognostic factor for OSCC.The expression level of VEGF in tumor tissues of OSCC patients was significantly higher than that in normal tissues(P<0.05).It was found that the high expression of VEGF was closely related to the high expression of IDO+CDl a+ and PD-L1+CD1a+,indicating that VEGF may induce high tol-DC.Express IDO and PD-L1.2.Flow detection of peripheral blood Mo-DC and pDC in patients with OSCC decreased compared with healthy controls.The analysis showed that the expression of Mo-DC and pDC in OSCC patients was significantly lower than that in healthy controls.These data indicate that OSCC microenvironment may promote DC and its Subgroups are reduced.The content of VEGF in serum of patients with OSCC was significantly higher than that of healthy people.The content of VEGF was negatively correlated with the content of Mo-DC(P<0.05).However,there was a negative correlation with the content of pDC,but there was no significant difference(P>0.05).In OSCC patients,Mo-DC expressed IDO and PD-LI higher than healthy people(P<0.0001).These data indicate that the OSCC microenvironment may promote the expression levels of IDO and PD-L1 molecules of Mo-DC,and convert DC to tol-DC.VEGF was positively correlated with the expression of IDO and PD-L1 in Mo-DC(P<0.05),which indicated that VEGF could promote the differentiation of Mo-DC into tol-DC and express IDO and PD-L1 in OSCC tumor microenvironment.3.The aggregation of DC after VEGF stimulation was poor.The expression of CD83,CD86,CD80 and HLA-DR on the surface after VEGF stimulation was significantly decreased by flow cytometry(P<0.05).The results indicate that VEGF factor can inhibit the differentiation of DC into maturation,which may cause it to differentiate into tol-DC.Compared with mDC group,PD-L1 and IDO were increased by VEGF group(P<0.05).4.After VEGF stimulation,the expression of p-JAK2,p-STAT3,IDO and PD-L1 protein increased significantly.After interfering with VEGFR1 and VEGFR2,p-p-JAK2,STAT3,IDO,PD-L1 The expression of protein decreased,and VEGF could activate JAK2/STAT3 pathway through VEGFR1/VEGFR2,which induced the increase of IDO and PD-L1.[Conclusion]VEGF in OSCC tissues and peripheral blood tumor microenvironment can induce DC to differentiate into tol-DC,high expression of IDO and PD-L1,and IDO+CD1a+ and PD-L1+CD la+ can be used as independent prognostic factors.VEGF can inhibit DC maturation and high expression of IDO and PD-L1,and its mechanism may be explored.It may be because the activation of VEGFR1/VEGFR2-JAK2/STAT3 pathway provides a new option for screening biotherapeutic targets for oral squamous cell carcinoma.