A Study On The Inhibition Of Sunitinib On The Expression Of PD-L1 On Dendritic Cells Induced By LPS Through STAT3 Pathway

Objective To investigate the effect of sunitinib on the expressions of 6 co‐inhibitory molecules including programmed death ligand1(PD‐L1), PD‐L2, CD80, CD86, B7‐H4 and herpes virus entry mediator(HVEM)on monocyte‐derived dendritic cells(DCs) from patients with renal cell carcinoma(RCC),and to explore whether sunitinib through STAT3 signaling pathway to regulate the expressions of these co‐inhibitory molecules, in order to supply a certain theoretical basis for the therapeutic regimen of sunitinib combined with DCs for RCC.Methods Monocyte‐derived DCs from patients with RCC were cultured in vitro and randomly divided into four groups: sunitinib combined with lipopolysaccharide(LPS), LPS only, STAT3 pathway specific inhibitor JSI ‐ 124 combined with LPS, or treated with DMSO.sunitinib‐combination group was pretreated with 200 ng/m Lsunitinibfor 12 hours, and then stimulated with 1 μg/m L LPSfor 24 hours; LPS group was pretreated with1 μL/m L DMSOand then stimulated with1 μg/m L LPSfor 24 hours; JSI‐124‐combination group was pretreated with 500 nmol/m L JSI‐124 for 12 hours, and then stimulated with 1 μg/m L LPSfor 24 hours; DMSO group was treated with 1 μL/m L DMSOfor 36 hours.Morphological changes were observed by an inverted microscope.Detectedthe cells viabilities on each group by Taiwan phenol blue staining method. Flow cytometry was used to detect three markers including HLA‐DR, CD83 and CCR7 of DCs, andthe co‐inhibitory molecules including programmed death ligand1(PD‐L1), PD‐L2, CD80, CD86, B7‐H4 and HVEMon DCs.The analyses of the un‐ and phosphorylated STAT3 proteins were used by Westren blot. Data analysis using SPSS19.0 version, all of the results are shown as mean ± Standard Deviation(x±s),Levene’s test was used for all the data, followed with the comparison of two groups were analyzed by Student’s t‐test. A P value of less than 0.05 was considered significant.Results 1.With increasingincubation time, the adherent cells of all groups appeared colony growth, increased gradually, relatived gradually suspension growth, became larger and had irregular shape.Induced by LPS for 24 h, sunitinib in combination with LPS group and LPS group were significantly larger, burr shape more obvious, showing a typical form of mature dendritic cells. The cells of JSI ‐ 124 ‐combination group changed significantly, burr shape was not obvious, and all of themwere in the state of suspension.In addition, morphological changes in DMSO group were not obvious. 2.DC cytoactive of each group was detected by trypan blue staining.According to the formula: living cells rate(%) =the number of living cells/(numbers of living cells and dead cells) x 100%, compared with LPS group, All the calculated resultsof living cells had no obvious difference. 3.The results were analyzed byflow cytometryshowed that the percentage of DC in the study was approximately 79.92%, so it was very high that the purity of DC in this experiment. Each DC phenotype including CCR7,CD83 and HLA‐DR analysis: compared with LPS group, the percentage of CCR7 insunitinib‐combination group and DMSO group decreased obviously(P<0.01); the percentage of CD83 on Sunitinib‐combination was higher than that of LPS group, while LPS group was significantly higher than DMSOgroup(P < 0.01); In addition,the average fluorescence intensity of HLA – DR was expressed on sunitinib‐combination group, JSI ‐ 124‐combination group had no significant difference, but DMSO group was significantly lower expression(P < 0.01). Analysis of the expressions of co‐inhibitory molecules on DCs, compared with LPS group, in Sunitinib‐combination group: the percentage of B7‐H4 and the average fluorescence intensities of PD‐L1 and CD80 were lower(P<0.01, P<0.05,P<0.01), but there were no significant difference among the average fluorescence intensities of PD‐L2, CD86 and HVEM.In JSI‐124 ‐combination group: the average fluorescence intensity of PD‐L1 was decreased(P<0.05), however, the expressions of CD80, CD86, PD‐L2, B7‐H4 and HVEM had no significant differencecompared with LPS group.In DMSO group, the expressions of all co‐inhibitorymolecules were lower(P<0.01). 4. The gray intensitiesof the p‐STAT3 protein among Sunitinib‐combination, JSI‐124‐ combination and DMSO group were lower than LPS group(1.24±0.11、0.51±0.1、0.02±0.004 vs 2.1±0.3).Conclusions 1.Monocytes‐derived dendritic cells from RCC matured quickly induced by LPS.Sunitinib did not affect the Morphological changes of DC. 2.LPS could promote DCsto express inhibitory signals.Sunitinib inhibited the expressions of co‐inhibitory molecules including PD ‐ L1, CD80 and B7 ‐ H4 on DCs induced by LPS to restrain inhibitory signals transmission. 3. Sunitinib inhibited STAT3 signaling pathway activation in DCs induced by LPS 4. PD‐L1 expression on DCs induced by LPS was controlled by STAT3 5.the inhibition of Sunitinib on the expression of PD‐L1 on dendritic cells induced by LPS maybe through STAT3 Signalingpathway.