The Chemokine CCL25 Mediated Activation Of Fibroblasts Induces Immune Escape Of Tongue Squamous Cell Carcinoma Cells And Its Related Mechanism

[Objective]The immunosuppressive state of the tumor microenvironment is conducive to the immune escape of tumor cells.The activated fibroblasts in the tumor microenvironment are called cancer-associated fibroblasts(CAFs),which can promote the occurrence and development of tumors and are closely related to the immune status of tumor cells,but the mechanism is still unclear.The previous research of the research group found that platelet-derived growth factor BB(PDGF-BB)can induce fibroblast activation,high secretion of chemokine CCL25,and promote tumor development.This study uses the chemokine CCL25 as the entry point to explore the regulatory effect and molecular mechanism of PDGF-BB-induced activated fibroblasts on the immune escape of tongue squamous cell carcinoma cells,with a view to providing experimental basis of targeted CAFs to regulate the immune status of tumor cells and play a role in tumor therapy.[Methods]1.30ng/ml PDGF-BB was used to induce fibroblast activation,immunoblotting method was used to detect the expression of activation marker protein ?-SMA,ELISA method was used to detect CCL25 secretion,and activated fibroblasts were cultured routinely to prepare conditioned medium(AFs-sup).2.Western blotting was used to detect tongue squamous cell carcinoma cells PD-L1,CCL25 receptor(CCR9),Akt,p-Akt,STAT3,p-STAT3 and AP1 expression after the activated fibroblast supernatant(AFs-sup)or CCL25 treatment with different volume concentrations and different time conditions.Real-time fluorescent quantitative PCR was used to detect the expression level of PD-L1 mRNA in tongue squamous cell carcinoma cells.Immunofluorescence staining was used to detect the expression of CCR9 and PD-L1 in tongue squamous cell carcinoma cells.3.Construct the low-expression CCR9 lentiviral vector LV-CCR9-RNAi,and transfect the tongue squamous cell carcinoma Cal-27 cell to obtain stable Cal-27-shRNA-hCCR9,and use the empty lentiviral vector Cal-27-shRNA-Scramble as the control Group,q-PCR and Western blotting were used to detect the expression level of CCR9 in tongue squamous cell carcinoma Cal-27 cells after transfection.4.Construct a transplanted tumor model of tongue squamous cell carcinoma in nude mice,observe tumor formation and immunohistochemical staining to analyze the expression of PD-L1,CCR9 and p-Akt protein in tongue squamous cell carcinoma tissue.5.Magnetic beads were used to sort CD8+T cells from normal human peripheral blood;to establish a direct co-culture model of tongue squamous cell carcinoma Cal-27 cells and CD8+T cells induced by different conditions,ELISA method to detect IFN-? secretion,The proliferation rate of CD8+T cells was detected by CFSE staining cytometry,and the apoptosis rate of tongue squamous cell carcinoma Cal-27 cells was detected by PI/Annexin V staining flow cytometer.6.All experimental date are expressed as "mean�standard deviation",unpaired t test and one-way analysis of variance,using Graphpad Prism 6.0 to analyze and graph the data,P<0.05 is statistically significant,P<0.01 is significant Statistical difference.[Results]1.After PDGF-BB induced fibroblasts,western blotting showed that the expression of?-SMA increased(P<0.05),showing an activated state.ELISA method was used to detect the concentration of chemokine CCL25 in the culture medium of fibroblasts induced by PDGF-BB Significantly increased(P<0.05).Activated fibroblast supernatant(AFs-sup)induced tongue squamous cell carcinoma Cal-27 cells,and the results of western blot showed that the expression of PD-L1 protein was up-regulated,and it was concentration-and time-dependent(P<0.01).2.When activated fibroblast supernatant induces tongue squamous cell carcinoma Cal-27 cells,after adding CCL25 antibody blocker and its receptor CCR9 inhibitor respectively,Western blotting showed that the up-regulation of PD-L1 protein expression was inhibited,line q-PCR further detected the expression of PD-L1 mRNA,and the results were similar(P<0.05).Compared with the control group,immunofluorescence staining observed that the expression of CCR9 and PD-L1 in the tongue squamous cell carcinoma Cal-27 cells in the activated fibroblast supernatant group and the CCL25 factor group was significantly increased,and the results of further immunoblotting were similar.3.After the chemokine CCL25 directly induces Cal-27 and HSC-4 cells,compared with the control group,the PD-L1 protein expression level of Cal-27 and HSC-4 cells is up-regulated.At the same time,after adding CCR9 receptor inhibitor,The up-regulation of PD-L1 protein expression in Cal-27 and HSC-4 cells was inhibited.q-PCR was performed to further detect the expression of PD-L1 mRNA in tongue squamous cell carcinoma Cal-27 cells,and the results were similar(P<0.05)4.Western blotting showed that before and after CCL25 treatment,the expression of Akt,STAT3,p-STAT3 and AP1 protein in tongue squamous cell carcinoma Cal-27 cells did not change significantly(P>0.05),while the expression of p-Akt protein increased with the treatment time High(P<0.05),and the expression level increased with the increase of CCL25 concentration(P<0.05).After adding Akt inhibitor,the up-regulation of PD-L1 mRNA expression was inhibited,and the results of western blotting were similar(P<0.05).5.Compared with the Cal-27-shRNA-Scramble control group,CCL25 induced tongue squamous cell carcinoma Cal-27-shRNA-hCCR9 cells,and the up-regulation of PD-L1 and p-Akt protein expression was inhibited(P<0.05).6.Activated fibroblasts and tongue squamous cell carcinoma Cal-27 cells were co-transplanted subcutaneously in nude mice.Compared with the tongue squamous cell carcinoma Cal-27 cell transplantation group and the normal fibroblast co-transplantation control group,the transplanted tumor weight increased,and The expression levels of PD-L1,CCR9 and p-Akt in transplanted tumor tissues were significantly increased(P<0.05).Furthermore,co-transplantation of stabilized tongue squamous cell carcinoma Cal-27-shRNA-hCCR9 cells with activated fibroblasts inhibited the increase in tumor weight and the up-regulation of PD-L1 and p-Akt expression.7.CD8+T lymphocytes were directly co-cultured with tongue squamous cell carcinoma Cal-27 cells induced by activated fibroblast supernatant or CCL25,Compared with the uninduced tongue squamous cell carcinoma control group,flow cytometry showed that The apoptotic rate of the tongue squamous cell carcinoma Cal-27 cells decreased,the proliferation rate of CD8T lymphocytes decreased,and the IFN-y secretion concentration detected by the ELISA kit decreased;after the addition of CCL25 neutralizing antibody,Akt inhibitor and PD-L1 inhibitor,The apoptosis rate of tongue squamous cell carcinoma Cal-27 cells increased,and the proliferation rate of CD8+T lymphocytes and the concentration of IFN-? in the supernatant were significantly increased compared with the control group(P<0.05).[Conclusions]1.PDGF-BB induced activation of fibroblasts is an important factor that makes tongue squamous cell carcinoma cells in an immune escape state,and the immune escape state of tongue squamous cell carcinoma cells promotes tumorigenesis and development.2.CCL25 is a key mediator that activates fibroblasts to induce immune escape of tongue squamous cell carcinoma cells.It may bind to the CCR9 receptor to activate the Akt signaling pathway,up-regulate the high expression of PD-L1 in cancer cells,and promote the immune escape of tongue squamous cell carcinoma Cal-27 cells.