Cancer-associated Fibroblasts Promote The Immune Escape Of Colorectal Cancer By Upregulating PD-L1 Expression Through Akt Phosphorylation

BackgroundColorectal cancer(CRC)is one of the most vital malignant diseases around the world,with a high morbidity and mortality rate.Although radical resection is one of the most important treatment methods for CRC,about 30-40%of patients will still have recurrence and metastasis after surgery.Local recurrence and distant metastasis are the main causes of death in CRC patients,and immune escape of tumor cells plays an important role in it.The overall survival of patients with advanced CRC is very poor.The mechanism of development of CRC has not been fully clarified.As an important immune checkpoints,programmed death receptor ligand 1(PD-L1)binds to programmed death receptor 1(PD-1)to inhibit T cell function and promote immune escape.PD-L1 is highly expressed in a variety of tumors and is associated with worse prognosis and inferior stage.The upregulation of PD-L1 expression in tumors is regulated by multiple mechanisms,in which tumor microenvironment plays an indispensable role.Cancer-associated fibroblasts(CAFs)are one of the most important components of tumor microenvironment,and it has been shown that CAFs can promote the growth and survival of CRC tumor cells,resulting in tumor progression and poor prognosis in CRC patients.CAFs have also been proved to up-regulate the expression of PD-L1 in tumor cells,but the mechanism has not been fully clarified.Therefore,our study plans to explore the effects of CAFs on the expression of PD-L1 and immune escape in CRC,and further to clarify the specific regulatory mechanism,in order to provide potential therapeutic targets for the treatment of CRC.Methods1.To investigate the effect of CAFs on immune escape of CRC cell lines,we cocultured MSCs with exosomes derived from CRC cell lines.The characteristics of CAFs were verified by Western Blot and multi-differentiation induction experiment.2.The killing effect of peripheral blood mononuclear cells(PBMCs)on CRC cell lines was tested to verify the influence of CAFs on immune escape capacity of CRC cell lines.CRC cell lines were cultured with CAFs conditioned medium(CAF-CM)and normal medium(control group),and then PBMCs from healthy volunteers were used to kill CRC cells.MTS assay was used to detect the survival of tumor cells,and flow cytometry was used to detect the apoptosis rate of tumor cells to evaluate the immune escape capacity of tumor cells.Western Blot was further used to verify the correlation between immune escape capacity and PD-L1 expression in CRC cell lines.3.Determination of key signaling pathway:Western Blot and RT-qPCR were used to detect key factors in the signaling pathways involved in regulating the expression of PD-L1.Subsequently,cell killing assay was used to verify the correlation between the above key signaling factors and immune escape in CRC cell lines.4.Colorectal cancer samples were retrospectively collected with informed consent from patients receiving surgery.Clinical and pathological data of the patients were collected,immunohistochemical staining was performed on tumor tissue samples,and the correlation between clinicopathological features,CAFs,the key signaling pathway that CAFs regulates PD-L1 expression in CRC,PD-L1 expression and prognosis were analyzed.Results1.Exosomes derived from CRC cell lines induced the differentiation of MSCs into CAFs.After induction into CAFs,the characteristic proteins a-SMA and FAPA were significantly up-regulated,while the ability of multi-differentiation was significantly decreased.2.The results of MTS assay and flow cytometry assay showed that the killing rate of PBMCs on CAF-CM group was significantly lower than that of the control group.The results showed that the immune escape capacity of the former was significantly enhanced.Western Blot and RT-qPCR results showed that the expression of PD-L1 in CAF-CM-treated CRC cells was significantly increased.The phosphorylation levels of Akt,ERK,NF-κB p65 and STAT3 in CAF-CM-treated CRC cells were also significantly up-regulated.3.After the addition of Akt,ERK,NF-κB p65 and STAT3 phosphorylation inhibitors in the culture system,only Akt phosphorylation inhibitor(pAkti)could significantly reduce the up-regulation of PD-L1 expression in CAF-CM-treated CRC cells,and the ability to resist the killing of PBMCs was also significantly reduced.After adding anti-PD-L1 monoclonal antibody(αPD-L1)into CAF-CM cultured CRC cells,there was no significant difference in the killing rate of PBMCs in the blocking PD-L1 group,the blocking Akt phosphorylation group and the co-blocking group,but they were all significantly higher than the control group(CAF-CM cultured group).Thus,CAFs promote immune escape through the up-regulation of PD-L1 expression caused by Akt phosphorylation in CRC.4.A total of 102 postoperative patients diagnosed with CRC were enrolled,including 49 males and 53 females.Among all the patients,40 patients(39.2%)were positive for PD-L1 expression,20(19.6%)were positive for p-AKT expression,and 25(24.5%)had high a-SMA expression in the stromal cytoplasm.Positive PD-L1 expression was associated with inferior tumor stage(χ2=7.808,P=0.005).There was no significant correlation of PD-L1,p-AKT or a-SMA expression with age,gender,tumor location and tumor differentiation(P>0.05).Correlation analysis suggested that the expression of PD-L1 was positively correlated with the p-AKT(Spearman R=0.213,P=0.031)and a-SMA levels(Spearman R=0.246,P=0.012).The DFS of patients with positive PD-L1 expression was significantly shorter than that of patients with negative expression(P=0.011).Patients with high a-SMA expression had shorter DFS than those with low expression(P=0.025).The DFS of p-Akt positive patients tended to be worse than that of negative patients,but the difference was not significant(P=0.132)The DFS of patients with combined PD-L1+,p-AKT+ and α-SMAhigh status was remarkably shorter than that of patients with triple negative expression(P<0.001).Conclusions:1.CAFs promote CRC immune escape by regulating Akt phosphorylation of CRC cells and up-regulating PD-L1 expression.2.The prognosis of colorectal cancer patients with co-expression of PD-L1,pAkt and a-SMAhigh is worse.