Exosomal PD-L1 And Immune Model Influence The Prognosis Of Advanced Tumor And Immunotherapy Response And The Mechanism

Objective:In the past,the treatment of advanced malignant tumors mainly focused on radiotherapy,chemotherapy and targeted therapy.Comprehensive treatment can improve the overall survival and quality of life of patients with advanced tumors,but the overall benefit is limited.Nowadays,immunotherapy,especially immune checkpoint inhibitors,has become a hotspot and innovation in anti-tumor therapy,providing a new treatment for advanced tumors.In the immune checkpoint pathway,programmed cell death 1(PD-1)and its ligand(PD-L1)are the main research objects.The combination of the two inhibits the activation and function of T cells and plays an important role in tumor immune escape,progression and metastasis.PD-1 or PD-L1 monoclonal antibodies can induce anti-tumor immune responses by blocking the connection between PD-1 and PD-L1.In recent years,various types of immunotherapies,such as immune checkpoint inhibitors,have carried out numerous clinical trials and been approved for the treatment of a variety of advanced malignancies.Overall,the response rate of single-agent PD-1 monoclonal antibody therapy is low,only 10%-25%.Current predictors of efficacy are limited,so research on biomarkers that can effectively predict the efficacy of anti-PD-1 therapy is urgently needed.As a key factor in the target pathway,the expression of PD-L1 in tumor tissues detected by immunohistochemistry(IHC)only serves as a predictor of efficacy in the first-line treatment of advanced lung cancer with Papolizumab.However,some studies have also shown that the benefits of anti-PD-1/PD-L1 therapy in patients with tumors with low or no PD-L1 expression are not significantly different from those in patients with high PD-L1 expression.In addition,according to the conclusions of different clinical trials,the cutoff value of PD-L1 positivity was set at 1-50%,and no unified standard was reached.Therefore,PD-L1 cannot be a stable predictive treatment for the efficacy of immunotherapy.The reason is mainly due to the diversity of PD-L1 expression forms and the complexity of the immune microenvironment.On the one hand,PD-L1 has a variety of manifestations,and the existing studies mainly include membrane-surface type PD-L1,soluble type PD-L1 and exosomal type PD-L1(exosomal PD-L1).Exosomes are a kind of extracellular vesicles with a diameter of 30-150 nm,which contain a large number of DNA,RNA,protein and other substances.Because of their advantages such as good stability,high content and easy enrichment,exosomes have become a research hotspot in liquid biopsy.Compared with membrane-surface and soluble PD-L1,exosomal PD-L1 is more stable and easily accessible,making it a potential biomarker for the efficacy of immunotherapy.Exosomal PD-L1 is not confined to local tumors,but can exert its immunosuppressive function at a distance.Recent studies have shown that exosomal PD-L1 can inhibit T cell activation,suppress immune memory function and promote tumor growth,and exosomal PD-L1 can also lead to resistance to immunotherapy by directly binding to PD-L1 antibody.Chen et al also found that patients with melanoma with high baseline exosomal PD-L1 expression had worse efficacy with PD-1 monoclonal antibody because exosomal PD-L1 secreted in metastatic melanoma inhibited the activation of CD8+ T cells and promoted tumor growth.In head and neck squamous cell carcinoma patients,exosomes with high expression of PD-L1 can significantly inhibit CD69 expression on CD8+ T cells,and this inhibition can be blocked by PD-1 antibody.In addition,inhibition of exosome production can enhance the effect of PD-1 mAb.Other studies have shown that melanoma patients with significantly elevated exosomal PD-L1 respond worse to PD-1 antibodies after treatment.On the other hand,due to the complexity of the immune system,a single indicator cannot completely and comprehensively reflect the efficacy and status of anti-PD-1 therapy.The characteristics of tumors,the functions of immune cells and the status of the immune microenvironment are closely linked and interacted in the course of immunotherapy.In addition to the immune microenvironment of tumor cells represented by PD-L1,there are many immunosuppressive and immunostimulatory factors involved in regulating the immune microenvironment.The depletion status of T cells represented by PD-1,LAG-3 and Tim-3,the activation status of T cells marked by CD8 and CD28,and the indicators of T cell killing function such as INF-gamma,granzyme B and perforin may all have important predictive value for the efficacy of immunotherapy.Therefore,this study intends to explore the predictive value of different forms of PD-L1 for the prognosis of advanced tumors and the efficacy of anti-PD-1 therapy through the detection of different forms of PD-L1,combined detection of immunotherapy-related factors and bioinformatics mining,as well as the predictive value of gene models characterizing the immune microenvironment for the response to immunotherapy.Therefore,it provides a scientific basis for the prediction of the efficacy of tumor immunotherapy and the screening of potential beneficiaries.Methods:1.Incorporate 103 cases of advanced gastric cancer patients with pathologically confirmed non-surgical resection or postoperative recurrence treated in the First Hospital of China Medical University from December 2012 to December 2015,and accept from January 2017 to April 2019 44 patients with advanced malignant tumors with pathologically confirmed unresectable or postoperative relapse were treated with single drug PD-1 monoclonal antibody.2.The exosomal PD-L1,exosomal PD-1 and immunerelated factors were detected by ELISA.3.The expression of PD-L1,PD-1,CD9,CD63 and other proteins was detected by protein immunoblotting.4.Flow cytometry was used to identify exosomal PD-L1 and detect the effect of exosomal PD-L1 expression on T cell apoptosis and exhaustion.5.The levels of CD8+T cytokines and tumor immune related factors were detected by MILLIPLEX magnetic bead analysis.6.Detection of plasmaenriched exosomes by electron microscopy and Nanosight technology.7.Use TCGA and GEO databases to download RNA sequencing expression datasets of lung squamous cell carcinoma.8.The data set samples were scored by ssGSEA method,and clustered by WGCNA method.9.Statistical processing: SPSS 22.0 statistical software was used for chisquare test,Spearman rank correlation test and Fisher’s exact calculation of probability method.T-test was used for comparison between groups.Each experiment was repeated 3 times,and the data were expressed as mean standard deviation(s).P < 0.05 was statistically significant.Univariate and multivariate survival analysis was performed using Cox model.Simplification of analysis results by LASSO COX regression.Results:1.Effect of exosomal PD-L1 on prognosis of patients with advanced gastric cancer and its mechanism:(1)Survival analysis showed that patients with high exosomal PD-L1 expression in advanced gastric cancer had shorter overall survival than those with low expression(p=0.007).(2)Univariate analysis showed that high expression of exosomal PD-L1 was an adverse factor for overall survival of patients with advanced gastric cancer(HR=2.299,95% CI 1.232-4.291,p=0.009).(3)Multivariate analysis showed that high expression of exosomal PD-L1 was an independent prognostic factor for overall survival of patients with advanced gastric cancer(HR=2.213,95% CI 1.183-4.137,p=0.013).(4)Validation centralized validation showed that exosomal PD-L1 overexpressing advanced gastric cancer patients had shorter OS(P=0.023),and timedependent ROC curve suggested that it had good predictive value.(5)Correlation analysis showed that exosomal PD-L1 was negatively correlated with CD4,CD8 and CD3 counts(r=-0.318,p=0.026;r=-0.428,p=0.002;r=-0.436,p=0.002),negatively correlated with the expression of stimulatory factor IFN-gamma(r=-0.407,p=0.004),and positively correlated with the expression of inhibitory factors IL-10 and TGF-beta(r=0.338,p=0.018;r=0.448,p=0.001).(6)Exosomes increased apoptosis after acting on activated T cells(P=0.0104),and apoptosis was further promoted after using exosomes overexpressing PDL1 on activated T cells(P=0.0002).(7)Exosomal PD-L1 is positively correlated with the proportion of exhausted T cells,and patients with a high proportion of exhausted T cells are not well treated with PD-1 monoclonal antibody.2.Exosomal PD-L1 combined with CD28 predicts the efficacy of anti-PD-1 therapy in patients with advanced malignant tumors:(1)The expression of exosomal PD-L1 and exosomal PD-1 was significantly higher in patients with PD-1 mAb ineffective group(p values were 0.010 and 0.022,respectively).(2)The expression of CD28,CD80,CD86,GITRL,ICOS and TLR-2 at baseline was higher in the effective group of PD-1 monoclonal antibody treatment(p values were 0.005,0.019,0.038,0.024,0.009 and 0.008,respectively).(3)X-tile selected the best cut-off value,the anti-PD-1 treatment progression-free survival was shorter in the exosomal PD-L1 high expression group(P=0.001),and the anti-PD-1 treatment progression-free survival was longer in the CD28,CD80,CD86,GITRL,ICOS and TLR-2 high expression group(p values were 0.005,0.048,0.017,0.017,0.045 and 0.007,respectively).(4)Univariate analysis showed that high expression of exosomal PD-L1 was an adverse factor for progression-free survival(HR=3.017,95% CI 1.439-6.325,p=0.003),high expression of CD28 was associated with prolonged progression-free survival(HR=0.394,95% CI 0.192-0.811,p=0.011),and high expression of CD86,GITRL and TLR-2 was also associated with prolonged progressionfree survival(HRCD86=0.409,95% CI 0.182-0.915,p=0.030;GITRL=0.409,95% CI 0.182-0.915,p=0.030;HR TLR-2=0.348,95% CI 0.149-0.812,p=0.015).(5)Multivariate analysis showed that exosomal PD-L1 was an independent predictor of poor efficacy of anti-PD-1 therapy(HR=2.746,95% CI 1.627-8.480,p=0.009).CD28 was an independent predictor of the effectiveness of anti-PD-1 therapy(HR=0.430,95% CI 0.206-0.897,p=0.025).(6)In subgroup analysis,both low expression of exosomal PD-L1 and high expression of CD28 in NSCLC group showed significant prolongation of progression-free survival(p values were 0.010 and 0.045,respectively).In other tumor groups,high expression of CD28 showed a prolonged progression-free survival(p=0.045),and high expression of exosomal PD-L1 also showed a trend of reduced progression-free survival(p=0.066).(7)Using ROC curve to evaluate the prediction efficiency,exosomal PD-L1 combined with CD28(AUC=0.850)was more efficient than single index(AUC exosomal PD-L1=0.678,AUCCD28=0.784).(8)Two-index positive patients had longer progressionfree survival(p=0.003),and the effective rate of PD-1 monoclonal antibody treatment was significantly higher than that of single-index or no-index positive patients(p<0.0001).This result was also validated in non-small cell lung cancer and other cancers(p values were 0.002 and 0.006,respectively).3.Bioinformatics was used to establish an immunoinflammatory gene model in lung squamous cell carcinoma and to predict the efficacy of immunotherapy:(1)Using ssGSEA score and WGCNA method,immune-related modules were screened from TCGA and GSE50081 lung squamous cell carcinoma data sets.(2)Enrichment of immune-related module function was correlated with immune cell function,and TCGA immune-related module gene was significantly positively correlated with tumor inflammatory type(r=0.69,p<1e-200;r=0.93,p<1e-200;r=0.57,p=3.5e-135)(3)Hub gene was identified by immune-related modules screened by TCGA and GSE50081,and 51 genes were obtained by taking the intersection,which was significantly positively correlated with tumor inflammation type(r=0.38,p=1.1e-18;r=0.92,p=4.4e-200;r=0.32,p=5.4e-13)(4)External data sets validated that 51 genes were highly expressed in immunoinflammatory patients(p=2.5e-6 and p=7.5e-14)and in the effective group of PD-L1 monoclonal antibody treatment(p=0.031).(5)The simplified 7-gene model of LASSO was highly expressed in immunoinflammatory patients(p=1.7e-6 and p=7.7e-14)and in the effective group of PD-L1 monoclonal antibody treatment(p=0.026).(6)The threshold of positive expression of each gene is 70% quantile,and at least four genes in the 7-gene model are positive,which can represent the positive of the model.(7)In TCGA data of lung squamous cell carcinoma,the expression of inflammatory representative molecules(IFNgamma,DC and PD-L1)was higher in 7 gene model-positive patients(p=3.7e-16,p<2.2e-16,p=6.4e-10).Conclusion: 1.Exosomal PD-L1 is an independent prognostic factor for advanced gastric cancer,which affects the number and function of T cells by promoting T cell apoptosis and exhaustion,IL-10,Increased release of TGF-beta and decreased release of IFN-gamma lead to poor prognosis in advanced gastric cancer.2.Baseline Exosomal PD-L1 combined with CD28 levels in patients with advanced tumors can be used as a predictive biomarker for the efficacy of PD-1 monoclonal antibody therapy,and the role of exosomal PD-L1 may be related to T cell activation status.Seven gene models reflecting the immunoinflammatory type of lung squamous cell carcinoma were obtained by bioinformatics methods,which could screen out the patients with lung squamous cell carcinoma who could potentially benefit from immunosuppressants.