The Function And Mechanism Study On PD-L1 Expressed In Macrophages Of Non-Small Cell Lung Cancer-Via The Landscape Analysis Of Macrophages

Objective:Lung cancer is the leading cause of cancer death in the world.Proto-oncogene activation and inactivation of tumor suppressor genes play a key role in the malignant biological behavior of lung cancer.The occurrence and development of non-small cell lung cancer and the behavioral characteristics of invasion and metastasis are not only related to the characteristics of lung cancer cells,but also closely related to the immune microenvironment of lung cancer.Although molecular targeted drugs targeting driver gene mutation significantly improved the overall survival?OS?,it is still great obstacle for improving the prognosis of advanced lung squamous cell carcinoma and wild-type lung adenocarcinoma.The advent of immune checkpoint inhibitors established milestone for the improvement of advanced lung cancers.PD-1?Programmed cell death protein 1?antibody approved by the US Food and Drug Administration?FDA?for advanced non-small cell lung cancer?NSCLC?has achieved significant clinical efficacy in the first-and second-line therapy.Therefore,immune checkpoint inhibitors made breakthroughs in the treatment of wild-type NSCLC patients who are not suitable for tyrosine kinase inhibitors?TKIs?therapy.However,PD-1/PD-L1,as the therapeutic target,is still controversial to be a predictor of efficacy.It was noticed that some patients with tumor cell PD-L1 negative could still benefit from anti-PD-1/PDL1 antibody therapy.It is related to the complexity of the microenvironment.In addition to the tumor cells,PD-L1 was also known to express in immune cells in TME.Futhermore,the number of tumor infiltrating lymphocytes?TIL?and the distribution of other immune cells in the microenvironment,such as myeloid-derived suppressor cells,macrophages,etc.,affect the immune status and prognosis of patients.Recently,tumor immune microenvironment?TIME?was classified into 4 subtypes based on the status of tumor cell PD-L1 and TILs.Type I:tumor cell PD-L1 positive with TILs driving adaptive immune resistance.Patients in this group have a relatively good prognosis,and are largely responding to checkpoint blockade;Type II:tumor cell PD-L1 negative without TIL indicating immune ignorance.Patients in type II are predicted to have very poor prognosis based on their lack of detectable immune reaction;Type III:PD-L1 positive without TIL indicating intrinsic induction.It highlights that tumor cell PD-L1 positivity alone cannot be taken as a predictive factor for response to anti-PD-1/PD-L1 therapies;Type IV:tumor cell PD-L1negative with TIL.The immune status and prognosis of patients of type IV are still uncertain.Immunosuppressive cells,such as myeloid derived suppressor cell?MDSC?,tumor-associated macrophage?TAM?,play an important role on the promotion of immune tolerance.It aroused our great interesting that some patients of type IV with negative expression of PD-L1 in tumor cell,could still benefit from anti-PD-1 therapy.Therefore,in the complicate TIME,to elucidate the PD-L1 expression in immune cells except for tumor cells is of great significance for the full understanding of TIME prognosis function and screening of the effective population for PD-1/PD-L1 inhibitors therapy.However,it is unclear that which kind of immune cells mainly express PD-L1and what function these PD-L1 positive immune cells exert on prognosis and anti-PD-1therapy in NSCLC patients,especially in patients with tumor cell PD-L1 negative.In this study,we aimed to clarify the factors that affect the PD-L1 expression in TME,and their functions on the NSCLC prognosis.Methods:1.RNA sequencing data of 1035 available patients with NSCLC were downloaded from the TCGA data portal?http://cancergenome.nih.gov/?.2.The association between PD-L1 and biological processes/signaling pathway gene set was analyzed using Gene set enrichment analysis?GSEA Java software?.3.Multiple quantitative fluorescence staining technique was used to present the immune microenvironment landscape of patients with NSCLC.4.The protein expression was analyzed by Western blotting.5.RT-q PCR was used to detect the level of target gene expression.6.Flow cytometry was used for cell sorting and surface protein expression.7.Liposome-mediated siRNA transfection was used to knockdown gene expression.8.Statistical analysis:Each cell experiment was repeated three times independently,and the results were expressed as mean±SD.T-test,Chi-square test,Spearman rank correlation test,non-parametric test and Fisher's precise calculation probability method were performed with SPSS 17.0 statistical software.Survival curves were determined using the Kaplan-Meier method and compared using the log-rank test.Univariate and multivariate analyses were performed by Cox proportional hazards regression models to estimate HR and 95%CI.Statistical significance was considered at P<0.05Results:1.PD-L1 expression is associated with MHC class II antigen presentation in NSCLC by TCGA online data analysis:To investigate PD-L1-related factors,the TCGA cohort including 1035 NSCLC patients was classified into PD-L1 high expression and PD-L1 low expression groups,and the GSEA functional enrichment analysis was carried out.The results showed that PD-L1 expression was significantly correlated with antigen recognition and presentation.Further pathway Gene Ontology analysis of antigen recognition and presentation-related 81 differential genes revealed that these genes are mainly enriched in the function of MHC class II antigen presentation.2.PD-L1 expression is associated with macrophages in NSCLC by TCGA online data analysis:Therefore,we speculate PD-L1 expression might be correlated with macrophage in NSCLC.As a result,this postulate was approved by the positive correlation between macrophage marker CD68and PD-L1 using spearman correlation analysis?R=0.46,P<0.01?.The correlation between M1 macrophage markers CD11c,NOS2 or M2 macrophage markers CD206,CD163 and the expression of PD-L1 in NSCLC was further analyzed by multiple linear regression method.The result showed that the M2 macrophage marker CD163?beta=0.332,P<0.01?is the most important indicator for the PD-L1 expression.3.Multi-quantitative immunofluorescence staining of non-small cell lung cancer tissue specimens confirmed positive correlation between macrophage and non-small cell lung cancer PD-L1 expression:137 patients with stage I-III NSCLC were enrolled to visualize the macrophage landscape in the microenvironment of NSCLC.The correlation analysis showed that there was a positive correlation between the number of macrophages and the expression of PD-L1 in NSCLC tissues?R=0.7066,P<0.01?,and the correlation between M2 macrophages and PD-L1?R=0.5636,P<0.01?was stronger than M1macrophages?R=0.4688,P<0.01?,which was consistent with the results of gene expression in 1035 NSCLC patients.It is demonstrated that macrophages,especially M2macrophages,are associated with the expression of PD-L1 in non-small cell lung cancer.4.Macrophages express PD-L1 abundantly in the microenvironment of non-small cell lung cancer:By observing the landscape of non-small cell lung microenvironment,It was found that macrophages did express PD-L1.The expression of PD-L1 in microenvironment of 137 patients with non-small cell lung cancer was analyzed in detail.It was found that about 88%of non-small cell lung cancer patients had PD-L1 positive expression in macrophages,and the expression of PD-L1 in M2 macrophages was higher than that in M1 macrophages.5.Distribution of macrophages in microenvironment of non-small cell lung cancer:The distribution of macrophages in the NSCLC microenvironment was analyzed.The results showed that the number of macrophages in the tumor tissues accounted for 15.84%of all cells in tumor tissue?27.8%of all stroma cells?.Among the macrophages,36.68%were M1 and 63.32%were M2,furthermore,77.18%of macrophages were located around the tumor interstitial region?these macrophages are defined as marginM?,whereas 22.82%of the macrophages infiltrated the tumor islets?defined as centralM?.6.The number and distribution of macrophages were correlated with the stage of lung cancer:The amount of total M2 macrophages and the M2 polarization status?M2/M?significantly increased from stage I to stage III.We also observed the sites of macrophage infiltration in tumors of various stages.CentralM2and marginM2 increased from state I to stage III.CentralM1?M1 macrophages infiltrating into tumor islets?rapidly increased while marginM1?M1 macrophages infiltrating in stroma?decreased in stage II,and then central and marginM1 both decreased in stage III.Moreover,with the development of tumors to late stage,most macrophages in microenvironment are polarized to M2 state,suggesting that M2macrophages may play an important role in the development of NSCLC.7.The number of macrophages was correlated with the pathological types,EGFR status and smoking status of lung cancer:Compared to patients with lung adenocarcinoma?LUAD?,patients with EGFR mutations,and non-smokers,higher densities of total macrophages were found in patients with lung squamous cell carcinoma?LUSC?,patients with wild-type EGFR,and smokers.M2 polarization?M2/M?levels were nearly identical between the subgroups.5.Infiltration of macrophages influenced the overall survival of patients with NSCLC:The overall survival?OS?of patients with high infiltration of M2 macrophages was significantly shorter than that of patients with low infiltration of M2 macrophages?P=0.003?,whereas the total numbers of macrophages and M1 macrophages did not affect the OS.Compared to patients with high infiltration of centralM and centralM2,patients with low infiltration of centralM?P=0.003?,especially centralM2?P=0.000?,had a better OS.Patients with high infiltration of marginM1 had a better OS than those with low infiltration of marginM1?P=0.034,?.In addition,high infiltration of marginM2 also decreased the OS?P=0.036?.Futhermore,patients with centralM2/marginM2^high had a worse OS than those with centralM2/marginM2^low?P=0.001?,suggesting that centralM2have a greater impact on the OS of NSCLC patients than marginM2.9.High infiltration of centralM2 is an independent prognostic factor for poor overall survival in patients with non-small cell lung cancer:Results from univariable and multivariable analyses revealed that the amount of centralM2 was an independent predictor of OS for NSCLC?HR=2.859,95%CI?1.125-7.262?,P=0.027?.10.Infiltration of macrophages influenced the disease-free survival time of patients with NSCLC:The macrophage density,total M1macrophage density and total M2 macrophage density did not significantly affect the patient's disease-free survival time.High infiltration of central macrophages led to poor disease-free survival?P=0.023?.Patients with low infiltration of centralM?P=0.023?,especially centralM2?P=0.006?had better disease-free survival time?DFS?than patients with high infiltration of centralM and centralM2.The patients with centralM2/marginM2^high had worse DFS than those with centralM2/marginM2^low?P=0.005?.These results indicate that centralM2 have an important impact on the DFS of NSCLC patients.11.High infiltration of centralM2 is an independent prognostic factor for poor DFS in patients with non-small cell lung cancer:Results from univariable and multivariable analyses revealed that the amount of centralM2 was an independent predictor of DFS for NSCLC?HR=1.809,95%CI?1.085-3.015?,P=0.023?.12.The combined analysis of macrophages and tumor PD-L1 could improve the accuracy of prognosis prediction:Combined analysis of M2 macrophage and tumor PD-L1expression in the prognosis of NSCLC was conducted.Patients with M2^lessPD-L1^-had the best OS while patients with M2^morePD-L1⁺had the worst OS?P=0.027?.Next,analysis of correlation between the prognosis and the combination of centralM2 and tumor PD-L1 was performed.The OS and DFS in the centralM2^lessPD-L1^-group were the best while the centralM2^morePD-L1⁺group had the worst OS and DFS?P=0.002,0.034,respectively?.The combined analysis of macrophages and tumor PD-L1 could improve the accuracy of prognosis prediction.13.M2 macrophages were the important immune cells expressing PD-L1 in tumor microenvironment and are important sources of PD-L1 in tumor-negative PD-L1 negative patients:88%of patients were found to express PD-L1 in macrophages,especially mostly in M2 macrophages,regardless of the PD-L1 expression in tumor cells.In patients with tumor cells PD-L1 negative,about the65%of PD-L1 positive cells in stroma cells are macrophages.Besides,it was found that PD-L1 in macrophages was more expressed in M2 macrophages.14.Overall macrophage PD-L1 expression did not affect the prognosis of NSCLC patients with tumor PD-L1negative:No difference of OS was found between MPD-L1^high and MPD-L1^low,M1PD-L1^high and M2PD-L1^low,M2PD-L1^high and M2PD-L1^low groups.15.High expression of PD-L1 in centralM2 leads to poor prognosis of NSCLC patients with tumor PD-L1 negative:The expression of centralM PD-L1 and centralM1 PD-L1 had no significant effect on the prognosis of NSCLC patients with tumor PD-L1 negative,while the high expression of centralM2 PD-L1 led to poor prognosis?OS:P=0.000;DFS:P=0.032?.16.High expression of PD-L1 in marginM1 leads to good prognosis of NSCLC patients with tumor PD-L1 negative:The expression of PD-L1 in marginM and marginM2 had no significant effect on the prognosis of NSCLC patients with tumor PD-L1 negative,while the high expression of PD-L1 in marginM1 resulted in a good prognosis?OS:P=0.000;DFS:P=0.032?.17.CentralM2 PD-L1 and marginM1 PD-L1are independent prognostic factors for NSCLC with tumor PD-L1 negative:Results from univariable and multivariable analyses revealed that the PD-L1 expression on centralM2?OS:HR=10.332,95%CI?2.863-37.291?,P=0.000;DFS:HR=2.607,95%CI?1.337-5.085?,P=0.005?and marginM1?OS:HR=0.190,95%CI?0.068-0.534?,P=0.002;DFS:HR=0.413,95%CI?0.213-0.801?,P=0.009?were independent predictors of OS and DFS for NSCLC with tumor PD-L1 negative.18.PD-L1 on macrophages inhibits T cells activation by binding to PD-1 on T cells,which leads to immunosuppression.A case of lung adenocarcinoma was randomly selected and stained with CD8 and PD-1.It was observed that PD-L1 on the macrophages binds to PD-1 on the CD8+T cell.PMA was used to induce human monocyte line THP-1 to differentiate into macrophage and knockdown PD-L1 of macrophages.At the same time,human peripheral blood lymphocytes were collected.Macrophages were co-cultured with activated T cells and then we found that macrophages expressing PD-L1 inhibited the activation of CD8+T cells,while had no significant effect on CD4+T cells.This further proves that PD-L1 on macrophage can induce immunosuppression by inhibiting the function CD8⁺T cells.19.The expression of PD-L1 in macrophage promotes its transformation to immunosuppressive phenotype.It was found that the inflammatory factors released by macrophages,including IL-1,IL-6,TNF-alpha,Arg-1,increased after silencing of PD-L1 expression in macrophages,which proved that the expression of macrophage PD-L1 inhibited the release of inflammatory factors and transformed macrophages into immunosuppressive phenotype.Conclusion:1.The macrophage infiltration in the microenvironment of NSCLC is abundant,and the number and distribution of macrophages are closely related to the occurrence and development of non-small cell lung cancer;2.The number of macrophages was correlated with the pathological types,EGFR status and smoking status of lung cancer;3.High infiltration of centralM2 is an independent prognostic factor for poor prognosis in NSCLC patients;4.PD-L1 expression is positively associated with macrophages in NSCLC;5.M2 macrophages were the important immune cells expressing PD-L1 in tumor microenvironment,and are important sources of PD-L1 in tumor-negative PD-L1 negative patients;6.CentralM2 PD-L1 is an independent prognostic factor for poor prognosis in NSCLC with tumor PD-L1 negative;7.MarginM1 PD-L1 is an independent prognostic factor for good prognosis in NSCLC with tumor PD-L1 negative;8.PD-L1 in macrophages induces immunosuppression by promoting autophenotypic transformation and inhibiting function of CD8+T cells.