| Background and purposeLung cancer is the most common cause of cancer death in the world,which is divided into small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC)according to clinicopathological classification,and approximately 85%of lung cancers are NSCLC.Clinically,only a small percentage of lung cancer patients can be diagnosed in the early stage(stage I or II)and treated by surgical resection,but more than 60%of lung cancer patients have local or distant metastasis(stage III or IV)at the time of diagnosis,so they can only be treated with radiotherapy and chemotherapy or neoadjuvant radiotherapy and chemotherapy combined with surgery,however,the five-year survival rate is still low.Over the past 20 years,with the emergence of various targeted therapies and immunotherapies,the overall cure and survival rates of lung cancer have improved,but still rebound in the late stage of treatment.Therefore,it is necessary to study new drugs and combined immunotherapy to resolve this problem and improve the survival rate of patients.The tumor microenvironment is the place where tumor cells or cancer stem cells live,which is composed of various cells,including tumor cells,immune cells and mesenchymal cells.Myeloid-Derived Suppressor Cells(MDSCs),one of the major immune cells in the tumor microenvironment,are a heterogeneous group of immature bone marrow cells,regulating immune responses.During tumorigenesis,MDSCs will be abnormally produced and recruited into the tumor microenvironment to establish an immunosuppressive microenvironment and promote tumor escape.In addition,MDSCs also promote tumor angiogenesis and metastasis.Meanwhile,in patients with lung cancer,MDSCs are closely related to tumor progression,treatment and prognosis,so it is crucial to find therapeutic targets for MDSCs clearance.G protein coupled receptors(GPCRs)are membrane proteins with seven transmembrane domains and the largest receptor superfamily in human.GPCRs transduces intracellular signals through heterotrimeric G protein or inhibin,causing a variety of physiological and pathophysiological reactions,which is an important target for drug development.G protein-coupled receptor 84(GPR84),a type of orphan G-protein-coupled receptor,is mainly expressed in a variety of immune cells,including monocytes,macrophages and neutrophils.There is no or low expression of GPR84 under normal physiological conditions,but its expression is significantly up-regulated under inflammatory conditions,which indicates that GPR84 has the potential to be used as a therapeutic target in inflammatory environment.However,there are no studies on the role of GPR84 in tumor progression,its expression in tumor microenvironment and the effect of GPR84 expression on tumor microenvironment.The purpose of this study is to screen the key GPCR regulating the function of MDSCs and its effect on the prognosis of lung cancer,and then to explore the combined therapy of targeting MDSCs,and finally to explore the molecular mechanism of GPR84 to enhance the immunosuppressive function of MDSCs,which provides a new idea and direction for clinical treatment of patients with lung cancer.Part Ⅰ.Screening of GPR84 and its regulatory effect on the prognosis of lung cancerMethod1.We analysis of differential genes of G protein-coupled receptor-related genes on MDSCs using three GEO datasets.2.Detecting the expression of GPR84 on different immune cells in peripheral blood of lung cancer patients by flow cytometry.3.Construction of GPR84 knockout mouse models by CRISPR/Cas9 technology.4.We detect the effect of GPR84 knockout on the immune system of mice by flow cytometry.5.Administration of LLC subcutaneously to C57BL/6N mice and GPR84-/-mice to compare the number and functional alterations of T cells and MDSCs and tumor progression.6.We analysis of GPR84 expression in different tumors by TCGA database.7.We analyze the effect of GPR84 expression in lung cancer on the survival prognosis of patients by TCGA database and clinical samples.Results1.We found that GPR84 was the only gene with common high expression.2.Flow cytometry results showed that the expression of GPR84 on MDSCs in peripheral blood of lung cancer patients was higher than other immune cells,such as T cells,B cells and NK cells3.GPR84-/-mice were successfully constructed by genotypic identification.4.No significant difference in the number and function of major immune cells in GPR84-/-mice compared to C57BL/6N mice.5.Compared with C57BL/6N mice,GRP84-/-mice had significantly higher proportions of CD3+CD8+ T cells in spleen peripheral blood and tumor tissues,and the number of MDSCs was not significantly altered,but the functional molecules of MDSCs,Arginasel(ARG1)and induction of nitric oxide synthase(iNOS)expression were significantly lower.Tumor volume was significantly lower in GRP84-/-mice compared to C57BL/6N mice.6.The expression levels of GPR84 were higher in tumors than in normal tissues in different tumors.7.In lung cancer patients,the survival of patients with high expression of GPR84 was significantly reduced.Summary1.Knockout of GPR84 promotes the number of CD8+T cells in LLC-bearing mice,weakens the immunosuppressive function of MDSCs,inhibits the formation of immunosuppressive microenvironment,and delays the occurrence of tumor.2.In patients with lung cancer,the high expression of GPR84 on MDSCs reduced the survival time of patients and promoted the progression of tumor.3.GPR84 can be used as a key index to evaluate the function of MDSCs and the prognosis of patients with lung cancer.Part Ⅱ:GPR84 promotes the function of MDSCs by regulating PD-L1 expressionMethods1.To analyze MDSCs surface molecules and immunosuppressive molecule-related differential genes,we sequenced mRNA from MDSCs cells in C57BL/6N mice(Wild Type,WT)and GRP84-/-mice(Knockout,KO)with LLC-bearing mice.2.Analysis of the correlation between GPR84 and PD-L1 in lung adenocarcinoma and lung squamous carcinoma by TIMER database.3.The expression of GPR84 and PD-L1 on MDSCs was detected by flow cytometry.4.The expression of GPR84 and PD-L1 on MDSCs in peripheral blood of cancer patients was detected and their correlation was analyzed.5.The expression of PD-L1 on MDSCs was detected by flow cytometry,imaging flow cytometry and immunofluorescence,and the function of MDSCs was detected by RT-PCR and Western Blot.6.The expression of PD-L1 on MDSCs in tumor,spleen and peripheral blood of LLC tumor-bearing WT and KO mice was detected by flow cytometry and Western Blot.7.MDSCs and CD8+T cells were co-incubated in vitro,and anti-PD-L1 antibody was given to detect the effect of MDSCs on CD8+T cell proliferation and function by flow cytometry.8.We used PDB(Protein data bank,https://www.Rcsb.Org)and MOE(Molecular Operating Environment)platform to simulate the 3D protein structure model of GPR84,determined the binding sites of small molecules by SiteFinder program,and used Chemdiv database for virtual screening of small molecule inhibitors.9.The effect of small molecular inhibitors on MDSCs functional molecules was detected by RT-PCR and ELISA.10.The small molecule inhibitors were added to the MDSCs induction system,and the regulation of small molecule inhibition on MDSCs induction and Ca2+influx was detected by flow cytometry.11.GPR84+MDSCs,GPR84-MDSCs and T cells were treated with small molecule inhibitor.The apoptosis of MDSCs and T cells were analyzed.12.GPR84 antaginst8 and small molecule inhibitor were added to MDSCs,and the effect on the expression of PD-L1 was analyzed by RT-PCR,flow cytometry and Western Blot.13.The effect of small molecule inhibitor combined with anti-PD-L1 antibody on the immunosuppressive function of MDSCs was observed by flow cytometry.Results:1.The difference analysis of sequencing results showed that the expression of CD274(PD-L1)in KO group was significantly lower than that in WT group.2.The TIMER database results showed that there was a positive correlation between the expression of GPR84 and PD-L1 in lung cancer.3.Dimensionality reduction analysis showed that there was a significant positive correlation between the expression of GPR84 and PD-L1 in CD11b+cell population,and the expression of GPR84 and PD-L1 was significantly higher in MDSCs.4.There was a significant positive correlation between the expression of GPR84 and PD-L1 in peripheral blood of tumor patients.5.Compared with WT group,the expression of MDSCs functional molecules and PD-L1 decreased in KO group at gene level and protein level.6.Compared with WT group,the expression of PD-L1 on MDSCs in tumor tissue,peripheral blood and spleen in KO group decreased significantly.7.GPR84+MDSCs had stronger immunosuppressive function than GPR84MDSCs and significantly inhibited the proliferation and function of CD8+T cells,but when anti-PD-L1 antibody was added,the immunosuppressive function of GPR84+MDSCs was significantly inhibited.8.The protein 2RH1(humanB2-adrenergicGprotein-coupledreceptor),which is similar to the primary structure of GPR84,was identified as the template for the construction of the target protein by PDB.Through the screening results,9 small molecular inhibitors with good binding effect to GPR84 were identified.9.From the gene level and protein level,it was found that small molecular inhibitor(ZINC09680069)could significantly reduce the expression of MDSCs functional molecules(TGF-β and IL-10)and inhibit the immunosuppressive ability of MDSCs.10.The flow results show that ZINC09680069 can significantly inhibit the formation of MDSCs and reduce the influx of Ca2+.11.ZINC09680069 can specifically promote the apoptosis of GPR84+MDSCs,but it has no significant effect on the production,apoptosis and function of T cells.12.Through the detection of MDSCs functional molecules at the gene level,we found that ZINC09680069 could better inhibit the function of MDSCs than GPR84 antigonist8.13.GPR84 small molecule inhibitor and anti-PD-L1 antibodies synergistically inhibit the immunosuppressive function of MDSCs.Summary1.There was a significant positive correlation between the expression of PD-L1 and GPR84 in MDSCs.2.GPR84 promotes the expression of PD-L1 in MDSCs,and GPR84 maintains the immunosuppressive function of MDSCs depends on PD-1/PD-L1.3.Targeted GPR84+MDSCs small molecule inhibitor and anti-PD-L1 antibodies synergistically inhibit the function of MDSCs.Part Ⅲ:GPR84 inhibits lysosome-dependent degradation of PD-L1 by GSK3β phosphorylation.Methods1.Purified MDSCs from the spleen of LLC-bearing WT and KO mice was sequenced by mRNA and analyzed by GSEA(Gene Set Enrichment Analysis).2.The activity levels of subcellular organelles(endoplasmic reticulum,Golgi apparatus and lysosome)in WT and KO groups were compared by flow cytometry.3.Detection of lysosomal associated membrane protein and PD-L1 expression in WT and KO by imaging flow cytometry.4.Lysosome inhibitors were added to MDSCs in vitro,and the expression level of PD-L1 was detected by imaging flow cytometry and Western Blot.5.Analyze the correlation between GPR84 and GSK3β through TCGA database;6.Detect the expression of GSK3β and PD-L1 in WT and KO groups by Western Blot,GSK3β inhibitor was added to GPR84+MDSCs,and the expression of p-GSK3β and PD-L1 was detected.7.Purified MDSCs from spleen of LLC-bearing WT and KO mice was sequenced by mRNA and analyzed by KEGG enrichment analysis.8.Detect the expression of PI3K/Akt in WT and KO by Western Blot,Akt inhibitor was added to GPR84+MDSCs,and the expression of p-GSK3β was detected.Results 1.The GSEA showed that lysosomal related genes were significantly enriched in KO group.2.The results showed that the activity of lysosome in KO group was significantly higher than that in WT group.3.The results showed that there was a significant negative correlation between lysosomal proteins(LAMP 1,Rab7)and the expression of PD-L1.4.With the addition of lysosome specific inhibitor,the expression of PD-L1 was significantly higher than that in the treatment group.5.There was a positive correlation between the expression of GPR84 and GSK3β.6.The expression of GSK3β,p-GSK3β and PD-L1 decreased in KO group,and the phosphorylation level of GSK3β and the expression of PD-L1 decreased after the addition of GSK3β inhibitor.7.The results showed that PI3K-Akt signaling pathway was significantly enriched in WT group.8.The expression of p-PI3K,p-Akt and GSK3β decreased in KO group,and the phosphorylation level of GSK3β decreased after GPR84+MDSCs was added with Akt inhibitor.Summary1.GPR84 inhibits the activity of GSK3β by activating PI3K-Akt phosphorylation;2.GPR84 inhibits PD-L1 degradation by GSK3β phosphorylation;3.GPR84 promotes the expression of PD-L1 depending on the decrease of PD-L1 degradation in lysosomes.Conclusion(1)The high expression of GPR84 in MDSCs is closely related to the survival and prognosis of lung cancer;(2)The high expression of PD-L1 in GPR84+MDSCs enhances the immunosuppressive function of MDSCs and promotes the progression of tumor;(3)GPR84 inhibits the activity of GSK3β through PI3K-Akt phosphorylation,which in turn inhibits the lysosome-dependent degradation of PD-L1,promote the expression of PD-L1. |
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