Study On The Mechanisms Of Targeting GPR84+MDSCs To Improve The Efficiency Of Tumor Immunotherapy

Background and objective:The combination of geographical environment and genetic factors leads to the activation of proto-oncogenes in the body,accompanied by the inactivation of tumor suppressor genes,and ultimately induces cancer.The results of the survey on the distribution of malignant tumor types in China show that esophageal cancer is the fourth and third cause of death in male and female patients,respectively.It’s epidemic features,risk factors and pathological types are greatly different from those in Western countries,and the lack of common mutations in esophageal cancer tissues prevents targeted therapy development.So it is urgent to develop other treatments to inhibit the growth of lung cancer and esophageal cancer.Based on the role of immune system in the occurrence and development of cancer,immunotherapy has made significant progress and breakthrough in the treatment of cancer.In recent years,both the approved checkpoint blockers(anti-PD-1 and anti-CTLA4)and chimeric antigen receptor T cell(CAR-T)adoptive-transferred therapy belong to enhance anti-tumor immune response in patients to inhibit tumor growth.However,the above immunotherapy approaches have great difference in the treatment of solid tumor such as esophageal cancer,gastric cancer and non-small cell lung cancer,which is closely related to the immune state of the local microenvironment in tumor site.The tumor microenvironment consists of tumor cells,various stromal cells,and extracellular matrix.Activation of effector T cells plays a key role in antitumor response,but tumor cells combined with myeloid-derived suppressive cells(MDSCs)or other immune regulatory cells form a strong defense system that interferes with the proliferation and function of effector T cells to attenuate the patient’s response to immunotherapy,thereby maintaining tumor growth.MDSCs are the main force in inhibiting the expansion and activation of effector T cells in tumor microenvironment.MDSCs are classified into M-MDSCs(monocytic MDSCs)and PMN-MDSCs(granulocytic MDSCs)according to the morphology and phenotype.In the non-small cell lung cancer,colorectal cancer and breast cancer tissues,PMN-MDSCs are mainly aggregated and promote the poor prognosis of cancer patients.Therefore,improving the tumor microenvironment by targeting MDSCs is an important research direction to enhance the prognosis of cancer patients and restore the efficiency of immunotherapy.With the identification of the mechanism regulating MDSCs aggregation and activation in tumor microenvironment,combined with PI3Kγinhibitor or all-trans retinoic acid to reduce the accumulation of MDSCs,partial restore the effect of anti-PD-1 or CAR-T therapy.However,these approaches of eliminating MDSCs lack specificity,as reducing the proportion of MDSCs followed by decreasing the proportion of effector T cells or increasing the proportion of other immunosuppressive cells such as tumor-associated macrophages(TAM).Moreover,it has no significance in improving the long-term effect.Due to the geographical distribution of esophageal cancer and the lack of research models,there is no reports about the role of MDSCs in the development of esophageal cancer.Therefore,the first part of this thesis will construct an orthotopic esophageal cancer model and analyze the correlation between the distribution of MDSCs in the tumor microenvironment and the progression of esophageal cancer.The second part combines multiple sequencing data to screen for specific molecules that regulate the aggregation and function of MDSCs and detect its expression patterns in the microenvironment of esophageal cancer and lung cancer;the third part is to deeply analyze the regulation and mechanism of the selected molecules on the aggregation and function of MDSCs,and to develop a combination therapy strategy to provide new ideas for improving the effect of immunotherapy.Part 1 Study on the effects of MDSCs on the prognosis of esophageal cancerFirstly,we used 4-NQO to construct an orthotopic esophageal cancer model.Flow cytometry was used to dynamically monitor the changes of immune cell subsets during the formation and development of esophageal cancer.The results showed that the proportion of effective immune cells such as CD8~+T cells gradually decreased.While,the infiltration of MDSCs increased gradually,and PMN-MDSCs were the main aggregation subsets.Then the peripheral blood and tumor tissues of patients with esophageal cancer were collected to detect MDSCs accumulation.The results demonstrated that the infiltration of PMN-MDSCs in tumor tissues was significantly higher than that in peripheral blood.And it is significantly associated with lymph node metastasis,depth of tumor invasion,tumor size and clinical stage.Combined tumor tissue investigation results and TCGA data show that the cumulative proportion of PMN-MDSCs is a key factor leading to poor prognosis.To determine whether MDSCs play an immunosuppressive role in esophageal cancer,we first investigated the CD8~+T cell infiltration by flow cytometry.And the results showed that CD8~+T cells in peripheral blood of patients with esophageal cancer was significantly lower than that of healthy volunteers,and tumor tissue infiltration was lower than that of peripheral blood.The correlation analysis showed that the proportion of MDSCs was negatively correlated with CD8~+T cells.The same results were obtained for detecting the spleen of esophageal cancer mice.In order to further verify the immunosuppressive function of MDSCs,magnetic bead sorting was used to purify MDSCs from peripheral blood of healthy volunteers and esophageal cancer patients.The results of real-time PCR and flow cytometry indicated that MDSCs derived from esophageal cancer patients had higher expression of immunosuppressive molecules such as NCF4,CYBB,NOS2,and ROS.Co-incubation of MDSCs from different sources and CD8~+T cells,it was found that MDSCs derived from esophageal cancer patients had stronger inhibitory effect on CD8~+T cell proliferation and function than healthy volunteers.In conclusion,the results of the first part indicate that MDSCs with strong immunosuppressive function promote the occurrence and progression of esophageal cancer,providing a basis for subsequent studies to improve the prognosis of patients with esophageal cancer by interfering with the aggregation and function of MDSCs.Part 2 Screening and preliminary validation of key molecules that promote the accumulation and function of MDSCsWe established a system for inducing the generation of MDSCs.The results of flow cytometry and real-time PCR showed that stimulating bone marrow cells with GM-CSF and G-CSF is the best way to induce MDSCs production,and the MDSCs induced by this method inhibit the killing efficiency of CD8~+T cells on tumor cells,thereby maintaining the survival and proliferation of tumor cells.To screen for the key molecules that maintain MDSCs phenotype and immunosuppressive function,we downloaded MDSCs sequencing data(three data sets)in GEO database and used the Omicsbean platform for secondary data analysis.Venn analysis showed that 10 genes had high expression in MDSCs with immunosuppressive functions.Sorting the expression levels of the 10 genes in MDSCs of esophageal cancer mice,it revealed that CD38 and GPR84 are the two highest expression genes.CD38 has been reported expressed in various cells such as T cells and multiple myeloma cells,which is not consistent with interference MDSCs as the target.However,published results show that GPR84,which is a medium-chain fatty acid receptor,is associated with the expression of inflammatory factors such as IL-6 and MIP2.And there is no report on the expression distribution of GPR84 in the tumor microenvironment.Firstly,TCGA data showed that GPR84 expression was higher in tumor issues compared with normal tissues,and it was negatively correlated with disease-free survival and overall survival.Immunohistochemical detection results demonstrated that GPR84 was expressed on stroma cells and patients with high expression of GPR84 had a worse prognosis.In order to clarify the distribution of GPR84 in the tumor microenvironment to determine whether it can be used as a specific target for interfering with the aggregation and function of MDSCs,firstly,the real-time PCR and flow cytometry results showed that in esophageal cancer mouse the expression of GPR84 in MDSCs was significantly higher than that of B cells,dendritic cells,macrophages,NK cells and T cells.Immunofluorescence detection of mouse with esophageal carcinoma showed that GPR84 expression co-localized with MDSCs.Moreover,flow cytometry,real-time PCR and western blot analysis showed that GPR84 was specifically expressed in MDSCs.The expression of GPR84 in tumor tissue was higher than that in peripheral blood,and esophageal cancer mice with late stage had a higher GPR84 expression than that in early stage.The subcutaneous tumor models and clinical samples with lung adenocarcinoma we used to analyze whether the characteristic of specific high expression in MDSCs is present in other types of tumors.The results of flow cytometry and immunofluorescence also indicated that GPR84 was expressed in MDSCs,which provide the basis for selecting GPR84 as the target to inhibit MDSCs aggregation and functional in clinical transformation.In order to investigate the mechanism underlying GPR84 expression in MDSCs,multi-factor screening was used and CXCL1 was identified as the key molecule for inducing GPR84 expression.The results of immunohistochemistry showed that CXCL1 expression was highly consistent with GPR84,and esophageal cancer patients with higher CXCL1 expression had a worse prognosis.In view of the characteristics of GPR84-specific expression,regulation of MDSCs amplification and function is an important basis for judging whether it can be used as a therapeutic target.This is the next important research content of this thesis.Part 3 Targeting GPR84 inhibits the production and function of MDSCs to restore the therapeutic effect of anti-PD-1To determine whether GPR84 regulates the expansion and function of MDSCs,we firstly analyzed the correlation between GPR84 expression and molecules associated with MDSCs phenotype or function using mRNA sequencing data of esophageal cancer and lung cancer on cBioPortal platform.And the results showed that GPR84 distribution was consistent with CD33,STAT1 and C/EBPB which represents the phenotype or amplification,and the expression level of GPR84 was also positively correlated with the expression of immunosuppressive functional molecules such as IL-10,NCF4 and CYBB.Sort GPR84~+and GPR84~-MDSCs by flow cytometry for mRNA sequencing,and GSEA analysis indicated that MDSCs amplification and activation-related genes were enriched in GPR84~+MDSCs.The results of real-time PCR,co-incubation assay and ELISA showed that GPR84~+MDSCs had stronger immunosuppressive properties than GPR84~-MDSCs.In order to facilitate the development of combination therapy,we selected antagonists that can be used in vivo and in vitro to inhibit the activity of GPR84 to further verify its effect on the expansion and function of MDSCs.Adding GPR84antagonists in MDSCs induction system could significantly reduce the proportion of MDSCs induced by GM-CSF combined with G-CSF.After treatment with antagonists,the results of real time PCR,western blot,immunofluorescence and ELISA showed that the expression of immunosuppressive molecules decreased,and the inhibitory effect on CD8~+T cell function was significantly attenuated.GPR84 antagonists were orally administered to mice with esophageal cancer.It was found that the antagonists delayed the progression of esophageal cancer,and the proportion of MDSCs in the spleen decreased.While,the proportion of CD8~+T cells was significantly restored.Magnetic beads sorting were used to purify MDSCs from different treatment groups.Further results indicated that the expression of ARG1 and iNOS in MDSCs derived from esophageal cancer mice after treatment was significantly decreased,and the regulatory effect on CD8~+T cells was inhibited.In order to clarify the mechanism by which GPR84 regulates the function of MDSCs,western blot was used to detect the changes of GPR84-related pathways,and then verified by small molecule inhibitors.The results showed that GPR84 activated NF-κB,mTOR or STAT3 pathways to regulate the expression of immunosuppressive functional molecules in MDSCs.In order to clarify whether GPR84 antagonists can increase the therapeutic effect of anti-PD-1,a lung cancer mouse cell line LLC with Luciferase and subcutaneous xenografts were successfully constructed.Treat LLC model with anti-PD-1,GPR84antagonists or combination regimens,and the animal imaging systems showed that tumor growth was significantly inhibited in the combination treatment group.Moreover,tumor growth curves showed that GPR84 antagonists significantly enhanced anti-PD1 effect on tumor growth.Therefore,based on the specificity of GPR84 expression and regulation on MDSCs,targeting GPR84 inhibits the aggregation and function of MDSCs to improve the therapeutic effect of anti-PD-1,which provids a new idea for the clinical transformation of the combination therapy.