The Mechanism Of COX2 Mediated GPR84~+MDSCs Immunosuppressive Function To Promote Tumor Progression

Background and objective:Malignant tumors are major diseases that seriously threaten human health with the incidence and mortality increasing globally,especailly in China.Traditiaonal strategies,including surgery,chemotherapy and radiotherapy,still exhibited limited efficacy in clinical practice.Recently,the important role of immune system in tumorigenesis and progression has been intensively researched,immune checkpoint blockade(ICB)therapy and genetically engineered T cells therapy such as chimeric antigen receptor T cells(CAR-T)and T cell receptor engineered T cells(TCR-T)have been developed.Despite the fact that CAR-T cells achieved great success in the treatment of leukemia,the clinical response of immunotherapy in solid tumors is extremely limited.The large number of immunosuppressive cells and immunosuppressive factors in the microenvironment of solid tumors have been regarded as the major contributors to the failure of immunotherapy,and this phenomenon has been convinced in a various of solid tumors,including lung cancer,liver cancer,colorectal cancer and breast cancer.Therefore,revealing the factors that modulate the function of immunosuppressive cells is desperately needed to improve the efficacy of immunotherapy.Myeloid-derived suppressive cells(MDSCs)are a group of important immune cells in the tumor immunosuppressive microenvironment.On the one hand,MDSCs could directly promot tumor cell proliferation,invasion and metastasis.On the other hand,MDSCs could suppress the function of effector T cells in the microenvironment.Increasing evidences have shown that MDSCs accumulate in large numbers in solid tumors and lead to poor prognosis,including breast cancer,esophageal cancer,lung cancer and gastric cancer.Therefore,targeting and regulating the accumulation and immunosuppressive function of MDSCs at tumor sites and exploring the specific molecular mechanisms are crucial to reverse the immunosuppressive state of the tumor microenvironment and improve the efficacy of immunotherapy.G protein-coupled receptors(GPCRs)are seven transmembrane structural domain receptors playing important roles in various physiological processes including metabolic regulation and organism development.This makes GPCRs as a promising target.Currently,40%of small molecule drugs are agonists or antagonists of GPCRs on the worldwide drug market.GPR84 is a member of the GPCRs superfamily and is expressed primarily in bone marrow cells,peripheral leukocytes and lung.The expression of GPR84 is limited in normal condition,however,in the context of inflammation,GPR84 is highly expressed in monocytes and adipocytes.Therefore,GPR84 plays an important role in neurological diseases,leukemia,ocular diseases and inflammatory bowel disease.Recently,GPR84 inhibitors for the treatment of inflammatory bowel disease are in phase Ⅱ clinical studies and show great potential as therapeutic targets.However,the role of GPR84 in solid tumors is still unclear.Our previous study found that GPR84 was specifically highly expressed on MDSCs in the tumor microenvironment,and GPR84+MDSCs possessed stronger immunosuppressive function,suggesting that GPR84 may be a specific target to regulate the immunosuppressive function of MDSCs.However,the underlying molecular mechanisms of GPR84 highly expressed on MDSCs and how GPR84 regulate the immunosuppressive function of MDSCs are still unclear.Therefore,utilizing esophageal cancer patient samples,GPR84 knockout mice model,primary esophageal cancer mice model and B16 cells melanoma mice model,we continue to explore the mechanisms that affect GPR84 expression on MDSCs and how GPR84 regulate the immunosuppressive function of MDSCs.Firstly,we aim to reveal the regulatory effect of GPR84 on the immunosuppressive function of MDSCs.Secondly,we aim to explore the key factors and mechanisms that promote the high expression of GPR84 on MDSCs in the tumor microenvironment.Finally,we aim to analyze the specific molecular mechanism of GPR84 regulating the accumulation and immunosuppressive function of MDSCs in the tumor microenvironment of esophageal cancer.Our study will expand the tumor immunomodulatory function of GPCRs represented by GPR84,reveal the intrinsic relationship between GPR84 and tumor immune microenvironment through regulating MDSCs,which may provide theoretical foundations for the research of immunotherapy targets and the development of drugs targeting MDSCs.Part 1 GPR84 maintains the immunosuppressive function of MDSCsObjectiveTo investigate the role of GPR84 in the maintenance and regulation of immunosuppressive function of MDSCs.Methods1)Constructing GPR84 knockout mice by CRISPR/Cas9 technology,inducing in situ esophageal cancer model in mice by 4-NQO,and constructing melanoma model by subcutaneous injection of B16 cell line to detect the effect of GPR84 on tumor progression in mice.2)Observation of tumor growth in Wild type and GPR84-/-B16 melanoma mice by small animal live imaging techniques.3)Detection of the ratio of MDSCs and CD3,CD4,CD8 T cells and functionally relevant molecules(IFN-y,Ki67,CD69,CD28,CD 107a)in the spleen,peripheral blood and tumors of Wild type and GPR84-/-hooded mice by flow cytometry and immunofluorescence techniques.4)Sorting of MDSCs and CD8+T cells in mouse spleen by magnetic bead sorting.5)Detection of the effect of spleen-derived MDSCs from Wild type and GPR84-/tumor-bearing mice on IFN-γ secretion capacity of CD8+ T cells by ELISA technique.6)Detection of differences in functionally relevant molecules on splenic-derived MDSCs from Wild type and GPR84-/-hormonal mice by RT-PCR and Western Blot techniques.7)To detect the ability of MDSCs to proliferate and specifically kill B 16-OVA tumor cells by CFSE staining technique on CD8+T cells.Results1)Among in situ esophageal cancer mouse model induced by 4-NQO and B16 melanoma mouse model,GPR84-/-mice showed slower tumor progression and longer survival time compared to wild type mice.2)A decreased proportion of MDSCs and an increased proportion of T cells in spleen,peripheral blood and tumor tissue in GPR84-/-tumor-bearing mice and GPR84 inhibitor-treated mice compared with control tumor-bearing mice.3)Compared with control tumor-bearing mice,the expression of functionally relevant molecules of MDSCs of spleen origin was decreased in GPR84-/-tumor-bearing mice and GPR84 inhibitor-treated mice,and the inhibition of CD8+T cell proliferation capacity,IFN-γ secretion capacity and specific killing capacity by MDSCs was restored.Part 2 Investigation of factors and mechanisms regulating GPR84 expression on MDSCsObjectiveTo investigate the key factors and related molecular mechanisms regulating GPR84 expression on MDSCs in the tumor microenvironment.Methods1)Obtaining wild-type C57 mouse bone marrow cells and inducing them to become MDSCs using G-CSF and GM-CSF.2)To detect the effect of G-CSF and GM-CSF on GPR84 expression on MDSCs by RT-PCR,flow cytometry and Western blot techniques.3)Probing the downstream signaling pathways activated by G-CSF and GM-CSF by Western blot technique.4)To detect transcription factor C/EBPβ transcriptional regulation of GPR84 expression by dual luciferase reporter assay.5)Verification of C/EBPβ binding to the GPR84 promoter region by ChIP assay.6)Detection of the effect of G-CSF and GM-CSF on C/EBPβ activation into the nucleus by immunofluorescence and Western blot techniques.7)Analyzing the correlation of GPR84/STAT3/C/EBPβ in tumor tissues of esophageal cancer patients and melanoma patients in TCGA database by GEPIA platform(http://gepia.cancer-pku.cn/index.html).Results1)G-CSF and GM-CSF can promote GPR84 expression while inducing the activation of MDSCs.2)G-CSF and GM-CSF can activate STAT3 and AKT signaling pathways to regulate GPR84 expression.3)G-CSF and GM-CSF can induce the activation of transcription factor C/EBPβ into the nucleus by activating STAT3.4)C/EBPβ can bind to the GPR84 promoter region and then transcriptionally regulate the expression of GPR84 after nuclear entry.5)In both esophageal cancer patients and melanoma patients,C/EBPβ showed a significant positive correlation with STAT3 and GPR84 expression in tumor tissues.Part 3 Study on mechanisms of GPR84 regulating the immunosuppressive function of MDSCsObjectiveTo demonstrate the key mechanisms downstream regulating the immunosuppressive function of MDSCs by GPR84.Methods1)Magnetic bead sorting method to sort MDSCs in the spleen of wild type and GPR84-/-tumor-bearing mice for RNA sequencing analysis.2)Magnetic bead sorting method to sort spleens of hormonal mice by GPR84+MDSCs,GPR84-MDSCs,and CD8+ T cells.3)RT-PCR,flow cytometry,and Western blot techniques to detect the effect of GPR84 activation on MDSCs on COX2 expression.4)CFSE,flow cytometry and ELISA to detect whether COX2 inhibitors mediate the effect of GPR84+ MDSCs on T cell proliferation and IFN-y secretion capacity.5)Probing the signaling pathways regulated downstream of GPR84 by Western blot.6)To detect the effect of GPR84 activation on transcription factor c-Fos entry into the nucleus by imaging flow and Western blot.7)Detection of transcription factor c-Fos transcriptional regulation of COX2 expression by dual luciferase reporter assay.8)Verification of the binding of c-Fos to the COX2 promoter region by ChIP assay.9)To analyze the correlation between GPR84/COX2 expression in tumor tissues of esophageal cancer patients and melanoma patients in the TCGA database through the GEPIA platform.10)To analyze the correlation between COX2 expression and the ratio of MDSCs and CD8+T cells in tumor tissues of esophageal cancer patients and melanoma patients by TIMER2.0 platform(http://timer.cistrome.org).Results1)COX2 expression was significantly downregulated in splenic-derived MDSCs from GPR84-/-tumor-bearing mice compared with wild type mice.2)Activation of GPR84 on MDSCs was able to promote COX2 expression.3)Activation of GPR84 on MDSCs was able to regulate COX2 expression through activation of the downstream ERK1/2 signaling pathway.4)GPR84 activation can induce transcription factor c-Fos into the nucleus.5)c-Fos can bind to the GPR84 promoter region and then transcriptionally regulate the expression of COX2 after entering the nucleus.6)GPR84 showed a significant positive correlation with COX2 expression in tumor tissues of both esophageal cancer patients and melanoma patients.7)COX2 expression was positively correlated with the proportion of infiltrated MDSCs and negatively correlated with the proportion of infiltrated CD8+ T cells.