The Role Of CD39 In The Formation Of Immunosuppressive Microenvironment In Gastric Cancer

【Background】Gastric cancer is one of the most frequent malignancies in China,which seriously threatens the life and health of the people.According to the latest epidemiological statistics,there are 358,700 new cases and 260,400 deaths of gastric cancer in China in2022,accounting for about 40%of the total number all over the world.Traditional treatments for gastric cancer mainly include surgery,chemotherapy,and anti-HER-2targeted therapy,but these treatments show low specificity and short disease control time,resulting in poor prognosis of patients.In recent years,immunotherapy targeting PD-1/PD-L1 pathway has shown great developments in gastric cancer.However,the real-word data indicates that only less than 20%of patients are sensitive to immunotherapy,and most patients sensitive to immunotherapy also gradually develop drug resistance in the later stage.At present,most studies have shown that tumor microenvironment(TME)plays an important role in immunotherapy resistance.Tumor cells can inhibit the activity of immune cells by utilizing a variety of strategies,including immunosuppressive cytokine production,nutrient competition,and inhibitory ligand expression,thus generating immune tolerance and escape.As a solid tumor of digestive tract,TME can gradually evolve into a state of continuous immune tolerance under long-term chronic inflammatory stimulation,which leads to the tumor progression and treatment resistance.Therefore,elucidating the specific mechanism of immunosuppressive TME in gastric cancer has important theoretical and clinical translational values.Regulatory immune cell populations,including FOXP3~+CD4~+regulatory T cells,myeloid-derived suppressor cells and tolerant dendritic cells,contribute to the formation of immunosuppressive TME.Notably,these cells showed high expression of extracellular nucleotidases CD39 and CD73.Both hydrolyze extracellular ATP in turn to produce adenosine,which prevents anti-tumor responses by binding to A2A receptors on lymphocytes.Therefore,the ATP-adenosine metabolic pathway regulated by CD39/CD73is involved in the formation of immunosuppressive TME.As a key rate-limiting enzyme in the ATP-adenosine metabolic pathway,CD39 is highly expressed in a variety of human tumors,including lung,breast,and ovarian cancers,and suggests poor prognosis in patients.In gastric cancer,studies have shown high CD39expression in gastric cancer tissues.However,the role of CD39 in the formation of immunosuppressive TME in gastric has not been fully explored.【Objectives】1.To clarify the expression pattern of CD39 in gastric cancer tissues and evaluate its association with patients’prognosis.2.To elucidate the correlation between the expression level of CD39 in gastric cancer and the immune characteristics and clinical therapeutic effect of patients.3.To reveal the effect of CD39 on the infiltration and function of CD8~+T cells in TME of gastric cancer.4.To explore the specific molecular mechanism of CD39-mediated CD8~+T cells exhaustion in gastric cancer.5.To explore the predictive value of CD39-regulated immune activation pathway for the efficacy of immunotherapy in gastrointestinal cancer.【Methods】1.Gene expression profiles and clinical data of multiple cohorts of gastric cancer in TCGA and GEO databases were collected to analyze the differences in m RNA expression levels of CD39 in gastric cancer and adjacent tissues,and to explore the correlation between CD39 expression levels and tumor stage and overall survival.Immunohistochemical and RNA sequencing were performed to verify the association between CD39 expression levels and clinicopathological features and overall survival of patients with gastric cancer.2.The expression levels of CD39 in patients with different subtypes of gastric cancer were compared in the TCGA and ACRG molecular typing systems.Based on the sequencing and clinical data of PRJEB25780 and Xijing cohort,ROC curve analysis and Kaplan-Meier survival analysis were conducted to explore the association of CD39expression level with the efficacy of immunotherapy,postoperative adjuvant chemotherapy,and targeted therapy,respectively.3.Single-cell transcription sequencing data of one gastric cancer cohort was included to analyze the expression of CD39 in different components of TME,and to clarify the relationship between the expression level of CD39 and the infiltration number and function of CD8~+T cells.The roles of upregulated expression of CD39 in mediating CD8~+T cells exhaustion and tumor growth in syngeneic gastric cancer models were examined by flow cytometry.4.The inhibitory effect of anti-CD39 antibody on tumor growth in syngeneic gastric cancer models was investigated by drawing the tumor volume curve,and the effect of anti-CD39 antibody on the infiltration number of CD8~+T,NK and DC cells and their anti-tumor effects were evaluated by flow cytometry.The correlation between the reverse effect of anti-CD39 antibody on CD8~+T cells exhaustion and the activation of c GAS-STING signaling mediated by DNA damage was investigated by immunofluorescence and immunohistochemical staining.5.Based on the above CD39-regulated immune network,a multi-gene signature was constructed to characterize the immune activation response to DNA damage.The sequencing and clinical data of six immunotherapy cohorts,including gastrointestinal cancer,were collected.ROC curve analysis and Kaplan-Meier survival analysis were used to evaluate the predictive role of the newly established signature on the objective response rate and survival prognosis of immunotherapy,and the predictive efficacy of the signature was compared with that of commonly used clinical biomarkers for immunotherapy.【Results】1.The expression level of CD39 in gastric cancer tissues was significantly higher than that in adjacent tissues,and CD39 was mainly expressed in tumor stroma region.The expression level of CD39 was significantly positively correlated with the depth of tumor invasion and the number of lymph node metastasis.Univariate and multivariate Cox regression analyses showed that high expression of CD39 was one of the independent adverse factors for the prognosis of gastric cancer.2.Gastric cancer with high CD39 expression is characterized by low tumor mutation burden,microsatellite stability status,and active angiogenesis and fibrotic pathways.These patients have more significant survival improvement after receiving postoperative adjuvant chemotherapy,and may benefit from anti-HER-2,anti-VEGFR-2,and anti-FGFR2 targeted therapies.Patients with low CD39 expression had a higher objective response rate to anti-PD-1 therapy.3.There is a high level of CD39 expression on infiltrating lymphocytes in gastric cancer,and the expression distribution of CD39 is highly similar to that of immunosuppressive receptors such as PD-1 and CTLA-4,suggesting that CD39 defines an exhaustion phenotype of lymphocytes.In the syngeneic gastric cancer models,with the gradual increase of tumor burden,the expression level of CD39 on CD45~+lymphocytes(mainly CD8~+T cells,but not NK cells)gradually increased,and the expression of PD-1and LAG3 on CD39~+CD8~+T cells were also gradually upregulated.4.Anti-CD39 antibody can significantly inhibit tumor growth in the syngeneic gastric cancer models.Infiltrating CD8~+T and NK cells and their secretion of cytotoxic factors(including IFN-γ,TNF-α,and Granzyme B)increase after anti-CD39 therapy.The infiltration of CD103~+DC cells,which exerted the synergistic effect of T cells to initiate transport,also increased.Mechanism exploration found that after anti-CD39 therapy,the content ofγ-H2AX(a marker of DNA damage)in tissues increased,and the activity of c GAS-STING signaling pathway was enhanced(manifested by increased expression levels of c GAS,STING and IFNB1).5.The DNA damage response-related immune activation(DRIA)signature(including CXCL10,IDO1 and IFI44L)was constructed based on CD39-regulated DNA damage pathways.In the gastric cancer cohort,the predictive efficacy of anti-PD-1therapy(AUC=0.838)was better than that of PD-L1,microsatellite instability and tumor mutation burden,and the objective response rate of DRIA-high group was significantly higher than that of DRIA-low group.Multivariate Cox regression analysis of the remaining five pan-cancer cohorts found that a higher DRIA score was an independent positive predictor for the prognosis of patients treated with immunotherapy.【Conclusions】1.This study confirmed that high expression of CD39 in gastric cancer tissues was significantly correlated with tumor progression and poor prognosis,indicating that the expression level of CD39 could be used as a biomarker to judge the prognosis of gastric cancer.2.For gastric cancer patients with high expression of CD39,postoperative adjuvant chemotherapy and anti-HER-2,anti-VEGFR-2 and anti-FGFR2 targeted therapy are the preferred treatment options.For gastric cancer patients with low expression of CD39,immunotherapy is the preferred treatment plan,and postoperative adjuvant chemotherapy can be an alternative treatment plan.3.The up-regulation of CD39 expression in gastric cancer can lead to the formation of immunosuppressive TME mediated by CD8~+T cells exhaustion,while anti-CD39therapy can reverse CD8~+T cells exhaustion by inducing DNA damage and mediating the activation of c GAS-STING pathway,thus playing an anti-tumor role.4.The DRIA signature based on immune activation mediated by DNA damage can stably and efficiently predict the response rate and survival prognosis of patients with gastrointestinal cancer treated with immunotherapy,indicating that DRIA can be used as a new biomarker to guide clinical immunotherapy decision-making for gastrointestinal cancer.