| BackgroundGastric cancer is one of the most common malignant tumors of the digestive system.Although the incidence of gastric cancer has declined in global,the age of onset has become younger and the mortality has not declined significantly.The survival rate of patients with early gastric cancer after treatment is high,but early gastric cancer is difficult to be diagnosed because there are no obvious symptoms in patients with early gastric cancer.Advanced gastric cancer is under a low 5-year survival rate due to metastasis and recurrence.Traditional cancer treatment strategies include surgery,radiotherapy,and chemotherapy.Although these methods have made great progress in recent decades,the prognosis of patients with gastric cancer has not been significantly improved.In addition,the life quality of patients after surgical resection,radiation and chemotherapy will be greatly negatively affected.At present,cancer immunotherapy has become an effective method for clinical treatment of cancer.Immunotherapy is the treatment of cancer by using an immuno-tumour vaccine or anti-tumour antibody to trigger the body's own immune systemAt present,cancer immunotherapy is a research hotspot in the clinical treatment of cancer,and Tim-3 has received more and more attention as an important immune checkpoint.Tim-3 is a member of the Tim family,with an extracellular domain consisting of a membrane-distal N-terminal immunoglobulin(IgV)domain,followed by a membrane-proximal mucin domain containing potential sites for O-linked glycosylation.The stalk domain lying between the imucin and transmembrane domain has sites for N-linked sugars,which is followed by a transmembrane domain and a cytoplasmic tail.Tim-3 is primarily activated by its widely expressed ligand galectin-9.Many studies have shown that the expression or activation of Tim-3 in a variety of tumor tissues is related to the development of tumors,and is an immuno-negative regulatory molecule.Active Tim-3 signaling can lead to depletion of interferon-producing T lymphocytes,which in turn lead to Thl inhibition and immune tolerance.Tim-3 can be co-expressed with PD-1 in tumor-infiltrating immune cells,synergistically mediating the depletion and dysfunction of T cells.However,the expression pattern of Tim-3 in gastric cancer and the influence of Tim-3 on immune infiltration and prognosis of patients have not been fully understood.In addition,the effect of Tim-3,the important immune checkpoint involved in the regulation of T lymphocyte immune response,on the biological function of T lymphocytes in the immune microenvironment of gastric cancer remains unclear.Objectives and significanceThe purpose of this study was to elucidate the expression of Tim-3 in T lymphocytes and the regulation of Tim-3 in the progression of gastric cancer in gastric cancer,and to explore the effect of Tim-3 expression on the biological function of gastric cancer infiltrating T lymphocytes.This study can improve the theoretical research of gastric cancer immunotherapy,and it is expected to provide a theoretical basis for innovative clinical immunotherapy strategies for gastric cancer.Methods(1)Clinical data of gastric cancer in TCGA database was analysed by TIMER online software:the expression level of Tim-3 coding gene,HAVCR 2,in gastric cancer tissues and paracancerous tissues was analysed in the "diff exp" module;the correlation between the expression levels of HAVCR2&tumor purity,PDCD1&tumor purity,LGALS9&tumor purity was analysed in the "correlation”module;the correlation between the expression levels of Tim-3&PD-1,Tim-3&Galectin-9 in gastric cancer was analysed in "correlation" module;the relationship between THAVCR 2 and the infiltration level of immune cells,B lymphocytes,CD8 + T lymphocytes,CD4 + T lymphocytes,macrophages,neutrophils,dendritic cells,was analysed in "gene" module;the effect of Tim-3 and Galectin-9 expression level,and immune cell infiltration level on the survival rate of gastric cancer patients was analysed in "survival" module.(2)22 patients with gastric cancer(from Janurary 30,2015 to December 30,2015)were collected in this study.Basic clinical information of all patients was recorded.The gastric cancer tissue,adjacent tissues(2 cm from the lesion)and peripheral blood(fasting blood samples on the second day of admission)were obtained.Peripheral blood mononuclear cells and tumor infiltrating lymphocytes were obtained by discontinuous density gradient centrifugation.T lymphocytes were isolated and purified using Pan T Cell Isolation Kit(Miltenyi Biotec GmbH,Bergisch Gladbach,Germany).(3)The expression levels of Tim-3 in T lymphocytes in different tissues(peripheral blood,gastric cancer tissues and adjacent tissues)and different stages(T1,T2,T3 and T4)were detected by qRT-PCR and flow cytometry(4)Galectin-9 was added to activate Tim-3,and the levels of IFN-? and TNF-? secreted by T lymphocytes were detected by flow cytometry.Anti-Tim-3 antibody was added to block Tim-3,and the levels of IFN-y,TNF-a,IL-4,IL-8,IL-17 and IL-10 secreted by T lymphocytes were detected by ELISA Anti-Tim-3 antibody and/or anti-PD-1 antibody were added to block Tim-3 and/or PD-1,and the changes in cytotoxicity of T lymphocytes to gastric cancer cells were detected by flow cytometry.(5)Gastric cancer cells were injected under the right flank subcutaneous of mice to establish a tumor bearing model of gastric cancer cells in mice.Mice were treated differently when subcutaneous tumor nodules grew to a volume of approximately 0.1 mm3.Mice were randomly divided into 3 groups(8 mice in each group)and treated with intraperitoneal inj ection:untreated T lymphocyte treatment group;Galectin-9 treated T lymphocyte treatment group;PBS buffer treatment group.The survival of the mice,and the volume and quality of the tumor growth were observed regularly.Results(1)The expression level of Tim-3 encoding gene,HAVCR 2,in gastric cancer tissues was significantly higher than that in adjacent tissues.There was a negative correlation between the expression level of HAVCR2 and the tumor purity.The results showed that the higher the purity of gastric cancer cells in gastric cancer tissues,the lower expression level of Tim-3.Taken together,the expression level of Tim-3 in gastric cancer tissues was significantly higher than that in adj acent tissues,and the increase in this expression level was not caused by the expression of Tim-3 in gastric cancer cells.(2)The correlation between the expression of Tim-3 and the degree of immune infiltration in gastric cancer tissues was analysed in this study.The results showed that the expression of Tim-3 was positively correlated with the infiltration level of CD8+T cells,CD4+T cells,macrophages.neutrophils and dendritic cells.However,the expression level of Tim-3 and the level of infiltration of B cells showed a negative correlation.The above results indicated that the higher the overall level of immune cell infiltration in tumor tissues,the higher the expression level of Tim-3 Combined with the results of correlation analysis between Tim-3 expression and gastric cancer purity,we concluded that the expression of Tim-3 in gastric cancer tissues was up-regulated,mainly due to the high expression of Tim-3 in immune cells in gastric cancer tissues.(3)The correlation between the expression of Galectin-9 and the degree of immune cell infiltration in gastric cancer was analysed in this study.The analysis showed that the correlation between galectin-9 expression and the infiltration degree of CD4+T cells and macrophages was not statistically significant.However,it was positively correlated with the immune infiltration degree of CD8+T cells,neutrophils and dendritic cells,and negatively correlated with the infiltration level of B cells.Combined with the correlation analysis between the expression level of Tim-3 and the infiltration degree of immune cells,we found that the correlation between galectin-9 expression&immune infiltration level and Tim-3 expression&immune infiltration level were consistent.(4)The effects of Tim-3 and Galectin-9 expression on the survival rate of gastric cancer patients were analysed in this study.The results reminded that the expression levels of Tim-3 and Galectin-9 in gastric cancer tissue had no significant effect on the prognosis of gastric cancer patients.Then,the correlation between immune cell infiltration and survival rate in gastric cancer tissues was analysed.The results showed that the prognosis of patients with low infiltration levels of B cells,CD8 + T lymphocytes,CD4 + T lymphocytes,neutrophils and dendritic cells was not significantly different from that of patients with high infiltration levels.However,macrophages showed a high degree of infiltration level corresponding to poor prognosis of patients.(5)The correlation between Tim-3 and Galectin-9 expression in gastric cancer tissues was investigated.The results showed that the Galectin-9 expression level was negatively correlated with the purity of gastric cancer cells or the expression level of Tim-3.This trend was consistent with the correlation between the expression level of Tim-3 and the purity of gastric cancer cells in tumor tissues.In addition,the analysis of the correlation between the expression levels of Tim-3 and Galectin-9 showed that the expression level of Tim-3 coding gene,HA VCR 2,was positively correlated with the expression of Galectin-9 encoding gene,LGALS 9.Subsequently,the correlation between the expression level of Tim-3 and PD-1 in gastric cancer tissues was analyzed.The results showed that the expression of PD-1 gene,PDCD1,was negatively correlated with the purity of gastric cancer cells.This trend was consistent with the correlation between Tim-3 expression and gastric cancer cell purity.Moreover,the expression level of Tim-3 coding gene,HAVCR2,was positively correlated with the expression level of PD-1 coding gene,PDCD1.(6)The expression levels of Tim-3 in peripheral blood,gastric cancer and paracancerous tissues were analyzed by qRT-PCR and flow cytometry.The results showed that the expression level of Tim-3 increased in gastric cancer tissues,adjacent tissues and peripheral blood,and there were significant differences among these tissues.Next,we assessed the relationship between the expression level of Tim-3 and gastric cancer stage.The data indicated that the expression level of Tim-3 increased significantly with the staging of gastric cancer.(7)Galectin-9 was added to activate Tim-3,and then the levels of IFN-? and TNF-a secreted by T cells were detected.The results showed that activation of Tim-3 significantly inhibited the secretion of IFN-y and TNF-a by T cells.(8)Blocking Tim-3 by adding Tim-3 blocking antibody,and then the levels of IFN-y,TNF-a,IL-4,IL-8,IL-17 and IL-10 secreted by T cells were measured.The data showed that Tim-3 blockade can significantly promote the secretion of IFN-y and TNF-a,inhibit the secretion of IL-4 and IL-17,but has no significant effect on the secretion of IL-8 and IL-10.In addition,cytotoxicity experiments demonstrated that Tim-3 blockade can significantly promote the cytotoxicity of T lymphocytes on gastric cancer cells,and has a synergistic effect with PD-1(9)By establishing a tumor-bearing model of mouse gastric cancer cells,we demonstrated that Tim-3 inhibited the inhibition of T cells on gastric cancer cells.ConclusionThe expression level of Tim-3 in gastric cancer infiltrating T lymphocytes was higher than that of adjacent tissues.The expression level of Tim-3 increased with the staging of gastric cancer.Furthermore,the upregulation of expression or activation of Tim-3 inhibited the anti-tumor ability of T lymphocytes. |
PDF Full Text Request