The Clinical Significance Of Tim-3and Its Biofunctions In Gastric Cancer

Tim-3is an important immune regulatory molecule and is critical in tumorprogression and immune regulation. However, the clinical significance and biologicaleffects of Tim-3expression in different immune cells is still poorly understood. This studyaims at analyzing the clinical significance and possible mechanisms of Tim-3expression ingastric cancer immune cells through clinical specimens and in vivo function assay.Part I Clinical significance and implication of abnormal Tim-3expression on NK cells in patients with gastric cancerObjective To detect the expression of Tim-3on immune cells in vivo in patientswith gastric cancer and analyze its correlation with gastric cancer progression. MethodsPeripheral blood of62patients with gastric cancer and32healthy controls were collectedfor analysis by flow cytometry to detect Tim-3expression on CD56+NK cells. Gal-9protein was used to study the effect of Tim-3on the biofunctions of NK cells in vitro. Tofurther confirm the clinical phenomena observed, we constructed a tumor-bearing mousemodel. Gastric cancer cell line MCF was used to build xenograft tumor-bearing mousemodel, and the dynamic expression of Tim-3in peripheral blood and tumor-infiltrating NKcells was evaluated at different tumor-bearing time points. Then T-bet-/-, Eomes-/-andT-bet-/-Eomes-/-mice were used to analyze the transcriptional regulation of Tim-3on NKcells. Results In either healthy controls or patients with gastric cancer, Tim-3was mainlyexpressed on peripheral NK cells but not on B cells or T cells. Further analysis revealedthat Tim-3expression on NK cells in patients with gastric cancer was significantly higherthan healthy controls. In vitro experiments demonstrated that Gal-9(rhGal-9; Gal Pharma)could activate Tim-3to enhance IFN-γ secretion. Animal experiments showed that with theincrease in tumor burden, Tim-3expression on NK cells from peripheral blood, spleen andtumor tissues significantly increased respectively. Molecular regulation study demonstrated that T-bet but not Eomes played an important role in regulating Tim-3expression on NK. Conclusions This study suggested Tim-3expression on NK might bea potential indicator in the development of gastric cancer. Part II Clinical significance and biological function of Tim-3onmonocytes/macrophages of patients with gastric cancer.Objective To study clinical significance and biological function of Tim-3inmonocyte/macrophages of patients with gastric cancer. Methods peripheral blood of62patients with gastric cancer and32healthy controls was collected and the levels of Tim-3on CD14+monocytes were analyzed by flow cytometry. And, clinical significance of Tim-3expression was subsequently examined. The effects on mononuclear macrophage-mediatedTh differentiation were analyzed by mixed culture system and ELISA. To further confirmthe expression of Tim-3, subcutaneous xenografts were constructed to dynamicallymonitor the expression of Tim-3on CD11b+F4/80+monocytes/macrophages. In vitroanalysis was used to study the regulation mechanism of induced Tim-3expression.Subsequently, T-bet-/-, Eomes-/-and T-bet-/-Eomes-/-DKO mice were used to analyze thetranscriptional regulation of the expression of Tim-3on monocyte/macrophages. ResultsTim-3was abnormally highly expressed on monocyte/macrophages from patients withgastric cancer. The expression of Tim-3on monocyte/macrophages of advanced patientsshowed a higher level compared to that from early stage patients. Tim-3intervention couldsignificantly influence the secretion of TNF-α and IL-12p70. In addition, both of animalexperiments and in vitro experiments have confirmed that tumor condition was animportant factor inducing Tim-3expression. Conclusions Tumor burden in patients withgastric cancer could induce Tim-3expression on monocyte/macrophages, and Tim-3mightcontribute to the progress of gastric cancer by regulating TNF-α.