| In recent years,immune checkpoint inhibitors,including blockades of programmed cell death protein-1(PD-1)and its legend(PD-L1),have play an important role in the immunotherapy in lung cancer,which inhibit the growth of tumors and improve the overall survival of patients.However,lung cancer can induce immunosuppression via expression of PD-L1,which then inhibit the activation of T cells via interaction with PD-1.Galectin-3(Gal-3)is a member of the lectin family,expressed in the cells or the cellular microenvironment.Moreover,research has found that Gal-3 can regulate the function of immune system,but also participate the progression of lung cancer via expression in the lung cancer cells.Besides,JAK-STAT pathway is one of the most important signal pathways in the cancer cells,by involving proliferation and migration of cancer cells,especially STAT3,which could be overexpressed in the cancer cells and then induce immunosuppression by promoting the expression of PD-L1.Previous studies have demonstrated that hypoxia is a common phenomenon in tumor microenvironment,which induces the progression of cancer.Therefore,in present study,we investigated whether hypoxia could induce the expression of Gal-3 in lung cancer,and whether Gal-3 could contribute immunosuppression by regulation of PD-L1 via STAT3 pathway.Then we investigated whether blocking Gal-3 can inhibit expression of PD-L1 in lung cancer cells,in turn relieve immunosuppression.Also,we investigated whether Gal-3 inhibitor participated the activation of T cells,improved the T cell activities.In addition,we investigated whether Gal-3inhibitor can suppress the growth of tumor and can enhance anti-tumor activity of PD-L1 blockade in mice xenograft model.There are three main components in present study,as following:Part 1 Gal-3 expression and its regulatory effect on PD-L1 through the STAT3 Pathway in non-small cell lung cancerObjective: to investigate the expression level of Gal-3 in lung cancer cells,the effect of Gal-3 on the activity of STAT3 and the expression of PD-L1Methods: lung cancer A549 cells were cultured in vitro,and then maintained in hypoxia condition.The m RNA level of Gal-3 in cells was measure by Realtime PCR,and the Gal-3 level secreted by cells were measured by the enzyme-linked immunosorbent assay(ELISA).Also the cells were treated with different concentrations of Gal-3 inhibitor,and cell viability was evaluated by MTT assay,to determine the IC50 values of Gal-3 inhibitor.Thereafter,the A549 cells were treated with Gal-3 or Gal-3 inhibitor respectively,and then the expression of STAT3 activities,PD-L1 level were evaluated by western blot.And the expression of STAT3 was knockdown by transfection of STAT3 si RNA to knockdown its expression,verified by western blot.Then the cells were treated Gal-3 inhibitor to investigate whether Gal-3 regulate the PD-L1 expression through STAT3 pathway.Results:1.Hypoxia induces the expression and secretion of Gal-3 in human lung adenocarcinoma A549 cells.After lung cancer A549 cells were cultured under hypoxia condition,the total RNA was collected from cells,and the results showed that m RNA level of Gal-3 was significantly increased,in which the level of Gal-3 in cells under hypoxia condition was 3.3 fold of that in controls(P<0.05).Using ELISA,the Gal-3 level in cell medium was measured,and the data showed the Gal-3 level was(1866.67±32.25)ng/L after cells treated by hypoxia,and(320.67±12.34)ng/L in cell medium in control group,which indicated that hypoxia significantly increased the secretion of Gal-3 by lung cancer cells(P<0.05).2.The IC50 of Gal-3 inhibitor on human lung adenocarcinoma A549 cells was detected.The results showed that the IC50 of Gal-3 inhibitor on human lung adenocarcinoma A549 cells was 1.04 μ M.3.Gal-3 regulates the expression of PD-L1 through STAT3 pathway.Firstly,according to other previous studies,the concentration of Gal-3was determined to be 0.5 μ g / ml.After the cells were treated with Gal-3,the data showed that Gal-3 can promote the phosphorylation level of STAT3.Although there was no significant difference in STAT3 level between the cells with or without Gal-3 treatment,phosphorylation level of STAT3 in cells treated with Gal-3 was 189.33% of that in cells without Gal-3treatment(P<0.01),while the level of PD-1 in cells with Gal-3 treatment was188.36% of that in cells without Gal-3 treatment(P<0.01).However,compared to placebos,there was no significant difference in STAT3 level in the cells with Gal-3 inhibitor,whereas Gal-3 inhibitor can decrease the phosphorylation level of STAT3,in turn inhibit PD-L1 expression.The data showed that phosphorylation level of STAT3 in cells treated with Gal-3 inhibitor was48.72% of that in controls(P<0.05),while the level of PD-1 in cells with Gal-3 treatment was 56.37% of that in controls(P<0.01).In addition,the expression of STAT3 was knockdown using Si RNA,and the data showed the STAT3 expression level after knockdown was only 23 % of scramble controls.Moreover,our data showed that in cells with STAT3 knockdown,Gal-3inhibitor failed to regulate the expression of PD-L1 after the STAT3 was blocked.There was no significant difference in PD-L1 level among the groups of cells with STAT3 knockdown,STAT3 knockdown+Gal-3,STAT3knockdown+Gal-3 inhibitor(P>0.05).Summary: Lung cancer cells can express and secret Gal-3,which can activate the STAT3 pathway,and then promote the expression of PD-L1 in lung cancer cells.However,inhibition of Gal-3 can inhibit the activity of STAT3 pathway,and then decrease the expression of PD-L1.Part 2 the regulatory effect of Gal-3 inhibitor in the activation and function of immune cellsObjective: To investigate whether Gal-3 inhibitor can regulate the function of human peripheral blood mononuclear cells(PBMCs).Methods:Human peripheral blood samples were obtained from human,the PBMCs were obtained by the attachment method.Lung cancer A549 cells were culture in vitro,and tumor antigens were prepared by the freeze-thaw method.Thereafter,using tumor antigens,Lipopolysaccharide and cytokines(GM-CSF,Recombinant Human Interleukin-4,Recombinant Human Interleukin-2)to treat PBMCs to induce PBMCs cells activation and proliferation,and then using flow cytometry and antibody targeted CD8 to evaluate the proliferation of CD8 positive T cells,and using ELISA to evaluate the cytokines secreted by PBMCs,including Interferon-γ(IFN-γ),Tumor necrosis factor α(TNF-α).Besides,the PBMCs were treated with tumor antigens,Gal-3 inhibitor,PD-L1 blockade,and cytokines,and then co-cultured with A549 cells to investigate the cytotoxic activity by 4h-51 Cr release assay.Results:1.Gal-3 inhibitor can promote the proliferation of T cells in peripheral blood monocytes(PBMCs).Our data showed that the PBMCs co-treated by Gal-3 inhibitor had much higher percentage of CD8 + cytotoxic T cells as 77.09%,compared to that without Gal-3 inhibitor co-treated as 10.51%(P < 0.05).2.Gal-3 inhibitor can enhance the secretion of inflammatory factors in PBMCs.In the culture medium of PBMCs with Gal-3 inhibitor co-treatment,the IFN-γ、TNF-a were(1233.00 ± 23.20)pg/ml and(1679.84 ± 40.20)pg/ml respectively,which was significantly higher than that of PBMCs without Gal-3 inhibitor co-treatment as(782.33 ± 21.20)pg /ml and(1257.47 ± 42.23)pg/ml respectively(IFN-γ,P < 0.01,TNF-α,P < 0.01).3.Gal-3 inhibitor can enhance the cytotoxic effect of human PBMCs against human lung adenocarcinoma A549 cells.Results of cytotoxicity assay showed that Gal-3 inhibitor(P < 0.05)or PD-L1 blockade(P < 0.01)could facilitate the cytotoxic effect of PBMCs repectively.Moreover,the cytotoxic activity of PBMCs treated with combination of a Gal-3 inhibitor and PD-L1 blockade was superior to the monotherapy of Gal-3 inhibitor or PD-L1 blockade(P < 0.01).Summary: Gal-3 inhibitor can enhance the activity and function of PBMCs,and the promote the cytotoxic activity against lung cancer cells.Moreover,co-administration of a Gal-3 inhibitor promoted the cytotoxic activity of PBMCs induced by PD-L1 blockade.Part 3 The study on anti-tumor effect of combination therapy with PD-L1 blockade and Gal-3 inhibitor in lung cancerObjective: To investigate the anti-tumor effect of Gal-3 inhibitor,and combinatory anti-tumor effect of Gal-3 inhibitor and PD-L1 blockade in mice xenograft model.Methods: Mice xenograft model were created by subcutaneous injection of A549 cells.And the human PBMCs were injected in the mice via tail vein when the tumors were developed,then the mice were randomly divided into different groups,including Gal-3 inhibitor,PD-L1 blockade,Gal-3inhibitor+PD-L1 blockade,and placebo.Thereafter the tumor growth was monitored by evaluating tumor volumes.And the inhibitor effect of treatment was calculated through tumor inhibitory rate of different treatment groups,including group of Gal-3 inhibitor,PD-L1 blockade,Gal-3 inhibitor+PD-L1 blockade,while the tumor volumes in placebo group were considered as controls.Also the combination effect was determined by determination of combination index through comparing the inhibition rate of monotherapy and combination therapy.In addition,the tumor tissues were collected and the tumor weights were measured.Then using immunohistochemistry,the expression of CD3 and granzyme B were investigated respectively to evaluate the tumor-infiltrating lymphocytes in tumors.And the expression levels of IFN-γand IL-2 were analyzed by ELISA.Results: Both Gal-3 inhibitor and PD-L1 blockade treatment can inhibit the tumor growth,compared to the placebos,in which treatment with the Gal-3 inhibitor resulted in a 23.94% inhibition of tumor growth,while PD-L1 blockade resulted in a 62.02% inhibition of tumor growth.In contrast,the combination of PD-L1 blockade with a Gal-3 inhibitor induced a 78.17%inhibition of tumor growth.Our results indicated a synergistic effect of the combination of PD-1 blockade with Gal-3 inhibition(the combination index was 0.21).The inhibitory effect of combination therapy was significantly superior to the monotherapy of Gal-3 inhibitor or PD-L1 blockade.The immunohistochemistry data showed that the combined therapy significantly increased the number of CD3+ tumor-infiltrating lymphocytes.Moreover,in the tumors derived from mice that received combination therapy,the number of cells with granzyme B-positive expression was significantly higher than that in the mice treated with monotherapy.In the tissue of lung cancer which received combination therapy,the IFN-γ、IL-2 were(987.23 ± 81.13)pg/ml and(1230.41 ± 71.23)pg/ml respectively,which was significantly higher than that of monotherapy(P < 0.05).Summary: Gal-3 inhibitor can inhibit the tumor growth,and increase the tumor-infiltrating lymphocytes.Moreover,the combination of a Gal-3inhibitor and PD-L1 blockade synergistically suppressed tumor growth by enhancement of functions of tumor-infiltrating lymphocytes.Conclusions: Lung cancer cells can express and secrete Gal-3.Hypoxia can promote the expression and secretion of Gal-3.Gal-3 regulate the expression of PD-L1 through STAT3 pathway in non-small lung cancer,but it can not complete inhibit the expression of PD-L1,suggesting that Gal-3 may regulate the expression of PD-L1 through other signaling pathway.Gal-3 can regulate the number and function of immune cells and increase the number of T cells in tumor tissue.Gal-3 inhibitor can inhibit the tumor growth,and can enhance anti-tumor activity of PD-L1 antibody in non-small lung cancer.Gal-3 may become a new therapeutic target. |
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