| Objectives: PD-L1 is a representative immune checkpoint.While binding to PD-1molecules on the activated T cells,PD-L1 can induce T cell inactivation and then inhibit anti-tumor immune response.In the tumor microenvironment,IFN-γ can promote the expression of PD-L1 in cancer cells,thereby enabling cancer cells to produce acquired immune tolerance.In this study,an adenovirus vector carrying SOCS3 gene is used to increase the expression of SOCS3 in malignant ovarian cells,and inhibits the PD-L1 expression induced by IFN-γ by inhibiting the activation of STAT1 pathway,thus enhances the T cells anti-tumor immunity.Methods: We utilized "Kaplan-Meier Plotter" database to analyze the nexus between the prognosis of patients with ovarian cancer and the PD-L1 expression.In Timer2.0 and GEPIA,We analyzed the correlation between IFN-γ and PD-L1 expression in ovarian cancer,and detected the effect of IFN-γ on PD-L1 expression on the surface of ovarian cancer cells by flow cytometry.Besides,we analyzed the correlation between IFN-γ and STAT1 and STAT3 in ovarian cancer in Timer2.0 and GEPIA.The changes of STAT1 and STAT3 expression after IFN-γtreated were evaluated by Western Blot.STAT1 or STAT3 expression was silenced by si RNA respectively prior to stimulation with IFN-γ,and then the PD-L1 expression was examined by flow cytometry.Under the condition of IFN-γinducing,flow cytometry was used to detect the expression of PD-L1 after Ad-SOCS3 treatment,and Western Blot was performed to determine the changes of STAT1 and p STAT1 expression.Meanwhile,the killing effect of Ad-SOCS3 with different MOI on ovarian cancer cells was performed through CCK8 in vitro.In C57BL/6 subcutaneous tumor model,the capability of Ad-SOCS3 on anti-tumor was assessed.The mouse tumor cells and tumor infiltrating lymphocytes were isolated.The PD-L1 expression on the surface of tumor cells and T cell function indicators Granzyme B and IFN-γwere discovered by flow cytometry.And the expressions of SOCS3、Granzyme B、PD-L1 and CD8 were revealed by immunohistochemistry.The expressions of SOCS3 in the normal epithelium of fallopian tubes and ovarian cancer tissues were evaluated by immunohistochemistry.The nexus between the SOCS3 expression and the prognosis of patients with ovarian cancer was estimated in both "Kaplan-Meier Plotter" and Progno Scan database.Results: PD-L1 expression is negatively correlated with the prognosis of patients with ovarian cancer.There is a positive correlation between IFN-γ and PD-L1 expression in ovarian cancer.IFN-γ can induce PD-L1 expression in ovarian cancer cells in vitro.Through database analysis,it was found that IFN-γ was positively correlated with the expression of STAT1 and STAT3.STAT1 and STAT3 pathways were both activated in tumor cells after IFN-γ treatment,silencing STAT3 could not inhibit PD-L1 induced by IFN-γ while silencing STAT1 can effectively inhibit PD-L1 induced by IFN-γ.SOCS3 is the main negative regulator of JAK/STAT pathway.Ad-SOCS3 can restrain the production of PD-L1 induced by IFN-γ by inhibiting the activation of STAT1.In addition,compared with the control group,Ad-SOCS3 has no killing effect on tumor cells directly in vitro.It has been verified in animal experiments that Ad-SOCS3 can control tumor development to a certain extent.This effect is through down-regulation of PD-L1 expression in tumor cell,thereby enhancing the T cells anti-tumor immune response.Consistent with this,the expression of SOCS3 in human ovarian cancer tissues is significantly lower than that of normal fallopian tube epithelium,and patients with low SOCS3 expression have shorter PFS,OS and DFS.Conclusion: Ad-SOCS3 inhibits PD-L1 induced by IFN-γ by restraining the activation of STAT1,reduces the acquired immune resistance brought by IFN-γ,and enhances the anti-tumor immune response mediated by T cells.Targeting SOCS3 may produce new method of treating ovarian cancer. |
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