The Role Of The Negative Costimulatroy Molecule PD-L1 In Immune Escape Of Ovarian Cancer

Ovarian cancer, with the highest incidence of gynecological malignancies, is the leading cause of death of women worldwide. Many factors are involved in the interaction of tumor tissue and immune system within the tumor microenvironment. Along with cognition about malignant tumor microenvironment in tumor occurrence and development, the tumor escape mechanism of ovarian cancer has become a frontier topic discussion. Recent research shows that the B7 family members of negative costimulatory molecules express in many cancer tissues. PD-L1 has attracted much attention in recent years. By arresting the cell cycle, PD-L1 ligation of T cells has a profound inhibitory effect on the growth, cytokine secretion, and development of cytotoxicity. The observations suggest that PD-L1 over-expression may reflect a more aggressive biologic potential and may play a role in tumor immune surveillance mechanisms.In this contribution, we focus on the study of PD-L1 mediated ovarian cancer immune escape in tumor microenvironment by histology, cell biology, immunology, animal model.Part I, To study the expression of negative costimulatroy molecule PD-L1 in ovarian cancer tissues and its relationship with the expression of K i67, the infiltration of CD8+T cells and CD68+cells by immunohistochemistry. It was found that the PD-L1 protein was mainly localized in the cytomembrane/cytoplasm(mem/pla) of ovarian cancer cells. The ovarian carcinoma could be classified by mem/pla expression of PD-L1. The memlow/negplahigh tumors accounted for 39.50% of cases( 32/81), memlow/negplalow/neg tumors accounted for 33.33% of cases(27/81), the memhighplahigh tumors accounted for 16.05% of cases(13/81), and memhi ghplalow/neg tumors accounted for 11.11% of cases(9/81). Instead of cytoplasmic PD-L1, a relationship was noted between cytomembranic PD-L1 expression and degree of tumor differentiation. Furthermore, cytoplasmic PD-L1 expression was also correlated directly with proliferation/differentiation antigen Ki-67 expression. C ytomembranic PD-L1 expression was correlated with CD68+ cells, cytoplasmic PD-L1 expression was correlated with tumor infiltration of CD8+T cells. Additionally, different from the other findings, there appeared no relationship between expression of PD-L1 and FIGO staging of ovarian carcinoma.Part II, In ovarian cancer, PD-L1 is a potent negative regulator of anti-tumor immunity, and facilitates tumor growth. PD-L1 silencing with si RNA could inhibit cell proliferation and migration. To elucidate the functional significance of cancer-related PD-L1 expression, we performed co-culture experiments of cancer cell with CD8+ T cells. Cancer-related PD-L1 was identified as a strong inhibitor of T-cell effect. Furthermore, PD-L1 m Ab had an ability to inhibit tumor growth in vivo. We established a mouse model of ovarian cancer ID8. Both PD-1 and PD-L1 monoclonal antibodies can effectively inhibit the growth of tumor. We analyzed the expression of PD-1 molecule in T cells by flow cytometry. CD8+T cells of each group do not express PD-1 in peripheral blood and lymph node. The PD-1 expression in CD8+T cells of tumor bearing mice was significantly increased in the spleen. Furthermore, we confirmed downregulated expression of PD-1 in CD8+T cells in the spleen of the antibody treatment compared with the control group mice.Part III, We investigated the expression and the function of PD-L1 in ovarian cancer in vitro. Although ovarian cancer line(SKOV3) constitutively expressed PD-L1 m RNA and protein in cytoplasm, PD-L1 was not expressed on the surface. In SKOV3 cell transplanted ovarian carcinoma model, the expression of membrane-bound PD-L1 on primary tumor cells was increased. Exposure to tumor-associated macrophage(TAM) also strongly induced membrane-bound PD-L1 expression on SKOV3, which was mediated though IL-6, IL-10, TNF-? and IFN-? produced by TAM. Western blot and real-time PCR analysis showed that the stimulation by different cytokines lead to an obvious increase of the total protein expression level of PD-L1, but not less effects on the level of transcription. AKT phosphorylation and ERK1/2 phosphorylation of SKOV3 cells were significantly increased after 15 min stimulation of IFN-? and TNF-?, not accompanied by an increased phosphorylation of STAT3. The stimulation of IL-6 can promote the phosphorylation of STAT3, not accompanied by increased ERK1/2 and AKT phosphorylation. TNF-α-increased PD- L1 expression was significantly blocked by ERK1/2 inhibitor, the protein expression caused by IFN-γ was partially blocked by AKT inhibitor. These inhibitors don’t affect the up-regulation of membrane-bound PD-L1 expression on SKOV3 by IL-6 and IL-10. The chemical STAT3 signal inhibitor AG490 can’t affect membrane-bound PD-L1 expression on SKOV3 with IL-6 and IL-10.Part IV, Compared with that on benign ovarian disease, PD-L1 is selectively over-expressed on some TAM of ovarian cancer. In peripheral blood, the proportion of PD-L1+CD68+ cells to C D68+ cells and the intensity of PD-L1 staining on CD68+ cells in healthy group were similar to those observed in ovarian cyst group. Instead, these two measures were significantly higher in ovarian cancer group, thereafter related to TNM stage. Interestingly, intracellular levels of IL-10, IL-6, TNF-α and IFN-γ in PD-L1+CD68+ macrophage were higher than those in PD-L1-CD68+ macrophage, especially IL-6 expression. Our data supported that expression of PD-L1 on TAM promoted apoptosis of T cells via interaction with PD-1 on CD8+T cells. Taken together, these results suggested that PD- L1-expressing macrophage represents a novel suppressor cell population in ovarian cancer, which contributes to immune escape of ovarian cancer.In summary, the ovarian carcinoma could be classified by mem/pla expression of PD-L1. Instead of cytoplasmic PD-L1, an inverse relationship was noted between cytomembranic PD-L1 expression and degree of tumor differentiation. Furthermore, PD-L1 expression was also correlated directly with proliferation/differentiation antigen Ki-67 expression, CD68+ cells and CD8+T cells. Exposure to tumor microenvironment also strongly induced membrane-bound PD-L1 expression, which was mediated though IL-6, IL-10, TNF-? and IFN-? produced by TAM. The PD-L1 expression relates to the AKT, the ERK1/2 signal. PD-L1 is a potent negative regulator of anti- tumor immunity, and facilitates tumor growth. PD- L1-expressing macrophage may represent a novel suppressor cell population in ovarian cancer, which contributes to immune escape of ovarian cancer.