Breast Cancer Cells Promote Tumor Development And Immune Escape Through The Expression Of Inhibitory Molecule CTLA-4 And IL-35

BackgroundBreast cancer is one of the most common malignant tumors in women,which seriously threatens the life and health of women.The incidence and mortality of breast cancer in China are increasing every year.Currently,the comprehensive treatment based on surgery,endocrinotherapy and chemoradiotherapy has made great progress.The biological characteristics of breast cancer include uncontrollable invasive growth,aggressiveness,chemoresistance,and special immunosuppressive microenvironment.Due to the undesirable characteristics of breast cancer,the recurrence and metastasis are still the main causes of survival and mortality.Tumor microenvironment is composed of tumor parenchyma and interstitium,which is chracterised by infiltrating immunocytes,unique cytokines and chemokines.Tumor microenvironment plays a critical role in the development,progression,invasion,metastasis and immune escape of the tumor.It is reported that the inhibitory molecules and cytokines expressed by tumor cells could also regulate the tumor immune escape through suppressing the function of immunocytes in tumor microenvironment.Therefore,it is one of the hotspots in breast cancer research to explore the interaction and mechanism between breast cancer cells and immunocytes in microenvironment.Dendritic cell(DC),the most potent antigen presenting cell,is the central link in regulating and maintaining the immune response of the body.CD83 is the maturation marker of DC,moreover,mature DC also have the high expression of HLA-DR,CD40,and costimulatory molecules CD80 and CD86.Normally,mature DC could effectively capture and process the antigens,then present them to specific T lymphocytes in order to activate the specific immune response and maintain the stability of the body function.The anti-tumor immunity is mainly dominated by mature DC and other immunocytes in the microenvironment.The polarization of naive T cells induced by mature DC is the key to regulate the immune response and plays a vital role in the interaction between tumor and immune system.The direct contact between tumor cells and DC in the local microenvironment affects DC function through a certain ways and inhibits the anti-tumor immunity.In the present study,we studied the effect of inhibitory molecule CTLA-4 and suppressive cytokine IL-35 that expressed by breast cancer cells on the maturation and function of DCs in the tumor microenvironment,as well as the effects and mechanisms of IL-35 on proliferation and apoptosis of breast cancer cells under H/SD condition.Our research provides a valuable complement for further understanding the interaction between tumor cells and immune cells in microenvironment,and helps to elucidate the mechanisms of tumor progression and immune escape.Meanwhile,it also provides a new insight into the immunotherapy and biological treatment for breast cancer.Section I:CTLA-4 positive breast cancer cells suppress dendritic cells maturation and functionObjectiveCytotoxic T-lymphocyte associated antigen 4(CTLA-4),one of the most important immunosuppressive receptor,is mainly expressed on effector T cells and regulatory T cells(Tregs).CTLA-4 expressed on Tregs can bind to the surface molecules B7 on DC and exerts immunosuppressive effects through inhibiting the proliferation and activation of T cells,the cell cycle progression,and secretion of pro-inflammatory cytokines.Currently,it is known that many types of tumor could express CTLA-4,such as breast cancer,colon cancer,kidney cancer,osteosarcoma.In our previous study,we have found that the high expression of CTLA-4 in breast cancer is closely related to the clinical stage and lymph node metastasis.However,we have little knowledge on the function and mechanism of CTLA-4 that expressed on breast cancer.Whether CTLA-4 expressed on breast cancer have the same function as on Tregs?What the effect of CTLA-4 positive expression is on the function of DC in the microenvironment?Whether CTLA-4 functional antibody have a direct effect on the biological behavior of CTLA-4+breast cancer cells?Based on the.above question,the co-culture system of human peripheral blood monocytes derived DC and CTLA-4+ breast cancer cells was established and the effect of CTLA-4+breast cancer cells on the maturation and function of DC was explored,including the maturation and the cytokine secretion level of DC,the ability to stimulate T cell proliferation,the capacity to induce naive CD4+T cell polarization,and the effect on CD8+T cell function.In addition,we also discussed the related signaling pathways of CTLA-4 on breast cancer cell surface affecting DC function and the direct killing effect of CTLA-4 functional antibody on breast cancer cells.Methods1.The expression of CTLA-4 on the surface and intracellular of breast cancer cells was detected by flow cytometry and the CTLA-4+ breast cancer cell was screened.2.A co-culture system was established between human peripheral blood monocytes derived DC and CTLA-4+breast cancer cells.The effects of CTLA-4+ breast cancer cells on the mature phenotype and the cytokines secretion of DC were detected by flow cytometry and Human High Sensitivity Panel.3.After co-culturing CTLA-4+breast cancer cell-treated DC and CD4+/CD8+T cells,CCK-8 assay was used to detect the proliferation of T lymphocytes.4.The flow cytometry and Human High Sensitivity Panel were performed to analyze the effect of CTLA-4+breast cancer cell-treated DC on the polarization of naive CD4+T cells and the function of CD8+T cells.5.Western blotting was used to determine the relevant signaling pathway on CTLA-4+breast cancer cell-treated DC.6.The direct killing effect of CTLA-4 functional antibody on the proliferation,apoptosis and cell cycle of CTLA-4+ breast cancer cells was detected by CCK-8 and flow cytometry.Results1.CTLA-4 was expressed on breast cancer cells,and the intracellular expression was higher than the surface expression.2.CTLA-4+breast cancer cells inhibited the maturation of CD14+monocytes-derived DC by down-regulating the expression of CD83,CD40,CD80,CD86 and HLA-DR.While blocking CTLA-4,the suppressed surface markers and decreased cytokine(IL-1?,IL-2,IL-12 and TNF-a)level were rebounded to different degree.3.CTLA-4+ breast cancer cells-treated DC suppressed the proliferation of CD4+/CD8+T cells.4.CTLA-4+breast cancer cells inhibited the ability of DCs to prime the differentiation of naive CD4+T-cells into Thl effectors.5.CTLA-4+breast cancer cells-treated DC suppressed CD8+T cells function through decreasing the production of Granzyme B and pro-inflammatory cytokines.6.Aberrant activation of ERK and STAT3 might be responsible for CTLA-4+breast cancer cells-induced DC maturation.7.CTLA-4 functional antibodies could directly inhibit CTLA-4+breast cancer cells proliferation and promoted cell apoptosis,but had little influence on cell cycle progression.ConclusionsOur study demonstrated for the first time that CTLA-4,a suppressive molecule expressed on the surface of breast cancer cells,could directly effect on DCs in the tumor microenvironment and achieve tumor immune escape through inhibiting the maturation and function of DCs,while blocking CTLA-4 could directly inhibit biological behavior of breast cancer cells.Our study provided evidence for a novel tumor-evading mechanism in breast cancer,and more importantly,suggested a crucial immune-modulatory target,which might help to provide novel immunotherapeutic strategies for breast cancer patients.Section ?;Tumor-derived IL-35 protects breast cancer cell survival via induction of autophagy in hypoxia/serum deprivationObjectiveAutophagy is a process that regulates the degradation and re-use of cellular contents to satisfy the metabolic needs.The special survival mechanism of energy reutilization plays a crucial role in maintaining self-healing,operating properly and escaping adverse environment,such as inflammation,hypoxia and starvation.Meanwhile,as a new mechanism of cell death,autophagy is closely related to apoptosis.That is to say,to a certain extent,cells can avoid apoptosis by enhancing autophagy and help cells resist the bad environment.Breast cancer is a typical solid tumor.With the uncontrolled proliferation of tumor cells,there is a wide range of hypoxic ischemic region within the tumor.Therefore,the breast cancer cells develop ubiquitous autophagy to overcome relatively barren tumor microenvironment.IL-35,a member of IL-12 family,is a novel immunosuppressive cytokine.It is made up of Epstein-Barr Virus Induced Gene 3(EBI3)and IL-12p35 in a heterodimeric form.Present studies have confirmed that IL-35 is not only secreted in Tregs and Bregs but also expressed in many tumors,such as lung cancer,rectal cancer,pancreatic cancer.The expression of IL-35 are involved in tumor development and progression.It has been reported that IL-35 which is expressed on breast cancer tissue and infiltrating lymphocytes,plays a protective role on breast cancer cells through promoting its proliferation,while the expression of IL-35 receptor on breast cancer cells is significantly upregulated under hypoxia/serum deprivation(H/SD)condition.However,there is no report on whether IL-35 is involved in the survival and autophagy process of breast cancer cells under H/SD condition.In the preaset study,we explore the influence and mechanism of IL/35 on the autophagy activation in breast cancer cells under hypoxia/serum deprivation(H/SD)condition.In vitro study of IL-35-induced autophagy effected the cell proliferation and apoptosis levels in breast cancer cells.Methods1.Immunohistochemistry and immunofluorescence staining were performed to analyze the IL-35 expression on breast cancer tissue and breast cancer cells.The flow cytometry assay was used to detect the expression of IL-35 receptor on breast cancer cells under normal or H/SD condition.2.CCK-8,cell cycle detection,apoptosis detection and western blotting were performed to investigate the effect of IL-35 on the proliferation and apoptosis of breast cancer cells under H/SD condition.3.MDC staining and western blotting were used to analyze the effect of IL-35 on the autophagy activation of breast cancer cells under H/SD condition.4.CCK-8 and flow cytometry were performed to detect the effect of IL-35 on the proliferation,apoptosis and cell cycle of breast cancer cells after blocking autophagy by 3-MA and Baf-A1.5.Western blotting was used to determine the relevant signaling pathway on IL-35-induced autophagy under H/SD condition.Results1.Immunohistochemical and immunofluorescence staining showed that the two subunits EBI3 and IL-12p35 were expressed and co-localization on the breast cancer cells.Flow cytometry assay demonstrated that IL-35 receptor was expressed on the breast cancer cells and the expression was up-regulated under H/SD condition.2.Under H/SD condition,IL-35 promoted the proliferation of breast cancer cells,reversed the H/SD-induced G1 arrest,inhibited apoptosis,up-regulated the expression of cyclinD1,cyclinE1,CDK2 and Bcl-2,and down-regulated the expression of p27,Bax and cleaved caspase 3 in a dose-dependent manner.3.IL-35 up-regulated the autophagy in breast cancer cells under H/SD condition.Treatment of breast cancer cells with autophagic inhibitor 3-MA and Baf-A1,the protective effect of IL-35 was obviously weakened,which showed inhibited proliferation and increased apoptosis.4.Western blotting assay revealed that IL-35 could down-regulated the expression of p-mTOR,p-AKT and PI3K under H/SD condition to promote the autophagy of breast cancer cells.ConclusionsIn this part of the study,we found that IL-35 was constitutively expressed on the breast cancer tissue and cells.We explored for the first time that IL-35 up-regulated the autophagy level of breast cancer cells through mTOR/AKT/PI3K pathway under H/SD condition,and then promoted the proliferation and inhibited the apoptosis of breast cancer cells.This discovery provided strong evidence for IL-35 to promote the development of breast cancer,and provided a new target for the treatment of breast cancer.Section ?:Interleukin 35:Inhibitory regulator in dendritic cell maturation and activationObjectiveAs an important inhibitory cytokine,IL-35 has a clearly known immunosuppressive function.IL-35 can directly inhibit the proliferation and cytokine secretion of effector T cells,suppress the polarization of Th17 and IL-17 secretion,and promote the proliferation of Tregs.In addition,IL-35 can also induce the transformation of naive T lymphocytes into Tregs secreting IL-35(IL-35 induced regulatory T cell,iTr35),thereby amplifying the immunomodulatory effect of Tregs.Tumor-derived IL-35 can promote tumor growth and inhibit the anti-tumor immunity through increasing the accumulation of myeloid-derived suppressor cells and promoting tumor angiogenesis.Our previous study have also found that IL-35 is highly expressed on breast cancer tissue and infiltrating lymphoid tissues of breast cancer and its expression level is closely related to clinical staging and lymph node metastasis.However,the effects of IL-35 on the function of dendritic cells and biological behavior of tumor cells in microenvironment have not been reported yet.In our present study,the effect of exogenous IL-35 on human peripheral blood monocyte-derived DC was used to mimic the function of tumor autocrine or paracrine IL-35 on DC in the tumor microenvironment,in order to observe the effect of IL-35 on DC differentiation,maturation,immunological functions and its relevant mechanism.Methods1.The flow cytometry was performed to analyze the expression of IL-35 and its receptor on monocytes-derived DC.2.IL-35 was added during the process of DC differentiation or maturation stage.The effects of IL-35 on DC phenotype and cytokines secretion were detected by flow cytometry and Human High Sensitivity Panel.3.After establishing the co-culture system between IL-35-treated DC and CD4+/CD8+T cells,CCK-8 assay was used to detect the proliferation of T lymphocytes.4.The flow cytometry and Human High Sensitivity Panel were performed to analyze the effect of IL-35-treated DC on the polarization of naive CD4+T cells and the function of CD8+T cells.5.Western blotting was used to determine the relevant signaling pathway on IL-35-treated DC.Results1.Neither DC nor IL-35-treated DC had the expression of IL-35,while both CD4+ monocytes and DC could express IL-35 receptor.2.IL-35 partially inhibited differentiation of CD14+ monocytes to DC by down-regulating the expression of CD1a and CD209.3.IL-35 significantly suppressed the maturation and cytokines secretion of DC,mainly in the down-regulated expression of HLA-DR,CD40,CD80,CD86 and CD83,and decreased secretion of cytokines IL-1?,IL-2,IL-12p70,IFN-? and TNF-?.4.IL-35-treated DC inhibited the proliferation of allogeneic CD4+/CD8+ T lymphocytes,suppressed the polarization of naive CD4+T lymphocytes towards Th1 phenotype and impaired CD8+T cells allogeneic responses.5.Suppression of DC maturation and function by IL-35 might be due to the aberrant activation of STAT1/STAT3 and inhibition of p38 MAPK/NF-?B signaling pathway.ConclusionIn our study,we demonstrated for the first time that in addition to T and B cells,IL-35 could also inhibit the maturation and function of DC,and IL-35-treated DC could further inhibit the proliferation of T cells,suppress the polarization of naive CD4+T cells towards Th1 cells,cytotoxic ability of CD8+T cells and further reduce the specific immunity.Additionally,IL-35 inhibited the maturation and function of DC through aberrant activation of STAT1/STAT3 and inhibition of p38 MAPK/NF-?B signaling pathway.The inhibitory effect of IL-35 on DC maturation and function might facilitate the development of promising therapeutic interventions in tumors and other diseases.