Mechanisms Of Breast Cancer Cells-derived IL-35 Promoting Immune Escape And Tumor Progression

BackgroundBreast cancer is one of the most malignant tumors that seriously threatens women’s physical and mental health.In China,the incidence and mortality of breast cnacer are increasing.Despite the development of treatment strategy of breast cancer,such as the comprehensive treatment based on surgery,chemotherapy and radiotherapy which has significantly improved the survival rate of patients,the outcome of patients with recurrence and metastasis is still not good.The "seed-soil”theory holds that the interaction between tumor cells and stromal cells is very important,and tumor microenvironment(TME)plays a central role in tumor development.The TME is the cellular environment in which the tumor exists,including tumor cells,surrounding blood vessels,immune.cells,fibroblasts,bone marrow-derived inflammatory cells,lymphocytes,signaling molecules and the extracellular matrix(ECM),which are essential for the initiation,invasion,metastasis and immune escape of tumor.Recent studies have found that tumor cells can induce inhibitory subpopulations and immune escape,leading to malignant progression of the tumor.Therefore,exploring the interaction between tumor cells and infiltrating immune cells,and the biomolecules involved in are important for uncovering the mechnasims of immune escape in breast cancer.Regulatory T cells(Tregs)are novel immunosuppressive CD4+ T cells subpopulations.The proportion of Tregs in TME is significantly higher than that in normal tissues.There are two different population of Tregs.nTregs(natural Tregs)and iTregs(induced Tregs).And iTregs are classified into three different types according to cytokines inducing them:iTr-TGF-β,iTr-IL-10 and iTr-IL-35(IL-35-producing regulatory T cells)which are induced by IL-35.These Tregs inhibit the anti-tumor function of Th1 and Th17 by secreting inhibitory cytokines,leading to tumor development and unfavourable prognosis.Tumor-associated macrophages(TAM),the second immune population in the TME,are original from peripherally macrophages in the context of TME.TAM is classified into two subpopulations,M1 macrophages which promote inflammation and M2 macrophages which inhibit inflammation.The TAM mainly express M2 markers,such as CD206,CD 163 and Arg-1.Infiltrating M2 macrophages can directly promote tumor growth by secreting suppressive cytokines,and play a tumor-promoting role via indirectly inducing Tregs and Th2 cells.This study aims to explore the mechanisms by which breast cancer cells-derived IL-35 promotes tumor progression and immune escape via promoting the proliferation of Tregs and induce iTr-35 directly,and inducing M2 cell polarization through exosomes indirectly.Our study provides new biomarker for the risk assessment of breast tumors,providing a solid theoretical basis for immunotherapy,with great clinical improtance and good prospect for clinical transformation.Chapter I Breast cancer cells-derived IL-35 is associated with poor prognosisObjectiveIL-35(Interleukin 35),a novel immunosuppressive cytokine,belongs to the IL-12 cytokine family and consists of two subunits,EBI3(Epstein-Barr virus-induced gene3)and p35,mainly expressed and secreted by Tregs and regulatory B cells(Bregs).Recent studies showed that in addition to immune cells,IL-35 can also be expressed and secreted by tumor cells.Moreover,high serum IL-35 level was significantly associated with poor prognosis in a variety of tumor.However,it is still not clear whether breast cancer cells can express IL-35.Also,the expression pattern and clinical significance of breast cancer cells-derived IL-35 are unknown.To clarify these issues,we included 86 breast cancer patients to detect IL-35 expression pattern in breast tumor,and then evaluated the correlation between IL-35 expression level and prognosis to establish a prognostic risk model for breast cancer patients.Methods:1.Eighty-six unrelated Chinese women with breast cancer who underwent lumpectomy or mastectomy were included.The clinicopathological characteristics were collected and analyzed.2.We detected the expression of EBI3 and p35 using serial sections by IHC(immunohistochemistry)respectively.The intensity and extent of stain were eveluated.The correlation of the expression of the two subunits was analyzed.3.According to the patient’s IL-35 score,the patients were divided into "high IL-35"group and" low IL-35" group,and the relationship between IL-35 expression level and clinicopathological characteristics was analyzed.4.Analyzed the relationship between the IL-35 expression and overall survival(OS)and progression-free survival(PFS).Univariate analysis and multivariate analysis were performed to determine whether breast cancer cells-derived IL-35 was an independent prognosis risk factor for breast cancer patients.Results:1.The immunohistochemistry results showed EBI3 and p35 were co-expressed in the cytoplasm of most cancer cells.Furthermore,the expression of EBI3 and p35 was correlated significantly.2.Higher level of IL-35 was signifcantly associated with poor differentiation,negative ER status,negative PR status and positive Her-2 status.3.IL-35 level was signifcantly correlated with the survival rate of patients.Both the OS rate and PFS rate of patients with low IL-35 expression were better than those with high IL-35 expression.4.The multivariate Cox’s regression analysis revealed that IL-35 expression was an independent unfavorable prognostic factor for breast cancer.Conclusions:This study confirmed that breast tumor cells could express the inhibitory cytokine IL-35.BCC-derived IL-35 was associated with important clinicopathological characteristics such as tumor stage and lymph node involvement.Furthermore,BCC-derived IL-35 was an independent unfavorable prognostic factor for breast cancer.This study provided new evidence for the tumor-promoting function of IL-35,new biomarkers for prognostic risk assessment,also new targets of immunotherapy for breast cancer.Chapter Ⅱ Breast cancer cells-derived IL-35 promotes tumor progression via induction of IL-35-producing induced regulatory T cellsObjectives:Regulatory T cells(Tregs)were immunosuppressive subpopulations of CD4+ T cells and played an important role in immune-related diseases such as tumors and autoimmune diseases.The increasing number of infiltrating Tregs indicated poor prognosis in various tumors,such as breast cancer,lung cancer,and rectal cancer.IL-35,mainly produced by Tregs,induced T cells into IL-35-producing regulatory T cells(iTr-35)to pronounce immunosuppressive functions.Our study found that IL-35 could be expressed by breast cancer cells(BCCs),and high expression of breast cancer cells-dernved IL-35 could lead to poor prognosis.Weather BCCs derived-IL-35 performed the same function as IL-35 derived from immune cells?Could BCCs derived-IL-35 induce surface inhibitory receptors(IRs)and change cytokine profiles of T cells?All of these was not clear.This study aimed to investigate the effects of BCCs derived-IL-35 on T cell proliferation,iTr-35 differentiation,surface IRs and cytokine profiles of T cell and signaling pathways involved in this procession.Methods:1.The mRNA expression level of two subunits of IL-35,EBIB and p35,were detected by qRT-PCR in four breast cancer cell lines.The expression level of secretory IL-35 in these four breast cancer cell lines were detected by ELISA.The mRNA expression level of EBIB and p35 and the expression level of secretory IL-35 were detected after knocking out two subunits of IL-35 by CRISPR/CAS9 method.2.The effect of BCC derived-IL-35 on T cell proliferation was detected by CCK-8 method.3.The expressions level of EBI3 and p35 in T cells induced by derived-IL-35 were detected by qRT-PCR,Western blotting and flow cytometry.4.Cytokine production of IL-1β,IL-4,IL-6,IL-10,IL-12p70,IL-17a,TNF-a and IFN-γ were determined by the commercially available Human High Sensitivity Panel.5.The surface IRs of T cell induced by BCC derived-IL-35 were detected by flow cytometry.6.The expression of singnal molecules STAT1/STAT3/STAT4 in T cell induced by BCCs derived-IL-35 were detected by Western blotting.Results:1.The two subunits of IL-35 were detectable in all of four cell lines MDA-MB-231(231),MCF-7,T47D and SKBR3,and the higher IL-35 level was observed in 231 and MCF-7 cells.2.Both supernatant(SN)from 231 and MCF-7 cells markedly suppressed the proliferation of T cells and IL-35-blocking could partially reverse these effects.3.Compared with control,conventional T cells(Tconv)activated with anti-CD3/CD28 in the presence of BCCs’ SN simultaneously upregulated the expression of IL-35 at both mRNA and protein levels.Single-cell analysis by intracellular cytokine staining also suggested that treatment with BCCs’ SN or rhIL-35 induced the expression of IL-35 in T cells.4.Secretion of IL-10 and IL-12p70 was obviously upregulated,whereas the secretion of Thl-type cytokine IFN-y and Thl7-type cytokine IL-17 was markedly depressed in both IL-35 groups and BCCs’ SN-treated groups.5.The percentage of CD73+ T cells is upregulated in BCCs’ SN-treated groups.However,BCCs’ SN treatment had no effect in the induction of LAG3 and CTLA-4.6.Increased p-STAT1 and p-STAT3 expressions were observed in IL-35-treated groups and BCCs’ SN-treated groups compared with control Tconv cells,and the addition of IL-35-blocking mAb in BCCs’ SN-treated groups could partially reverse these effects.Conclusions:Breast cancer cells secreted IL-35,and these tumor-derived IL-35 inhibited T cell proliferation,induced suppressive iTr-35 cells,upregulated IRs expression and inbitory cytokines secretion of T cells by activating T cell STAT1/STST3 signaling pathway.This study clearly illustrated the biological mechanism by which breast tumor-derived IL-35 induced the immunosuppressive microenvironment to promote tumor progression,providing a potential immunotherapeutic target for breast cancer.Chapter Ⅲ Exosomes from IL-35-stimulated breast cancer cells facilitate tumor growth by polarizing M2 MacrophagesObjective:Tumor-associated macrophages(TAM),mainly expressing markers of suppressive M2 macrophages,was significantly correlated with the poor prognosis of virous tumors.Exosomes(Exo),40-150 nm nanovesicles derived from multivesicular endosomes and secreted by the cells,played an important role in cell development,immune cell differentiation,and tumor development.Exosomes secreted by tumor cells can promote tumor invasion,organotropic metastasis,fibroblast activation,and inhibition of T cell function.However,it is unclear whether exosomes derived from breast cancer cells effect the differention of TAM.Our previous study found that there was a high level of IL-35 stress in the TME.Whether exosomes from breast cancer cells stimulated by IL-35 are involved in the polarization of M2 macrophages?The mechinisms involved and the role of induced M2 macrophages in tumor development are unknown.The aim of this chapter was to explore the role of exosomes from IL-35-stimulated breast cancer cells in M2 polarization,and to uncover the role of exosome-induced M2 polarization in tumor development.Methods:1.The morphology and size of tumor exosomes were measured by transmission electron microscopy(TEM)and nanosized zs90.The exosome surface markers HSP90,TSG101 and CD9 were detected by Western blotting.2.To detect the direct transfer of tumor exosomes into macrophages,exosomes were labeled using the DiI and incubated with macrophages for 24h,and then the fluorescence signals were detected by Confocal microscopy.3.The macrophages were treated with breast cancer exosomes or IL-35-breast cancer exosomes.Then,observed the morphology of macrophage,and detected M1 marker HLA-DR and IL-6,M2 marker Arg-1,CD206 by qRT-PCR and flow cytometry.4.Established breast cancer mouse model,pretreating mouse with breast cancer exosomes and IL-35-breast cancer exosomes for 2 weeks followed by subcutaniously injection of breast tumor cells.Measured the size of tumor every other day.Detected the expression of CD68 and CD 163 by immunofluorescence chemistry method.5.Detected the expression of moleculers involved in STAT3,JNK,p38,ERK,p65 and IκB singnal pathway by Western blotting.Results:1.The exosomes from breast cancer cells were about 110 nm in diameter,cup-shaped,and positive for exosomal markers HSP90,TSG101 and CD9.2.In the coculture of breast cancer exosomes and macrophages,directional transfer of exosomes to macrophages was observed.Most of the red fluorescence was distributed in the cytoplasm.3.More spindle-shaped M2 macrophages were observed after the treatment of exosomes,especially IL-35-breast cancer exosomes.Futhermore,the expression of Arg-1 and CD206 in macrophages were upregulated by IL-35-breast cancer exosomes.Flow cytometry results showed that exosomes induced macrophages to express higher levels of the M2 biomarker CD206 and IL-35-breast cancer exosomes had stronger effects than breast cancer exosomes.4.In vivo study showed that,more CD68+CD163+M2 macrophages and larger tumor volume were observed in exosomes groups,compared with control group.Furthermore,IL-35-breast cancer exosomes were more capable of inducing CD68+CD163+M2 macrophages and promoting tumor growth than 231exo.5.The exosomes could activate the STAT3,JNK,and p38 pathways of macrophages,and the effects of IL-35-breast cancer cell exosome was much stronger.Conclusions:Exosomes from IL-35-stimulated breast tumor cells could induce a strong M2 phenotype by activating macrophage STAT3 and JNK/p38 signaling molecules,and leading to immune escape,which contributed tumor malignant progression.This work clarified the tumor-promoting function of IL-35 in the tumor microenvironment,and elucidated the new mechanism of IL-35 induced immune escape,providing theoretical basis for immunotherapy of breast cancer.