Study On Clinical Significance Of Myeloid-Derived Suppressor Cells In Breast Cancer Patients And Immunosuppression Mechanisms

Objective:To examine clinical significance of myeloid-derived suppressor cells(MDSC)in breast cancer patients,and induce MDSC in vitro by cocultureing tumour cells and normal human CD33+ myeloid cells to study the STAT3/IDO pathway in MDSC-dependent immunosuppression.Methods:35 breast cancer samples were collected from Tianjin Cancer Hospital.The proportion of MDSC(Linl-CD33+CD13+CD14-CD15-)were determined by flow cytometry.Then,the correlation of MDSC proportion and patients’ clinical characteristics was analyzed.CD33+ cells of healthy individuals peripheral blood were collected by the magnetic cell sorting system.CD33+ cells were cultured alone as negative control.CD33+ cells and MDA-MB-231 were cocultured together to induce MDSC in vitro.After 2 days,CD33+ cells were harvested to do phenotypes examination.Autologous T cells were purified by the magnetic cell sorting system.After coculture T cells with normal CD33+ cells or MDSC overnight,Annexin V staining method was applied to detect apoptosis of T cells and study the IDO-dependent MDSC immunosuppression.Supernatant of various T cells was collected to detect secreting of multiple cytokines,including IL-4,IL-10,IL-12,IFN-Y,TGF-β.Expression of IDO,STAT3 mRNA and protein,as well as p-STAT3 protein were respectively detected by Real-time quantitative PCR assay and Western blotting method.Results:The level of MDSC in breast cancer patients was correlated with the clinical stage and the number of metastasis lymph nodes.The proportion significantly increased in stage Ⅲ breast cancer patients of(11.70±7.85)%compared to stage I/II patients of(5.32±4.59)%(P<0.05).Patients with more than 3 metastasis lymph nodes had a higher proportion of MDSC.After coculturing breast cancer cell line MDA-MB-231 with normal human CD33+ cells for 2 days,a population of Linl-CD33+CD13+CD14-CD15-MDSC was observed which could induce apoptosis of autologous T cells.Apoptosis rate of T cells stimulated by MDSC was(38.20± 1.70)%,much higher than that of T cells alone and T cells stimulated by normal CD33+ cells,(6.10±1.76)%and(18.53±2.58)%respectively,meanwhile 1-MT group had a lower level of(22.52±2.48)%(P<0.05).MDSC induced T cells tended to secret a higher level of TGF-β,IL-10 and a lower level of IFN-γ,when compared with control groups.There was no statistic differences of IL-4,IL-12 secretion(P>0.05).MDSC induced in vitro expressed high level of IDO mRNA,STAT3 mRNA and IDO,STAT3,p-STAT3 protein.In addition,JAK2-STAT3 inhibitor JSI-124 treated MDSC showed a dramatic decrease of IDO expression.Conclusion:Our study thus implied that MDSC may be significant for lymph nodes metastatis and disease progression of breast cancer patients.Tumour cells could induce MDSC with immunosuppressive activity in non-membrane dependent manner.Upregulation of IDO expression resulted from increasing STAT3 phosphorylation may be considered as an important mechanism of MDSC-dependent immunosuppression.