Breast Cancer Cell Exosomes Differentiate Bone Marrow Cells Into Myeloid-derived Suppressor Cells And Its Mechanisms

Background and aims:Breast cancer is one of the most common malignant tumors in women.In recent years,the mean age of breast cancer patients is getting younger and younger,which has seriously affected women’s health.At present,surgery,radiotherapy and chemotherapy are the main means of breast cancer treatment,but it is difficult to achieve the cure effect.Previous studies found that there are higher levels of myeloid-derived suppressor cells(MDSCs)in patients with metastatic diseases such as breast cancer,and the increase of MDSCs level in tumor microenvironment is closely related to the resistance therapy and poor prognosis of metastatic breast cancer.Tumor cells produce a large number of exosomes that provide an immunosuppressive microenvironment for tumor growth by influencing immune cells in the tumor microenvironment.However,the effects of tumor-derived exosomes on MDSCs and its mechanisms are still unclear.This study was designed to investigate the effects of breast cancer cell exosomes on the generation and expansion of MDSCs,and to explore its mechanism,so as to provide a theoretical and experimental basis for targeting MDSCs levels in anti-breast cancer treatment.Methods:1.Infiltration of immune cells in a breast cancer model:The orthotopic model of mouse breast cancer was established and flow cytometry was used to detect the number of MDSCs and CD8~+T lymphocytes in bone marrow and spleen.2.The effect of breast cancer cell-derived exosomes on the differentiation of bone marrow-derived macrophages and bone marrow cells:1)The supernatant of breast cancer cells was collected,and the exosomes were prepared by ultracentrifugation or exosome separation kit.The morphology of the exosomes was observed by transmission electron microscopy,the surface molecules were detected by Western blot,and protein concentration was determined by BCA protein quantitative method.2)Bone marrow-derived macrophages were treated with breast cancer cell-derived exosomes for 24 hours,and the changes of related genes were screened by gene chip technology and verified by q-PCR.3)The differentiation of bone marrow cells was stimulated by breast cancer cell-derived exosomes in vitro,and the changes of cell morphology were observed.The expression level of MDSCs-related molecules and chemokine receptors were detected by q-PCR and Western blot,and the numbers of MDSCs and the expression levels of chemokine receptors were further detected by flow cytometry.3.Bone marrow cell-mediated T cell suppression induced by breast cancer cell-derived exosomes:CD3~+T lymphocytes were isolated from mouse spleen cells using magnetic bead sorbent kit,labeled with CFSE and co-cultured with bone marrow cells induced by exosomes in a 1:1 ratio for 72 hours,and T cell proliferation was detected by flow cytometry.4.The effect of breast cancer cell-derived exosomes on immune cells in vivo:Breast cancer cell-derived exosomes were injected into mice via tail vein,and the spleen and bone marrow cells were obtained 24 hours later.The numbers of MDSCs and CD8~+T lymphocytes were detected by flow cytometry.5.The mechanism of MDSCs production and expansion induced by breast cancer cell-derived exosomes:1)Bone marrow cells were treated with chemokine receptor inhibitor AMD3100,ligand CXCL12 and exosomes.The expression levels of MDSCs-related molecules were detected by q-PCR and Western blot to determine whether CXCR4 affected the generation of MDSCs.2)Bone marrow cells were treated with breast cancer cell derived-exosomes for 24 hours.In order to explore the related pathways involved in MDSCs expansion induced by breast cancer cell-derived exosomes,western blot assay was used to detect the expression levels of P-STAT3,PCNA and Bax.Results:1.The numbers of MDSCs(mMDSCs and gMDSCs)in bone marrow and spleen of breast cancer-bearing mice were significantly increased compared to the control mice.In contrast,the CD8~+T lymphocytes in spleen were significantly reduced.2.The macrophages treated with breast cancer cell derived-exosomes showed a mixed M1/M2 phenotype.The expressions of CXCR4,CX3CR1 and immune co-stimulatory molecule CD28 were down-regulated,and inflammatory factors such as Arg-1,IL-6,IL-10 were up-regulated.The morphology of bone marrow cells treated with breast cancer cell-derived exosomes was changed significantly,the numbers of MDSCs were significantly increased.The expressions of MDSCs-related functional proteins Arg-1,iNOS and IL-6,IL-10 were up-regulated,CXCR4 was down-regulated.3.Bone marrow cells treated by breast cancer cell derived-exosomes inhibited the proliferation and activation of T cells compared with the PBS control group.4.The numbers of MDSCs in the bone marrow treated with exosomes were increased,and the CD8~+T lymphocytes in the spleen were significantly decreased,and the expression of CXCR4 was down-regulated compared wth the PBS control group.5.CXCL12,CXCR4 ligand,rescued the althernations of the expression levels of MDSCs-related functional proteins Arg-1,iNOS and inflammatory factors IL-6,IL-10induced by breast cancer cell-derived exosomes,and AMD3100,CXCR4 inhibitor,could not cause the up-regulation of MDSCs-related molecules.Breast cancer-derived exosomes increased the phosphorylation level of STAT3 and inhibited the expressions of PCNA and Bax in bone marrow cells.Conclusion:Breast cancer cell-derived exosomes promote the differentiation of bone marrow cells into MDSCs,induce the production and proliferation of MDSCs,and inhibit CD8~+T lymphocytes proliferation and activation,which promote the immuno-suppression microenvironment in breast cancer.MDSCs production induced by breast cancer cell-derived exosomes is partly related to the down-regulation of CXCR4.In addition,the activation of STAT3 pathway inhibits the proliferation and apoptosis of bone marrow cells and promotes their differentiation into MDSCs.