| Objective:Triple-negative breast cancer(TNBC)is a particular type of breast cancer characterized by strong invasion,easy metastasis and poor prognosis.Since TNBC dose not express estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER2),it is not sensitive to endocrine therapy and HER2-targeted therapy.Currently,surgery and chemotherapy are the main treatment methods for TNBC patients,but the prognosis is poor.Therefore,new treatment options are needed to improve the clinical efficacy of TNBC.Immunotherapy may be a more meaningful treatment for TNBC.The anti-PD-L1inhibitor atezolizumab or the anti-PD-1 inhibitor pembrolizumab in combination with chemotherapy have been approved in the United States to treat patients with metastatic PD-L1 positive TNBC.Furthermore,immune cells infiltration in the tumor microenvironment play a critical role in TNBC progression and metastasis.Tumor infiltrating lymphocytes(TILs)are significantly associated with distant recurrence and overall survival of TNBC.In addition,tumor-associated macrophages(TAMs)and myeloid-derived suppressor cells(MDSCs)can affect TNBC growth by secreting effective proteins or regulating T cell functions in the process of TNBC development.Group 2 innate lymphoid cells(ILC2s)are innate immune cells that reside mainly in the lung,skin,and gut mucosal surfaces.Following activation by their classical activators IL-33,IL-25,and thymic stromal lymphopoietin(TSLP),ILC2s can secrete a great quantity of type 2 cytokines,including IL-4,IL-5,and IL-13,to contribute to Th2-type immune response-mediated diseases.In recent years,studies have found that ILC2s also play a key role in a variety of tumors,ILC2s can inhibit tumor progression and metastasis in mouse malignant melanoma,subcutaneous lymphoma and lung cancer;while ILC2s promoted tumor progression in prostate cancer,bladder cancer,acute promyelocytic leukemia.The role of ILC2s in cancer appears to depend on the specific tumor microenvironment.In different microenvironments,ILC2s exhibit anti-tumor or pro-tumor effects by producing different cytokines,recruiting different types of immune cells and expressing immune-related surface molecules,and participate in the occurrence and development of cancer.The frequency of ILC2s in human malignant breast tissue is higher than that in benign breast tissue.In addition,IL-33 can accelerate the growth and distant metastasis of 4T1 tumors in mice,and significantly increase the amount of IL-5~+ILC2s and IL-13~+ILC2s in tumor tissues of tumor-bearing mice,suggestting that activated ILC2s may be involved in the progression of breast cancer.However,the role and potential mechanism of ILC2s in the distal metastasis of TNBC,especially in lung metastasis,which is highly likely to occur and has a high mortality rate due to difficult treatment,remains unclear.In this study,the role of ILC2s in TNBC lung metastasis was investigated by using the experimental mouse TNBC lung metastasis model,in the hope of providing experimental evidence to elucidate the mechanism of tumor occurrence and development and clinical immunological treatment.Method:1.6-8-week-old BALB/c female mice were inoculated intravenously with 2×1054T1 cells via the tail vein to establish the mouse model of TNBC lung metastasis.The frequencies and absolute numbers of total ILC2s,IL-13-producing ILC2s and IL-5-producing ILC2s in the lungs of tumor-bearing mice were detected by flow cytometry on days 0,7,and 14 after 4T1 cell implantation;ILC2s were isolated from lungs of tumor-bearing mice by immunomagnetic bead sorting,the relative mRNA expression of IL-13 and IL-5 in pulmonary ILC2s was determined by qRT-PCR.2.ILC2s were isolated from the lungs of normal BALB/c mice by immunomagnetic bead sorting.A total of 2×10~4 ILC2s were injected via the tail vein into each mouse 2h before 4T1 implantation.On day 14,after 4T1 cell implantation,HE staining and Ki67immunohistochemical staining were performed to compare the numbers of metastatic nodules,the percentages of metastatic area and Ki67-positive tumor cells in the lungs of ILC2-transferred tumor-bearing mice and non-ILC2-transferred tumor-bearing mice,the survival of the mice was monitored.3.qRT-PCR and ELISA were performed to detect the mRNA and protein levels of IL-13 and IL-5 in the lungs of ILC2-transferred tumor-bearing mice and non-ILC2-transferred tumor-bearing mice;mice were treated intranasally with anti-IL-13m Ab to neutralize IL-13,HE staining and Ki67 immunohistochemical staining were performed to compare the numbers of metastatic nodules,the percentages of metastatic area and Ki67-positive tumor cells in the lungs of antibody-treated ILC2-transferred tumor-bearing mice and non-antibody-treated ILC2-transferred tumor-bearing mice,the survival of the mice was monitored.4.qRT-PCR and Western blot were performed to detect IL-13Ra1 mRNA and protein levels of pulmonary MDSCs in ILC2-transferred tumor-bearing mice and non-ILC2-transferred tumor-bearing mice;the percentages and numbers of MDSCs in the lungs of model mice were assayed by flow cytometry,the mRNA expression levels of Arg1,iNOS,TGF-βand IL-10 in pulmonary MDSCs in each group were determined by qRT-PCR.5.On day 14,after 4T1 cell implantation,ILC2s and MDSCs were sorted from the lungs of tumor-bearing mice by immunomagnetic bead sorting,they were co-cultured by transwell in vitro in the presence or absence of an anti-IL-13 m Ab.The mRNA levels of Arg1,iNOS,TGF-βand IL-10 in MDSCs were determined by qRT-PCR,and the protein levels of Arg1,iNOS,TGF-βand IL-10 in the supernatant of MDSCs were determined by ELISA.6.Mice were treated intranasally with anti-Gr-1 m Ab to deplete MDSCs.On day 14,after 4T1 cell implantation,HE staining and Ki67 immunohistochemical staining were performed to compare the numbers of metastatic nodules,the percentages of metastatic area and Ki67-positive tumor cells in the lungs of antibody-treated ILC2-transferred tumor-bearing mice and non-antibody-treated ILC2-transferred tumor-bearing mice,the survival of the mice was monitored;the numbers of CD4~+T cells,CD8~+T cells,IFN-γ~+CD4~+T cells,IFN-γ~+CD8~+T cells and Tregs were assayed by flow cytometry.7.The mRNA expressions of IL-33,IL-25 and TSLP in lungs were determined by qRT-PCR on days 0,7,and 14 after 4T1 cell implantation.Mice were treated intranasally with anti-TSLP m Ab to neutralize TSLP in tumor-bearing mice.On day 14,after 4T1 cell implantation,the percentages and absolute numbers of ILC2s in the lungs of tumor-bearing mice were determined by flow cytometry,ILC2s were isolated from lungs of tumor-bearing mice by immunomagnetic bead sorting,the relative mRNA expression of IL-13 and IL-5 in pulmonary ILC2s was determined by qRT-PCR.Results:1.The numbers of total ILC2s and IL-13-producing ILC2s were increased significantly in the lungs of tumor-bearing mice:compared with normal controls,the percentages and absolute numbers of total ILC2s and IL-13-producing ILC2s were increased significantly in the lungs of tumor-bearing mice,especially at day 14 after 4T1implantation,while the numbers of IL-5-producing ILC2s were remarkably unchanged;the relative mRNA expression of IL-13 and IL-5 in pulmonary ILC2s was also enhanced at the indicated time points.These findings suggest that pulmonary ILC2s may be related to the process of TNBC lung metastasis.2.Adoptive transfer of pulmonary ILC2s promotes 4T1 lung metastasis:compared with the control group,the numbers of ILC2s in the lungs of ILC2-transferred mice were significantly increased,indicating that the adoptive transfer experiment was successful.Compared with non-ILC2-transferred tumor-bearing mice,the numbers of metastatic nodules,the percentages of metastatic area and ki67-positive tumor cells in the lungs of ILC2-transferred tumor-bearing mice were increased,while the survival rates were decreased.These results suggest that pulmonary ILC2s may accelerate TNBC lung metastasis.3.ILC2-accelerated TNBC lung metastasis depends on ILC2-derived IL-13:compared with non-ILC2-transferred tumor-bearing mice,the mRNA and protein levels of IL-13 in the lungs of ILC2-transferred tumor-bearing mice were increased,but the IL-5 levels were remarkably unchanged;blocking IL-13 significantly inhibited the promoting effect of ILC2s on TNBC lung metastasis,the numbers of metastatic nodules,the percentages of metastatic area and ki67-positive tumor cells increased due to ILC2s implantation decreased significantly after IL-13 blockade,while the survival rates of tumor-bearing mice increased.These results suggest that ILC2-derived IL-13 is indispensable for the promotion of 4T1 lung metastasis by ILC2s.4.The function of pulmonary MDSCs is regulated by ILC2-derived IL-13:compared with non-ILC2-transferred tumor-bearing mice,the numbers of pulmonary MDSCs and IL-13Ra1 expression in pulmonary MDSCs were enhanced significantly by in vivo ILC2 transfer.A significant increase in the relative mRNA expression of Arg1,iNOS,TGF-βand IL-10 in pulmonary MDSCs was observed in ILC2-transferred tumor-bearing mice compared with non-ILC2-transferred tumor-bearing mice.Blocking IL-13 significantly reduced ILC2-elevated expansion and activation of pulmonary MDSCs,the numbers of pulmonary MDSCs and the mRNA levels of Arg1,iNOS,TGF-βand IL-10 in pulmonary MDSCs increased due to ILC2s implantation decreased significantly after IL-13 blockade.These results suggest that ILC2-derived IL-13 might be necessary for ILC2-augmented MDSC activation in the experimental metastatic mouse 4T1 tumor model.5.In vitro experiments determine the effect of ILC2-derived IL-13 on MDSC proliferation and activation:the numbers of MDSCs and the mRNA expression levels of Arg1,iNOS,TGF-βand IL-10 in MDSCs were increased significantly by coculture with ILC2s,the protein levels of Arg1,iNOS,TGF-βand IL-10 in supernatant of MDSCs were also significantly increased.However,in the presence of anti-IL-13 m Ab,the numbers of MDSCs and the levels of Arg1,iNOS,TGF-βand IL-10 in MDSCs were decreased.In vitro experiments further confirmed that ILC2-derived IL-13 may contribute to the expansion and activation of MDSCs in the lungs of experimental metastatic 4T1 tumor bearing mice.6.ILC2-regulated MDSCs promote TNBC lung metastasis by inhibiting CD4~+T cells and CD8~+T cells and inducing Tregs:compared with non-antibody-treated ILC2-transferred tumor-bearing mice,the numbers of metastatic nodules,the percentages of metastatic area and ki67-positive tumor cells in the lungs of anti-Gr-1 antibody-treated ILC2-transferred tumor-bearing mice were decreased,while the survival rates were increased.Moreover,depletion of MDSCs enhanced the numbers of CD4~+T cells,CD8~+T cells,IFN-γ~+CD4~+T cells and IFN-γ~+CD8~+T cells,but diminished the number of Tregs in the lungs of ILC2-transferred tumor-bearing mice.These results suggest that ILC2-regulated MDSCs could promote TNBC lung metastasis by inhibiting CD4~+T cells and CD8~+T cells and inducing Tregs.7.TSLP up-regulated the number and activity of ILC2s in lung of tumor-bearing mice:the expression level of TSLP mRNA in the lungs of mice increased significantly on day 14 after 4T1 implantation,while IL-33 and IL-25 were remarkably unchanged;blocking TSLP in vivo with anti-TSLP m Ab,the percentages and absolute numbers of ILC2s were decreased in the lungs of tumor-bearing mice,and the mRNA expressions of IL-13 in pulmonary ILC2s were decreased.These results showed that TSLP played a key role in the proliferation and activation of ILC2s in TNBC lung metastasis.Conclusion:In the murine TNBC lung metastasis model,TSLP in tumor microenvironment promoted the activation of pulmonary ILC2s,and the activated ILC2s promoted TNBC lung metastasis via the ILC2-derived IL-13-activated MDSC pathway. |
PDF Full Text Request