CCL5-Mediated Th2 Immune Polarization Promotes Metastasis In Luminal Breast Cancer

Luminal breast cancer is typically estrogen receptors positive?ER⁺? and represents the largest proportion of women with breast cancer?around 70%?.Although Luminal breast cancer patients respond to endocrine therapy,those with high ER? levels inevitably develop early and late relapses and metastasis.Discovery of new therapeutic targets for the treatment of these patients represents a big unmet medical need.In the present study,we investigated the role of CCL5 in tumorigenesis and matastasis of Luminal breast cancer.We used MMTV-PyMT transgenic mouse line and generated PyMT mice bearing homozygous null CCL5(CCL5^-/- PyMT).Compared to CCL5^+/+ PyMT mice,the tumorigenesis,pulmonary metastasis and circulating tumor cell of CCL5^-/- PyMT mice decreased significantly.The specific transcriptional factors and effector cytokines indicative of Th1,Th2,Th17 and Treg response were examined by RT-PCR,ELISA.GATA3?Th2?expression was dramatically decreased in CD4⁺ T cells from both TILs and LNs of CCL5^-/- PyMT mice.Intracellular flow cytometry revealed that the percentage of IL4⁺ CD4⁺ T cells?Th2? was markedly decreased in CCL5^-/- PyMT mice.Blocking of CD4⁺ T cells resulted in a significantly reduced number of pulmonary metastatic foci in CCL5^+/+ PyMT mice compared to the controls,and the difference of pulmonary metastasis and circulating tumor cell induced by CCL5 deficiency disappeared,indicating that Th2 CD4⁺ T cells were crucial for pulmonary metastasis of Luminal breast cancer.There is on great difference of invasion acini of organoid between CCL5^+/+ mammary epithelial cell and CCL5^-/- mammary epithelial cell.TAM and CD4⁺ T cells from CCL5^+/+ PyMT promotes invasion acini of organoids.The EMT rate of organoid from CCL5^-/- mammary epithelial cell is lower than that from CCL5^+/+ PyMT,indicating that CD4⁺ T cells promotes EMT through TAM,and further promotes metastasis of Luminal breast cancer.Th2 CD4⁺ T cells induced from na?ve CD4⁺ T cells of CCL5^-/- PyMT were lower than that of CCL5^+/+ PyMT,but addition of rm CCL5 reduce the difference,indicating that CCL5 promotes Th2 differentiation.CCL5 was found to promote Gfi1 expression through RT-PCR,Western Blotting and so on.Gfi1 was overexpressed by lentivirus and then infects na?ve CD4⁺ T cells of CCL5^-/- PyMT.The percentage of Th2 CD4⁺ T cells increased significantly after Gfi1 overexoression.The functional receptor of CCL5 in the progress of inducing Th2 differentiation is CCR3,but not CCR1 or CCR5,identified by RT-PCR,immunofluorescence and receptor inhibitor addition.We verify the results in clinical samples of Luminal breast cancer.The expression of both CCL5 and IL4 were significantly higher in more aggressive tumors than less malignant tumors or the normal breast tissue,and there is a positive correlation between CCL5 and IL4 expression.Moreover,serum CCL5 levels and numbers of Th2 CD4⁺ T cells in peripheral blood are positively correlated with aggressiveness of human Luminal breast cancer.In summary,we investigated the role of CCL5 in tumorigenesis and matastasis of Luminal breast cancer.We found that CCL5 promotes Gfi1 expression through binding CCR3 on CD4⁺ T cells,and then induce Th2 CD4⁺ T cell polarization.Th2 CD4⁺ T cells promote EMT through TAM,and then promote metastasis of Luminal breast cancer.Moreover,we found that CCL5 expression in tumor tissue and serum are positively correlated with aggressiveness of human Luminal breast cancer,indicating that CCL5 can be a therapeutic target for the treatment of luminal breast cancer,but aiso serum CCL5 level might be new noninvasive prognostic indicators for monitoring the progression of luminal breast cancer.