Mechanism Of Ruyiping Formula Inhibit Breast Cancer Lung Metastasis By Regulating The Polarization Of Tumor Associated Macrophages

Objective:Based on the role of tumor associated macrophages in the microenvironment of tumor metastasis.Explored the mechanism of Ruyiping Formula(RYP)to inhibit the polarization of tumor associated macrophages and inhibit lung metastasis of breast cancer.Methods:1.The 4T1 cell line was used to establish the mouse model of breast cancer lung metastasis.The inhibitory effect of RYP on M2 type macrophages in the model of breast cancer was detected by flow cytometry,transmission electron microscopy,HE staining and Western blot.2.Human breast cancer cell line MD-MB-231 and mouse breast cancer cell line 4T1 were selected to investigate the effects of RYP(Euctus oryzae,Zedoary turmeric,honeycomb,Zalea augustinii and Semen coicis)on the malignant biological behavior of different breast cancer cells through MTT,wound healing experiment,Transwell chamber experiment and apoptosis experiment.To determine the inhibitory ability of breast tumor proliferation,invasion and metastasis and promote cell apoptosis effect of RYP.3.M0,M1 and M2 conditional media were extracted to intervene human breast cancer cells MDA-MB-231 and mouse breast cancer cells 4T1.The effects of different phenotypes of macrophage conditional media on the malignant biological behavior of different breast cancer cells were evaluated.At the same time,M0,M1 and M2 macrophages were directly co-cultured with MDA-MB-231 and 4T1 to observe the change of M2 specific macrophage protein CD206,and to evaluate the effect of RYP on inhibiting M2 polarization.Results:1.RYP could inhibit the tumor size,the number of lung metastatic nodules and the weight of spleen(P < 0.05 or P < 0.01).Flow cytometry and Western-blot immunoblotting showed that RYP increased the expression of M1 type macrophages in the microenvironment of tumor metastasis by regulating the Stat signaling pathway,and otherwise decreased the expression of M2 type macrophages.2.Through MTT cell proliferation assay,wound healing assay,Transwell chamber assay and apoptosis assay,it was found that the RYP could effectively inhibit the proliferation,migration,invasion and metastasis ability of breast cancer cells 4T1 and MDA-MB-231.It promoted the early and late apoptosis of breast cancer cells(P < 0.05 or P < 0.01).3.Different surface macrophages were induced by primary bone marrow cells extracted from mice and verified by flow cytometry,RT-PCR and immunofluorescence.The induction ratio of M0,M1 and M2 could reach more than 80%.After co-culture with different breast cancer cell lines,it was found that M2 type macrophages were positively correlated with the invasion and metastasis ability of breast cancer cells,while the expression of M2 type macrophages was decreased(P < 0.05 or P < 0.01),thereby reducing the invasion and metastasis ability of breast cancer cells.In the mechanism study,it was found that M1-type macrophages were positively correlated with Stat1,while M2 type macrophages were positively correlated with Stat6(P < 0.05 or P < 0.01).Conclusions:RYP can inhibit lung metastasis of breast cancer by inhibiting the polarization of related macrophages M2 in the tumor microenvironment.In vitro,RYP inhibits the biologic behavior of different breast cancer cells.At the same time,the invasion and metastasis ability of M2 conditioned medium to breast cancer cells and the expression of M2 specific protein were inhibited.In vivo,RYP can reduce the expression of M2 specific protein in tumor tissues and lung metastases,and play a role in anti-lung metastasis of breast cancer.