XIAOPI Formula Inhibits Breast Cancer Stem Cells via Suppressing Tumor-Associated Macrophages/C-X-C Motif Chemokine Ligand 1 Pathway

Shengqi Wang, Xiaoyan Liu, Renlun Huang, Yifeng Zheng, Neng Wang, Bowen Yang, Honglin Situ, Yi Lin, Zhiyu Wang, Shengqi Wang, Xiaoyan Liu, Renlun Huang, Yifeng Zheng, Neng Wang, Bowen Yang, Honglin Situ, Yi Lin, Zhiyu Wang

Abstract

Macrophages are the most abundant stromal cells associated with the host immune system in multiple malignancies including breast cancer. With proven clinical efficacy and no noticeable adverse effects, XIAOPI formula (XPS) has been approved for breast hyperplasia treatment by the State Food and Drug Administration of China (SFDA) in 2018. The existing knowledge about the anti-breast cancer activities and mechanisms of XPS has been very limited. The present study aimed to investigate whether XPS could exert an anti-breast cancer effect by regulating tumor-associated macrophages (TAMs) in tumor microenvironment. Herein, breast cancer cells and TAMs were co-cultured using the transwell co-culture system to simulate the coexistence of them. XPS could significantly inhibit the proliferation, colony formation, breast cancer stem cells (CSCs) subpopulation, mammosphere formation abilities as well as stemness-related genes expression in both human and mouse breast cancer cells in the co-culture system. Additionally, XPS could suppress M2 phenotype polarization as well as C-X-C motif chemokine ligand 1 (CXCL1) expression and secretion of TAMs. Notably, further mechanistic explorations verified TAMs/CXCL1 as the critical target of XPS in inhibiting breast CSCs self-renewal in the co-culture system as the exogenous CXCL1 administration could abrogate the inhibitory effect of XPS on breast CSCs self-renewal. More importantly, XPS significantly inhibited mammary tumor growth, breast CSCs subpopulation, and TAMs/CXCL1 activity in mouse 4T1-Luc xenografts in vivo without any detectable side effects. Taken together, this study not only uncovers the immunomodulatory mechanism of XPS in treating breast cancer but also sheds novel insights into TAMs/CXCL1 as a potential molecular target for breast CSCs elimination.

Keywords: C-X-C motif chemokine ligand 1; M2 phenotype polarization; XIAOPI formula; breast cancer stem cells; tumor-associated macrophages.

Copyright © 2019 Wang, Liu, Huang, Zheng, Wang, Yang, Situ, Lin and Wang.

Figures

Figure 1
Figure 1
XIAOPI formula (XPS) significantly inhibits the proliferation and colony formation abilities of breast cancer cells in the co-culture system. (A) The cytotoxicity of XPS in nonmalignant mammary epithelial cell line MCF-10A and breast cancer cell lines MDA-MB-231 and 4T1 was investigated by MTT method. (B) XPS (100∼500 μg/ml) could dramatically inhibit the colony formation abilities of breast cancer cell lines MDA-MB-231 and 4T1. (C) Both human and mouse macrophages were successfully induced into M2 phenotype TAMs when they were treated with the conditional medium (CM) of breast cancer cells for 48 h. (D) XPS treatment for 48 h could dramatically inhibit the proliferation of breast cancer cell lines MDA-MB-231 and 4T1 in the co-culture system. The IC50 values were calculated by Bliss method. (E) XPS could significantly suppress the colony formation abilities of breast cancer cell lines MDA-MB-231 and 4T1 in the co-culture system. All values are presented as the mean ± SD, n = 3, **P < 0.05.
Figure 2
Figure 2
XIAOPI formula (XPS) inhibits the self-renewal activity of breast cancer stem cells (CSCs) in the breast cancer cells and tumor-associated macrophages (TAMs) co-culture system. (A) XPS treatment for 48 h could significantly decrease the proportions of ALDH+ subpopulations in both the MDA-MB-231 cells and 4T1 cells when they were co-cultured with TAMs. (B) XPS treatment for 48 h significantly decreased the mammospheres’ numbers of the co-cultured breast cancer cells. Scale bar = 100 μm. (C) XPS treatment for 48 h strongly attenuated the mRNA expression levels of β-catenin, OCT4, and Nanog in the co-cultured breast cancer cells. (D) XPS treatment for 48 h could dramatically attenuate β-catenin protein expression levels in the co-cultured breast cancer cells. All values are presented as the mean ± SD, n = 3, **P < 0.05.
Figure 3
Figure 3
XIAOPI formula (XPS) inhibits M2 phenotype polarization, C-X-C motif chemokine ligand 1 (CXCL1) expression and secretion of tumor-associated macrophages (TAMs). (A) MTT assay indicated that XPS (100∼500 μg/ml) could strongly inhibit the proliferation of Raw264.7-derived TAMs while exhibiting no obvious cytotoxicity in Thp1-derived TAMs. (B) XPS treatment for 48 h could dramatically inhibit M2 phenotype polarization of TAMs induced by CM of breast cancer cells. (C) Elisa assay indicated that XPS treatment for 48 h could significantly inhibit CXCL1 secretion from TAMs in a dose-dependent manner. (D–E) Western blot and QPCR results further verified that XIAOPI formula treatment for 48 h could dramatically inhibit CXCL1 protein expression levels (D) and CXCL1 mRNA transcription levels (E) in both human and mouse TAMs. (F) Double luciferase reporter gene assay suggested that XPS treatment for 48 h could suppress the promoter activity of CXCL1 gene in Raw264.7-derived TAMs. All values are presented as the mean ± SD, n = 3, **P < 0.05.
Figure 4
Figure 4
XIAOPI formula (XPS) suppresses breast cancer stem cells (CSCs) by modulating tumor-associated macrophages (TAMs)/C-X-C motif chemokine ligand 1 (CXCL1) pathway. (A-B) CXCL1 stimulation for 48 h could abrogate the inhibitory effect of XIAOPI formula on breast CSCs subpopulations and mammospheres formation abilities in the co-cultured breast cancer cells. Scale bar = 100 μm. (C) QPCR assay suggested that CXCL1 stimulation for 48 h could abrogate the inhibitory effect of XIAOPI formula on mRNA expression levels of stemness-related genes including β-catenin, OCT4, and Nanog in the co-cultured breast cancer cells. (D) Western blot assay further convinced that CXCL1 treatment for 48 h could reverse the inhibitory effect of XIAOPI formula on β-catenin expression in the co-cultured breast cancer cells. All values are presented as the mean ± SD, n = 3, **P < 0.05.
Figure 5
Figure 5
XIAOPI formula (XPS) suppresses breast tumor growth and breast cancer stem cells (CSCs) activity in vivo. (A) Representative pictures of the in vivo imaging experiments of mouse 4T1-Luc xenograft model. Breast cancer xenografts were established by implanting luciferase-labeled 4T1-Luc cells into the mammary glands of Balb/c mice. Mice bearing 4T1-Luc xenografts received either saline or XPS (1 g/kg/day) by intragastric perfusion. (B) XPS administration could effectively suppress mammary tumor growth in the mouse 4T1-Luc xenograft model in vivo. n = 6. (C) There were no significant differences in mouse body weights between the saline group and XIAOPI formula group, implying no additional toxic and side effects of XPS. n = 6. (D–E) XPS administration significantly decreased the infiltration degree of macrophages as well as their M2 phenotype polarization and C-X-C motif chemokine ligand 1 (CXCL1) expression in the 4T1-Luc xenografts in vivo. n = 3. (F–G) XPS administration significantly decreased the proportions of ALDH+ subpopulations (F) and suppressed the expression levels of ALDH1A1 (G) in the 4T1-Luc xenografts in vivo. n = 3. Scale bar = 20 μm. All values are presented as the mean ± SD, **P < 0.05.

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