The Effects Of Tumor Associated Macrophage In Breast Cancer Metastasis And Its Related Mechanism

Background and objective:As the processing of aging, the incidence of malignant tumor is uprising accordingly. Breast cancer has been one of the most common malignant tumors among women in the developed countries, like Europe and the United States. However, there are an obvious increasing number of cases in our country, which is especially common in the coastal modern cities. The overall prognosis of patients with breast cancer is good compared with other malignant tumors, however, in case of metastatic diseases, the5-year survival rate was reduced significantly. Tumor associated macrophages derived from monocytes, which is induced by the cytokines released by cancer cells, usually share the alternatively activated macrophage (M2) phenotype. Research has shown that tumor associated macrophage infiltration in some specimens of breast cancer is quite common, whose abundance is associated with poor prognosis, while the mechanism is still unknown. The aim of this study is to explore the role of tumor associated macrophage in breast cancer stem cell (BCSC) maintenance and breast cancer metastasis, and to investigate how breast cancer cell effects the differentiation of moncytes and possible intervention measures.Method:First of all, we collected120cases breast cancer tissue samples between January2008and May2010from Fudan university Shanghai Cancer center through stratified randomization. We applied immunohistochemical technique to analyze the distribution of CD163positive cancer cells in different subtypes of breast cancer. U937cells were cultured in RPMI1640medium with phorbol12-myristate13-acetate (PMA)3days and macrophage colony stimulating factor (M-CSF) for7days, whose phenotypes were identified under by microscopy, and confirmed by RT-PCR and quantitative RT-PCR. We dyed the differentiated U937cells and breast cancer cells with cell tracker dyes CMTMR and CMFDA respectively. Hybrids were generated by fusing the differentiated U937with each breast cancer cell line using50%polyethylene glycol (PEG)1450, and were isolated by fluorescence-activated cell sorting (FACS). We compared the biological behaviors between the fused cells and their corresponding breast cancer cells both in vitro and in vivo. Secondly, we wanted to investigate whether breast cancer cells with different estrogen receptor (ER) can induce the differentiation of U937. So we set up a serial of stable cell lines with GFP-PURO and RFP-NEO dual-markers by lentivirus particles under the selection of antibiotics puromycin and neomycin accordingly. We cocultured U937cells with breast cancer cells to investigate the breast cancer conditioned medium on U937cells differentiation, and chemotactic migration and invasion using Transwell niche system to mimic the process of tumor associated macrophage infiltration in vivo. We also compared the cytokines (MCP-1, M-CSF, IL6and GM-CSF) related with monocyte differentiation between breast cancer cell lines with different ER status by Bio-Plex multiplex human cytokine assay kit. Finally, we paid attention to the effects of arsenic trioxide on the cytokines secretion of breast cancer cells and its impact on the tumorigenicity of breast cancer cells.Results:There were different expressions of CD163among different subtypes of breast cancer, which was higher in the ER-breast cancer specimens compared with ER+specimens. What is more, the abundance of CD163was an independent poor prognostic factor. U937differentiated into M2macrophages after the stimulation by PMA and M-CSF stepwise, which was confirmed morphologically and identified by CD163, CD204immune phenotype. There were some fusion events between U937cells and breast cancer cell lines MCF-7and MDA-MB-231after PEG stimulation. The isolated hybrids showed different biological properties compared with their corresponding breast cancer cell lines, such as higher percentage of CD44+CD24-breast cancer stem cell, and higher expression of epithelial mesenchymal transition (EMT) related genes. They also showed relatively stronger migratory and invasive ability, but weaker proliferation ability in vitro, and more tumorigenic and metastatic in vivo.ER-breast cancer cell line MDA-MB231could induce U937cells differentiate into tumor associated macrophages with M2phenotypes in a relatively short time (one week) when cocultured in Transwell niche system, while ER+breast cancer cell line MCF-7can not. We also confirmed the different level of cytokines related to monocyte differentiation (M-CSF, IL6and so on) among breast cancer cell lines.Arsenic trioxide (AS2O3) is a mineral drug. Its effect on the differentiation of acute promyelocytic leukemia (APL) has been well-documented. We found that sublethal dosage of AS2O3partially blocked the secretion of cytokines related to monocyte differentiation in vitro, and reversed the ER status. In NOD/SCID mouse MDA-MB231xenografts, we found that administration of As2O3decreases level of cytokines related to monocyte differentiation in the peripheral blood, and causes tumor necrosis and reexpression of ER.Conclusion:M2macrophages can promote breast cancer metastasis through fusion with breast cancer cells to form hybrids. The hybrid cells show some characteristics of breast cancer stem cells. Different breast cancer cell lines have varied cytokines profile, which determines their capability to educate monocyte differentiation. As2O3can partially block breast cancer cells secrete cytokines required for monocyte differentiation and reverse the ER status.