Tumor-Associated Macrophages of the M1/ M2 Phenotype are Involved in the Regulation of Malignant Biological Behavior of Breast Cancer Cells Through the EMT Pathway

Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. More and more studies have shown that the tumor immune microenvironment (TME) of TNBC is closely related to its poor prognosis and early metastasis. We try to explain how tumor-associate macrophages (TAMs ） , an important component of the TME, function in the matrix of TNBC. Therefore, we induced THP-1 cells to become M1-TAMs and M2-TAMs, investigated their influence on breast cancer cells. 82 TNBC paraffin samples were made into tissue microarrays. The expression of macrophages makers were measured by immunohistochemistry. Scratch assay, Transwell assay, CCK-8 cell proliferation assay were performed in the co-culture system of breast cancer cells lines and macrophages to observe the invasion and proliferation ability of breast cancer cell lines. Western Blot (WB) was performed to detect the expression of E-cadherin (CDH1) and N-cadherin (CDH2). M2-TAMs were more numerous than M1-TAMs in the matrix of TNBC cancer nests and associated with poor prognosis. M2-TAMs promoted the invasion, migration and proliferation of TNBC cells. M1-TAMs had inhibitory effects. In MCF-7 cells, WB showed a decrease in CDH1 and an increase in CDH2. In MDA-MB-231 cells and BT549 cells, CDH2 expression was reduced and CDH1 expression was increased. All of the above results were statistically significant, p < 0.001. M2-TAMs were more numerous in TNBC and associated with poor prognosis. M2-TAMs promoted the invasion, migration and proliferation of breast cancer cells. The mechanism may be related to the epithelial-mesenchymal transition (EMT). b c Western blot for E-cadherin and N-cadherin in ฀


Abstract
Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. More and more studies have shown that the tumor immune microenvironment (TME) of TNBC is closely related to its poor prognosis and early metastasis. We try to explain how tumor-associate macrophages (TAMs), an important component of the TME, function in the matrix of TNBC. Therefore, we induced THP-1 cells to become M1-TAMs and M2-TAMs, investigated their influence on breast cancer cells. 82 TNBC paraffin samples were made into tissue microarrays. The expression of macrophages makers were measured by immunohistochemistry. Scratch assay, Transwell assay, CCK-8 cell proliferation assay were performed in the co-culture system of breast cancer cells lines and macrophages to observe the invasion and proliferation ability of breast cancer cell lines. Western Blot (WB) was performed to detect the expression of E-cadherin (CDH1) and N-cadherin (CDH2). M2-TAMs were more numerous than M1-TAMs in the matrix of TNBC cancer nests and associated with poor prognosis. M2-TAMs promoted the invasion, migration and proliferation of TNBC cells. M1-TAMs had inhibitory effects. In MCF-7 cells, WB showed a decrease in CDH1 and an increase in CDH2. In MDA-MB-231 cells and BT549 cells, CDH2 expression was reduced and CDH1 expression was increased. All of the above results were statistically significant, p < 0.001. M2-TAMs were more numerous in TNBC and associated with poor prognosis. M2-TAMs promoted the invasion, migration and proliferation of breast cancer cells. The mechanism may be related to the epithelial-mesenchymal transition (EMT).

Background
TNBC is the most aggressive subtype of breast cancer, which is defined as no expression of estrogen receptor (ER) , progesterone receptor (PR) and no amplification or overexpression of HER2. Early recurrence, easy transfer and most importantly, lack of therapeutic targets are major problems in TNBC [1]. Multiple mechanisms are associated with poor prognosis of TNBC, include the TME. But patients with TNBC have little benefits [2]. This may be due to the lack of understanding the TME in TNBC. TME plays an important role in the study of TNBC immunogenicity [3], tumor-associated macrophages (TAMs) are the most inflammatory immune cells in TME, accounting for about 30% [4].
TAMs, have influence on tumor progression and distant metastasis by regulate tumor cells invasion, migration, and angiogenesis [5]. TAMs can be divided into many subtypes, of which the classical classification method is divided into the classical activated M1 subtype and the alternately activated M2 subtype, according to their receptor, cytokine and chemokine expression and their effect function Other studies have shown that TAMs can express a variety of cytokines that stimulate the proliferation and survival of tumor cells, such as epithelial growth factor (EGF), platelet-derived growth factor TGF-β1, hepatocyte growth factor (HGF) and epithelial growth ligand of the EGFR family [10]. In addition, other studies have shown that M2-TAMs promote angiogenesis, forms stem cell characteristics, promotes distant metastasis [11]. In comparison, most studies on M1-TAMs suggest that they recognize and kill tumors cells directly by secreting some tumor killing molecules including ROS and NO [12]. Tumor cells can also be killed through the antibody dependent cell-mediated cytotoxicity (ADCC) mechanism [13].
In this study, we intend to analyze the differences between M1-TAMs and M2-TAMs in the matrix of TNBC cancer nests by detecting the differences in the expression levels of antibodies CD163, CD206, CD80, CD86, IL-10 and IL-6 in tissue microarrays. Co-culture system of breast cancer cells (MCF-7, MDA-MB-231, BT549) and TAMs were established. Cell Scratching assay, Transwell invasion and migration assay, CCK-8 proliferation assay and WB were used to explore the effect of M2 on invasion, proliferation and preliminary mechanism in TNBC.

Tissue microarrays and IHC
All patients received surgical intervention for primary breast tumors. All specimens were formalin fixed, paraffin embedded, and cut into 4 μm thick sections for hematoxylin and eosin (H&E) staining.

Cell Scratch Assay
Mark on the back of 6-well plate, about every 1.0 cm, draw at least 5 lines. Each hole with 5×10 5 cells, add 2 ml medium, incubate overnight. Then, drew 2 or 3 straight lines with tips, wash the plate with PBS three times, incubate for 24 hr with serum-free culture medium, take pictures at 0 hr, 24 hr.

Transwell Migration Assay
Plate cells (5×10 5 ) in the upper chamber of 8 μm Transwell (Corning, NY), and allowed to migrate for 8 hr at 37 • C. Induce cell ability to migration by placing 1640 THP-1 cells，THP-1-M1 cells and THP-1-M2 cells in the lower chamber of Transwell inserts. Count the number of cells that migrated across the filters in 4 high-power fields per insert, and average values afterwards. For each migration condition, three identical replicates should be performed.

Transwell Invasion Assay
Thaw the stock 10 ml Matrigel (BD, Biosciences) overnight at 4°C by placing inside a refrigerator or cold room. Once thawed, always keep Matrigel chilled on wet ice. Diluted to 1/8, put 100 μl on the upper chamber, Incubate the plates at 37°C for at least 30 min before starting the invasion assay. The rest of the steps are to reference Transwell Migration Assay.

The CCK-8 cell proliferation Assay
Prepared cell suspension, counted 5×10 4 cells /ml, mixed evenly, took about 100ul, inoculated it into a 96-well plate. The same samples were repeated 5 times and put into 37 • C incubators. After cell adherence, 10 μl CCK8 was added, and continued culture for 4 hr. The absorbance was measured at 450nm.
For densitometry analyses, protein bands on the blot were measured by use of Image J.

Statistical analysis
All experiments were repeated three times, and data were expressed as mean ± standard deviation. The data were tested for significant differences by paired two-tailed t test, after a Bartlett test had shown that variances were homogenous using GraphPad Prism 6.01 software (GraphPad Software Inc, La Jolla, CA, USA). The difference was considered statistically significant at p < 0.05.

TAMs were induced and identification in vitro
Co-culture system for breast cancer cells and TAMs (Fig.4a). Observe the changes in THP-1 cell morphology after induction. THP-1 cells were full in shape and had good refractive index, a single cell grew in suspension. THP-1-M1 cells showed diversity in morphology, and some cells were in long spindle shape. THP-1-M2 cells were polygons , increased in volume, the refractive index became worse and the cytoplasm increased and became rough (Fig.4b). The qRT-PCR results showed that the markers of M1 macrophages (HLA-DR, TNF-α, IL-6, IL-1β) were increased in THP-1-M1 cells. The markers of M2 macrophages (CD206, IL-10, MRC-1, DC-SIGN, SOCS1, Arg-1, TGF-β) were increased in THP-1-M2 cells ( Fig.4c and 4d).

M2-TAMs enhance the migration and invasion of breast cancer cells
In the Scratch assay, the migratory ability of MCF-7 cells co-cultured with M2-TAMs was significantly enhanced, and co-cultured with M1-TAMs was not significantly changed (Fig.5a). MDA-MB-231 cells and BT549 cells co-cultured with M2-TAMs had significantly enhanced migratory ability, while co-cultured with M1-TAMs had significantly reduced ( Fig.5b and 5c). In Transwell migration and invasion assays, the migration and invasion abilities were enhanced for all these cell lines co-cultured with M2 and diminished for those co-cultured with M1 (Fig.5d, 5e and 5f).

M2-TAMs promote proliferation of breast cancer cells
In CCK-8 proliferation assays, the proliferation abilities of MCF-7, MDA-MB-231, BT549 cells co-cultured with M2 was enhanced, whereas the proliferation abilities of those cell lines co-cultured with M1 was diminished (Fig.5j).

M2 promotes EMT of breast cancer cells
The expression of CDH1, which was originally highly expressed, was significantly reduced after co-culture with M2, and CDH2 was expressed instead in MCF-7 cells (Fig.6a and 6b). In MDA-MB-231 and BT549 cells, CDH1 was expressed to a certain extent, while CDH2 was weakened, after co-culture with M1-TAMs. Compared with M2-TAMs showed strong expression in both CDH1 and CDH2 (Fig.6c -6f).

Discussion
TNBC is recognized as a subtype of breast cancer with poor prognosis. Multiple factors are associated with poor prognosis, among which tumor-associated macrophages (TAMs) have an irreplaceable position [4]。 The polarization of TAMs is continuously changing state. Most studies divide TAMs into two polarization tops, M1-TAMs and M2-TAMs [14]。The results of this study showed that the number of M2-TAMs in TNBC was positively correlated with tumor size, Nottingham Classification, and lymph node metastasis, suggesting a poor prognosis. It has been shown that a large number of CD206 + M2-TAMs is associated with high proliferation and low differentiation of tumors and these cells are more frequently found in TNBC [15]. It was also investigated to assess the status of TAMs in the tumor microenvironment using the number of M2-TAMs/the number of M1-TAMs, the TAMs balance fraction (MBF). Confirmed that in breast cancer, high MBF was associated with poor prognosis and could promote tumor metastasis [16,17]. Macrophages can be differentiated into subtypes of macrophages in different directions after stimulated by cytokines in vitro. In this paper, a model of THP-1 mononuclear cell line differentiation into macrophages induced by PMA was used [18]. Pathological histological observations showed that M2-TAMs were predominant in the matrix of TNBC cancer nests. In vitro, experiments revealed that M2-TAMs promoted the migration, invasion and proliferation of breast cancer cells, supporting the pro-tumor progressive role of M2-TAMs. It was 3shown that M2-TAMs were involved in the whole metastatic process of breast cancer, including local invasion, promotion of tumor cells into blood vessels, and extravasation from distant sites [19]. It has been shown that the mechanism is that they go through the production of matrix metalloproteinases (MMPs), cysteine histone proteases and serine proteases that destroy the cell matrix and subsequently, and then invade tumor cells into the surrounding tissue [20]. TAMs also secrete acidic substances, including chemokine ligands (e.g., CCL18) and epidermal growth factor (EGF), all of which have tumorigenic effects [21]. This study found that breast cancer cells may promote the polarization of M1 phenotype to M2 phenotype in co-culture mode. Some studies have reported that the polarization status of TAMs is influenced by several factors in breast cancer [22]，as the lactate metabolism [23].
It was also confirmed that changes in adenosine deaminase 2 (ADA2) activity may contribute to the differentiation of TAMs to the M2 phenotype in TNBC [24]. In addition, M1-TAMs inhibited the migration, invasion and proliferation of breast cancer cells in this study and had some inhibitory effect on tumor progression. Most studies have shown that M1-TAMs show anti-tumor capacity mainly through the release of pro-inflammatory cytokines such as TNF and interleukin (IL)-2, as well as reactive nitrogen and oxygen intermediates [22,25]. It has also demonstrated that the AMPK-NF-κB signaling pathway is involved in regulating the polarization of macrophage into the anti-tumor M1 phenotype [26]. invasiveness of TNBC cells [28]. It was also shown that miR-200c, a potent EMT suppressor, upregulated cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), promoted the polarization of TAMs to M1 phenotype [29]. In addition, we observed that some breast cancer cells aggregated into spheres after co-culture with M2-TAMs. The study confirmed that exosomes secreted by mesenchymal stem cells (MSCs) drive accelerated breast cancer progression by inducing monocytes to differentiate into M2 phenotype macrophages [30]. MSCs were also shown to differentiate into S100A4-secreting cancer-associated fibroblasts (CAFs), and S100A4 stimulates cells to secrete CCL2, which polarizes macrophages into M2-TAMs, promoting their metastatic potential through a bidirectional interaction between MSCs/CAFs and M2-TAMs [31]. Jia et al. used M2-conditioned medium to culture mesenchymal stem cells (cMSCs) and found that their ability to promote tumor growth was greatly enhanced in vivo. Moreover, IL-6 secreted by cMSCs could polarize infiltrating TAMs to the M2 phenotype [32]. Therefore, targeting TAMs is an extremely promising therapeutic strategy. Either to reverse the EMT process or to reduce the stemness of TNBC cells.