The tumor microenvironment is rich in multiple cell types that influence tumor development. Macrophages infiltrate tumors, where they are the most abundant immune cell population and secrete a number of cytokines. Aspirin acts as a chemopreventive agent against cancer development. This study investigated whether aspirin regulates crosstalk between breast cancer cells and macrophages. To study these interactions in a tumor microenvironment, a conditioned media was employed using 4T1 breast cancer cells cultured in RAW 264.7 cell-conditioned medium (RAW-CM), and a cocultured model of both cells was used. When 4T1 cells were cultured in the RAW-CM, there were increases in cell viability and secretion of the cytokines VEGF, PAI-1, TNF-
Breast cancer is the most frequently occurring cancer in women worldwide, especially in developed countries, and the incidence is increasing globally. In 2015, the World Health Organization performed a statistical analysis that revealed approximately 570,000 women die from breast cancer annually, indicating that up to 15% of all deaths in women are due to cancer [
The complicated interactions between tumors and the immune system have attracted the attention of scientists over the past decade. Briefly, the dynamic interactions between innate and adaptive immunity play an important role in tumor progression and inhibition [
Macrophages that produce mediators are crucial initiators of chronic inflammation in the tumor microenvironment. Macrophage heterogeneity includes categorization into M1 and M2 macrophages based on two distinct phenotypes that are a result of macrophage polarization and the development of different characteristics [
Aspirin, acetylsalicylic acid, is a nonsteroidal anti-inflammatory drug commonly used to reduce inflammation and prevent heart attack and stroke [
Currently, little is known about the role of aspirin in immune regulation of tumors, especially in terms of the tumor microenvironment. The main goal of this study was to better understand breast cancer chemoprevention by aspirin, which may regulate immune responses in both malignant cells and macrophages in the tumor microenvironment, as well as interfere with crosstalk between these cells. These insights might provide potential strategies for ameliorating triple-negative breast cancer, such as 4T1 cells, which is a highly aggressive type of breast cancer with resistance to treatments [
The murine breast cancer 4T1 cell line was purchased from the American Type Culture Collection (Manassas, VA, USA), and macrophage RAW 264.7 cell line was purchased from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). Both cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Caisson, Smithfield, UT, USA) containing 10% fetal bovine serum (FBS, Genedirex, Las Vegas, NV, USA) with 1% penicillin/streptomycin/amphotericin B (Caisson) in a humidified atmosphere with 5% CO2 in a 37°C incubator. Both cell lines were used to prepare conditioned medium and cocultures in this study. Aspirin (Sigma, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO, Sigma) to generate a stock solution. The final concentration of DMSO in the vehicle group was 0.1%, which is equivalent to the highest dose (2 mM) received by cells during aspirin treatment.
RAW 264.7 cells, 2.5 × 104 cells/well, were seeded in 6-well plates containing 10% FBS/DMEM and cultured overnight. The cells were then cultured for 24 h in the presence or absence of 100 ng/mL lipopolysaccharide (LPS, Sigma) in 1% FBS/DMEM according to a previous study, with modifications [
The 4T1 cells were seeded into 96-well plates at a density of 2 × 103 cells/well (Becton Dickinson, Franklin Lakes, NJ, USA) and were concurrently treated with 0.5, 1, or 2 mM of aspirin in media containing 20, 50, or 75% unstimulated or LPS-stimulated RAW-CM and 1% FBS/DMEM for 24, 48, and 72 h. After treatment, the cells were incubated in a 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT (Sigma) solution for 3 h. Supernatants were aspirated, DMSO was added to solubilize the formazan crystals, and absorbance was measured at 540 nm using a spectrophotometric microplate reader (BioTek, Winooski, VT, USA). The control was considered to be 100%, and cell viability of each sample is presented as percentage of control based on the formula
Migration of 4T1 breast cancer cells was measured using wound-healing assays. To determine the optimal concentration of RAW-CM for 4T1 cell migration, 4T1 cells were cultured in media containing 20, 50, or 75% RAW-CM and 3% FBS/DMEM for 24 h. Cells were seeded in 24-well plates and incubated until 80% confluence was reached. This monolayer of cells was gently scratched using a 20
The 4T1 cells, 2 × 104 cells/well, were seeded in a 48-well plate overnight and then treated with 2 mM aspirin in complete medium or 50% RAW-CM for 72 h. Culture supernatants were collected, and levels of cytokines, including MCP-1 (BioLegend, San Diego, CA, USA), VEGF (Peprotech, Rocky Hill, NJ, USA), PAI-1, TNF-
To define the role of the mammary microenvironment in tumorigenesis, the experimental models consisted of 4T1
Macrophages were incubated in the presence or absence of aspirin for 72 h and cultured in either control medium, the presence of LPS for the last 24 h of the incubation, or cocultured with 4T1 cells for 72 h. To assess surface marker expression, RAW 264.7 and 4T1 cells were collected after 72 h of coculturing and stained by incubating with fluorescein FITC anti-mouse CD11c and Alexa Fluor 647 anti-mouse CD206 monoclonal antibodies (Sony Biotechnology Inc.) at 4°C in the dark for 30 min. After washing, viable cells were stained with Hoechst 33342 (ChemoMetec, Allerød, Denmark) and subjected to FlexiCyte fluorescence-activated cell sorting analysis. The frequency of cells expressing each surface marker was determined by NucleoCounter NC-3000 (ChemoMetec) and analyzed using NucleoView NC-3000 software (ChemoMetec). Expression was quantified using median fluorescence intensity for the marker of interest.
Results are presented as mean ± SEM and are a compilation of at least three independent experiments. Statistically significant differences among groups were identified by one-way ANOVA with least significant difference post hoc tests using IBM Statistical Product and Service Solutions (SPSS version 19). A
To mimic the physiological tumor environment of macrophage infiltrates into tumor tissues and to study the effect of macrophage mediators on 4T1 cell viability, breast cancer 4T1 cells were cultured in RAW 264.7 cell-conditioned media (RAW-CM), as shown in Figure
Viability of 4T1 cells cultured in different amounts of RAW 264.7 macrophage-conditioned medium (RAW-CM). Different concentrations of (a) unstimulated and (b) LPS 100 ng/mL-stimulated macrophage-conditioned medium (RAW-CM) at 25, 50, and 75% were used to culture 4T1 cells. Cells were cultured for 24, 48, and 72 h, and cell viability was measured using MTT assays. Data are from at least three independent experiments and presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way ANOVA and least significant difference (LSD) post hoc tests.
A progressive increase in the number of 4T1 cells occurred with an increase in concentration of unstimulated RAW-CM. This increase in cell number, compared to the control (0% RAW-CM), occurred in a dose-dependent manner with the incubation time (Figure
Wound-healing assays were used to analyze cell migration, which is an indicator of cancer metastasis. Cells were grown until a confluent monolayer and scraped, and then the distance of healing by the cell layer was measured. The 4T1 cells cultured in 3% FBS/DMEM, that is, control, exhibited apparent healing, while the cells cultured in serum-free media, that is, negative control, did not. The distance of 4T1 cell migration over 24 h was measured for each treatment condition, including cells incubated in 20, 50, and 75% RAW-CM. RAW-CM was collected from cells that were not stimulated with LPS as a spontaneous condition and was found to have no effect on cell migration (Figure
Migration of 4T1 cells cultured in different amounts of RAW-CM. Migration patterns of 4T1 cells were assessed in scratched areas by culturing cells for 24 h in 20, 50, and 75% (a) unstimulated or (b) LPS-stimulated RAW-CM and then monitoring wound healing. (c) Distance was measured by microscope under a microscale and presented as percentage inhibition relative to the control. Data are shown as mean ± SEM and are from three independent experiments. Statistical analysis was performed using one-way ANOVA and LSD post hoc tests.
Subsequently, we investigated whether aspirin treatment influences 4T1 breast cancer cell growth when cultured under different macrophage-related conditions. The 4T1 cells were cultured in RAW-CM to mimic a microenvironment with macrophage infiltration into areas surrounding breast cancer cells, and then cell viability and migration were assessed. The 4T1 cells treated with 1 and 2 mM of aspirin had decreased cell viability when incubated in both unstimulated and LPS-stimulated RAW-CM for 24 h, while 4T1 cell numbers were not affected by aspirin in the complete medium (Figure
The effect of aspirin on viability of 4T1 cells cultured in RAW-CM. Different doses of aspirin were used to treat 4T1 cells cultured in 50% unstimulated or LPS-stimulated RAW-CM. Cells were cultured for (a) 24 or (b) 72 h, and cell viability was assessed using MTT assays. Data are shown as mean ± SEM and are from three independent experiments. Statistical analysis was performed using one-way ANOVA and LSD post hoc tests.
To investigate the effects of aspirin on 4T1 cell migration in RAW-CM, wound-healing assays were utilized. The 4T1 cells were cultured in fresh medium (Figure
The effect of aspirin on migration of 4T1 cells cultured in RAW-CM. Different doses of aspirin were used to treat 4T1 cells, which were cultured for 24 h in (a) 3% FBS/DMEM, (b) 50% unstimulated RAW-CM, and (c) 50% LPS-stimulated RAW-CM for 24 h, and wound-healing assays were performed. (d) Distance was measured by microscope under a microscale and is presented as percentage inhibition relative to the control. Data are shown as mean ± SEM and are from three independent experiments. Statistical analysis was performed using one-way ANOVA and LSD post hoc tests.
Therefore, the unstimulated RAW-CM, which mimicked the tumor microenvironment, promoted growth of 4T1 cells and was suitable to use for future experiments. Meanwhile, LPS stimulation triggered RAW 264.7 cells to exert an acute inflammatory response that inhibited growth and migration of 4T1 cells. On the basis of these studies, aspirin is an effective chemopreventive agent in the tumor microenvironment but did not exert an anticancer effect during the acute inflammatory stage.
Cytokines related to breast cancer carcinogenesis in the cultured supernatants were measured by ELISA. Cytokine levels are listed in Supplementary
Effect of aspirin on carcinogenic cytokine production by 4T1 breast cancer cells cultured in control medium and RAW-CM. (a) Effect of RAW-CM on cytokine production of 4T1 cells. Cells were cultured in 50% RAW-CM for 72 h, and then cytokines in the supernatants were measured by ELISA. (b) Aspirin was used to treat 4T1 cells, which were cultured in control medium (1% FBS/DMEM) for 72 h, and cytokine levels in the supernatants were measured. (c) Aspirin was used to treat 4T1 cells, which were cultured in 50% RAW-CM for 72 h, and then cytokine levels in the supernatants were measured. Data are shown as mean ± SEM. Statistical analysis was performed using independent sample
To investigate the effects of aspirin treatment on secretion of these cytokines, cytokine levels relative to tumor characteristics were analyzed (Figures
We determined whether aspirin treatment affects M1 and M2 macrophage subpopulations based on surface marker expression. Cluster of differentiation (CD)11c is a marker of M1 macrophages, while CD206 is a marker of M2 macrophages. RAW264.7 cells were cultured in control medium, LPS-stimulated RAW-CM, or cocultured with 4T1 cells and then characterized. Histograms and fluorescence intensity plots are presented in Figures
Effect of aspirin on M1 and M2 macrophage subtypes following LPS stimulation and coculture with 4T1 cells. Macrophages were incubated in the presence or absence of aspirin for 72 h and cultured in either fresh medium as a control, the presence of LPS for the last 24 h of the incubation, or cocultured with 4T1 cells for 72 h. (a) Histogram plots, (b) fluorescent intensity plots, and (c) quantitative data were presented. The immunofluorescent intensity of CD11c (M1) and CD206 (M2) on macrophages was analyzed using a NC-3000. Data are shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA and LSD post hoc tests. The comparisons between different culture mediums were done by
To further confirm the production of potential mediators of interactions between cells in culture supernatants, 4T1 and RAW 264.7 cells were cocultured together to mimic the physiology of the tumor microenvironment. Cytokine levels are listed in Supplementary
Aspirin inhibited angiogenic and inflammatory cytokines in supernatants of 4T1 and RAW 264.7 cell cocultures. 4T1 cells were cultured in the presence of macrophages for 72 h, supernatants were collected, and cytokine levels relative to tumor characteristics were measured by ELISA. (a) Angiogenic cytokines VEGF, MCP-1, and PAI-1. (b) Inflammation-related cytokines TNF-
The effects of aspirin treatment in the coculture model were apparent compared to the RAW-CM model, suggesting cocultures containing both types of cells can effectively crosstalk. These data indicate that aspirin disrupted secretion of mediators associated with carcinogenesis in both RAW-CM and cocultures. A schematic of factors with a possible active role in aspirin treatment is proposed in Figure
The schema of possible mechanism of chemoprevention of aspirin. In 4T1 breast cancer cell environment, RAW264.7 macrophage infiltration increased VEGF, PAI-1, TNF-
Breast cancer is the most prevalent malignant tumor currently found in women. The breast tumor microenvironment includes neoplastic, neighboring stromal, and recruited immune cells, such as macrophages and lymphocytes, where crosstalk among these cells is involved in tumor progression and metastasis [
The breast cancer cell line 4T1 is triple-negative, which is a form of breast cancer associated with a poor prognosis because the cells lack effective therapeutic targets, behave aggressively, and are accompanied with overexpression of inflammation-related mediators [
Normally, macrophages have a critical role in host defense that involves connecting innate and adaptive immune responses, as well as tissue repair. Macrophages secrete multiple cytokines that participate in inflammatory responses, tissue damage, pathogen clearance, tissue homeostasis, and disease development [
In a previously published study, mice were inoculated with 4T1 cells and implanted with sponge discs for 1 or 24 days to create acute and chronic inflammatory environments [
Our previous study demonstrated that aspirin treatment significantly inhibits the proliferation and migration of 4T1 cells, as well as causes an associated decrease in MCP-1 and VEGF production [
MCP-1, that is, CCL-2, is a chemokine that recruits and activates monocytes during inflammation. In tumor progression, MCP-1 plays an important role through facilitation of macrophage infiltration, which is involved in tumor progression and immunosurveillance [
In this present study, aspirin suppressed MCP-1, PAI-1, and IL-6 production by 4T1 cells cultured in fresh medium and RAW-CM, suggesting to inhibit proliferation and migration of breast cancer cells. In the coculture model, treatment with aspirin significantly inhibited MCP-1, IL-6, and TGF-
Macrophages can divide into two distinct phenotypes of M1 and M2. M1 macrophages are promoted by T-helper cell type 1 (Th1) cytokines and produce proinflammatory cytokines that evoke an adaptive immune response. Meanwhile, Th2 cytokines polarize monocytes into M2 macrophages that promote angiogenesis, clean injured tissues, and suppress adaptive immune responses [
Interestingly, aspirin treatment increased M1 marker expression, but decreased M2 marker expression in cocultures of the present study, suggesting that aspirin influences the macrophage profile in the neoplastic microenvironment away from a suppressive immune response, thus contributing to breast cancer cell suppression. Recently, it was demonstrated that macrophage phenotypes are regulated by aspirin in a model of RAW 264.7 cells cultured in pancreatic cancer cell line Panc02-conditioned medium. Aspirin significantly decreased protein and RNA levels of the M2 marker CD206 and prevented pancreatic carcinogenesis [
Clinical trials have revealed that aspirin is an effective chemopreventive agent. Observational studies have shown that regular aspirin use reduces the incidences of several cancers, as well as distant metastases of these cancers [
Based on accumulating evidence, macrophages play a crucial role in the tumor microenvironment, which includes intricate crosstalk involving a series of inflammatory chemokines and cytokines and angiogenic mediators secreted from neoplastic cells and infiltrating macrophages. The findings of this study indicate that aspirin has chemopreventive properties that function through both 4T1 breast cancer cells and macrophages. Aspirin interfered with the connection between various cells by decreasing communication through proinflammation and angiogenic mediators and modulating M1/M2 macrophage subtypes, suggesting that aspirin is a promising agent to prevent tumor progression.
The data used to support the findings of this study are all provided in the manuscript and supplementary file.
The authors declare that there is no conflict of interest regarding the publication of this paper.
The authors acknowledge the funding from Ministry of Science and Technology, Taiwan (103-2320-B-003-003-MY3, 106-2311-B-003-005-MY3).
Supplementary 1: effect of aspirin on carcinogenic cytokine production by 4T1 breast cancer cells cultured in control medium and RAW-CM. Supplementary 2: aspirin inhibited angiogenic and inflammatory cytokines in supernatants of 4T1 and RAW 264.7 cell cocultures.