Triple-negative breast cancer influences a mixed M1/M2 macrophage phenotype associated with tumor aggressiveness

Abstract

Triple-negative breast cancer (TNBC) is characterized by excessive accumulation of tumor-infiltrating immune cells, including tumor-associated macrophages (TAMs). TAMs consist of a heterogeneous population with high plasticity and are associated with tumor aggressiveness and poor prognosis. Moreover, breast cancer cells can secrete factors that influence TAM polarization. Therefore, this study aimed to evaluate the crosstalk between cancer cells and macrophages in the context of TNBC. Cytokine-polarized M2 macrophage were used as control. Distinct from the classical M2 macrophage, TAMs generated from TNBC-conditioned media upregulated both M1- and M2-associated genes, and secreted both the anti-inflammatory cytokine interleukin IL-10 and the proinflammatory cytokine IL-6 and tumor necrosis factor- α. Theses TNBC-induced TAMs exert aggressive behavior of TNBC cells. Consistently, TCGA and MTABRIC analyses of human breast cancer revealed upregulation of M1- associated genes in TNBC comparing with non-TNBC. Among these M1-associated genes, CXCL10 and IL1B were revealed to be independent prognostic factors for disease progression. In conclusion, TNBC cells induce macrophage polarization with a mixture of M1 and M2 phenotypes. These cancer-induced TAMs further enhance tumor cell growth and aggressiveness.

Fig 1. Generation of TNBC-induced TAMs.

(A) THP-1 cells were PMA-treated for 24 h and washed with RPMI media for another 24 h. The conditioned media collected from breast cancer cells were used to treat differentiated THP-1 and incubated for 48 h to generate TAMs. The illustration was created with BioRender.com. (B) Flow cytometry-based analysis of TAMs and M2-induced THP-1-derived macrophages for the expression of the surface marker CD163 as compared with an isotype control. Control, THP-1-derived macrophages with RPMI media alone; TAMs, THP-1-derived macrophages treated with MDA-MB-231 CM and MDA-MB-468 CM; and M2, THP-1-derived macrophages treated with IL-4 (20 ng/mL) and IL-13 (20 ng/mL) cytokines. (C) The bar graph represents the mean and SEM from four different experiments with significance level at *p < 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001. TAMs, tumor-associated macrophages; CM, conditioned media; PMA, phorbol 12-myristate 13-acetate; TNBC, triple-negative breast cancer, RPMI, Roswell Park Memorial Institute.

Fig 2. TNBC-induced TAMs contain a mixed population of M1 and M2.

(A-B) THP-1 derived macrophages were treated with MDA-MB-231 conditioned media (TAMs) or IL-4/IL-13 (M2) for 48 h. The mRNA expression level of (A) M1 markers (CXCL10, IL-1β, and TNF-α) and (B) M2 markers (TGFβ1, TGFβR2, CCL22, CCL18, and IL10) were quantified relative to THP-1-derived macrophages (control). The relative expression level was normalized to the level of the human β-actin gene. (C) Morphological observation under microscope of THP-1 cells treated under various conditions; MDA-MB 231 conditioned media (TAMs), IL-4 and IL-13 (M2), PMA stimulated THP-1 alone (control). Bright-field images were displayed at 20× magnification of phase contrast microscopy. Scale bar, 100 μm. (D) The culture supernatant harvested from PMA-stimulated THP-1 treated with MDA-MB 231 conditioned media (TAMs MDA-MB-231), MDA-MB-468 conditioned media (TAMs MDA-MB-468), PMA-stimulated THP-1 treated with IL-4 and IL-13 (M2), and PMA stimulated THP-1 alone (control) were analyzed by CBA assay to measure the concentrations of TNF-α, IL-6, IL-4, and IL-10. Data are represented as the mean ± SEM of three independent experiments, with significance level at *p < 0.05, *p < 0.05, **p < 0.01, and ***p < 0.001. TAMs, tumor-associated macrophages; TNBC, triple-negative breast cancer; IL, interleukin; TNF, tumor necrosis factor; PMA, phorbol 12-myristate 13-acetate.

Fig 3. TAMs increase TNBC cell proliferation.

(A) Schematic diagram of the Transwell assay. THP-1 monocytes were seeded on the Transwell insert and differentiated to macrophage. TAMs were generated by treating THP-1-derived macrophage with CM for 48 h. Transwell inserts containing TAM were co-cultured with MDA-MB-231 seeded in 6-well plate and incubated for 72 h. (B) Cell proliferation rate evaluated by WST-1 assay. Data are calculated as fold change to time 0 and represented as mean ± SEM of 3–4 independent experiments, with significance level at *p < 0.05. TAMs, tumor-associated macrophages; TNBC, triple-negative breast cancer; CM, conditioned media.

Fig 4. TAM promotes the migratory activity of TNBC cells.

(A) Representative images of the wound migration assay of MDA-MB-231 cells measured at 0 h, 6 h, 12 h, and 24 h under a phase-contrast microscope. (B) Time course of wound closures expressed as the remaining wound area relative to time point 0 h. Data are represented as mean ± SEM of 3–4 independent experiments, with significance level at *p < 0.05 vs control. TAMs, tumor-associated macrophages; TNBC, triple-negative breast cancer.

Fig 5. TNBC but not HR⁺ BCA secrete high amounts of IL6.

(A) mRNA expression levels of M1 markers (CXCL10, IL-1β, and TNF-α) and M2 markers (TGFβ1, TGFβR2, CCL18, CCL22, and IL10) of MCF-7 CM-induced TAMs. (B) Heat map of the mRNA expression levels of TAMs induced by the conditioned media from MDA-MB-231, MDA-MB-468, or MCF-7, and IL-4/IL-13 polarized macrophages (M2). (C) Quantitative detection of cytokines IL4, IL10, IL6, and TNF-α from the conditioned-media of MDA-MB-231, MDA-MB-468, and MCF-7 using CBA assay. (D) The mRNA expression levels of CXCL10, IL-1β, TNF-α, and CCL22 were measured in TAMs generated from MCF-7-CM supplemented with rhIL-6 1000 pg/mL. Data are represented as mean ± SEM of three independent experiments, with significance level at *p < 0.05, **p < 0.01, ***p < 0.001, ****, and p < 0.0001. TAMs, tumor-associated macrophages; TNBC, triple-negative breast cancer; CM, conditioned media; TNF, tumor necrosis factor; IL, interleukin.

Fig 6. Upregulation of M1-associated genes in human breast cancer.

(A) The differential gene expression analyses of M1- and M2-associated genes in tumor tissues from patients with breast carcinoma retrieved from METABRIC and TCGA studies. Boxplot analyses of indicated genes in TNBC versus non-TNBC. Mann−Whitney U-test, two-sided p-value; ****p < 0.0001; *p < 0.05; NS, p ≥ 0.05. (B) Kaplan-Meier survival curves of indicated genes categorized as low and high mRNA levels comparing to median expression. Median disease-free survival (red, low; black, high) and Logrank p-value are showed.