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.
Table 1.
Univariate and multivariate cox regression model using METABRIC database.