Attenuated TGFB signalling in macrophages decreases susceptibility to DMBA-induced mammary cancer in mice

Abstract

Background: Transforming growth factor beta1 (TGFB1) is a multi-functional cytokine that regulates mammary gland development and cancer progression through endocrine, paracrine and autocrine mechanisms. TGFB1 also plays roles in tumour development and progression, and its increased expression is associated with an increased breast cancer risk. Macrophages are key target cells for TGFB1 action, also playing crucial roles in tumourigenesis. However, the precise role of TGFB-regulated macrophages in the mammary gland is unclear. This study investigated the effect of attenuated TGFB signalling in macrophages on mammary gland development and mammary cancer susceptibility in mice.

Methods: A transgenic mouse model was generated, wherein a dominant negative TGFB receptor is activated in macrophages, in turn attenuating the TGFB signalling pathway specifically in the macrophage population. The mammary glands were assessed for morphological changes through wholemount and H&E analysis, and the abundance and phenotype of macrophages were analysed through immunohistochemistry. Another cohort of mice received carcinogen 7,12-dimethylbenz(a)anthracene (DMBA), and tumour development was monitored weekly. Human non-neoplastic breast tissue was also immunohistochemically assessed for latent TGFB1 and macrophage marker CD68.

Results: Attenuation of TGFB signalling resulted in an increase in the percentage of alveolar epithelium in the mammary gland at dioestrus and an increase in macrophage abundance. The phenotype of macrophages was also altered, with inflammatory macrophage markers iNOS and CCR7 increased by 110% and 40%, respectively. A significant decrease in DMBA-induced mammary tumour incidence and prolonged tumour-free survival in mice with attenuated TGFB signalling were observed. In human non-neoplastic breast tissue, there was a significant inverse relationship between latent TGFB1 protein and CD68-positive macrophages.

Conclusions: TGFB acts on macrophage populations in the mammary gland to reduce their abundance and dampen the inflammatory phenotype. TGFB signalling in macrophages increases mammary cancer susceptibility potentially through suppression of immune surveillance activities of macrophages.

Keywords: Cancer; Macrophage; Mammary gland; Transforming growth factor beta.

Fig. 1

Attenuation of TGFB signalling in macrophages from Cfms-TbrII transgenic mice. Representative immunofluorescent staining of the mammary glands with F4/80 (red channel; a, d, g) and pSMAD2 (green channel; b, e, h) antibodies, and merged images showing co-localisation and DAPI (blue channel) nuclear stain (c, f, i) from single transgenic control Cfms-rtTA (a–c) mice, TetO-TbrII (d–f) mice and double transgenic Cfms-TbrII (g–i) mice. Co-localisation was quantified and expressed as a percent of F4/80-positive macrophages that are pSMAD2 positive (n = 4/gp; j). The spleens collected from doxycycline-treated single transgenic control Cfms-rtTA, TetO-TbrII and double transgenic Cfms-TbrII mice, were analysed for ΔTgfbrII mRNA by RT-PCR and normalised to Actb expression (n = 12–13/gp; k). The result is given in arbitrary units where the average of the single transgenic control Cfms-rtTA is 1. Data are presented as mean + SEM with statistical analysis using a Kruskal-Wallis test, *p < 0.05, ***p < 0.005

Fig. 2

The effect of attenuated TGFB signalling in macrophages on mammary gland morphogenesis. Representative mammary gland carmine alum-stained wholemounts of doxycycline-treated single transgenic control Cfms-rtTA (n = 10; a), TetO-TbrII (n = 8; b) and double transgenic Cfms-TbrII (n = 12; c) mice at dioestrus. The number of branch points/mm was calculated from the analysis of wholemounts (d). Data are presented as mean + SEM with statistical analysis using a one-way ANOVA test

Fig. 3

The effect of attenuated TGFB signalling in macrophages on mammary gland morphogenesis. Representative mammary gland H&E-stained sections of doxycycline-treated single transgenic control Cfms-rtTA (n = 10; a), TetO-TbrII (n = 8; b), and double transgenic Cfms-TbrII (n = 12; c) mice at dioestrus. Examples of ductal epithelium are identified by arrows, and examples of alveolar epithelium are identified by arrowheads. The alveolar epithelium was quantified and expressed as a percentage of the total epithelium (d). Data are presented as mean + SEM with statistical analysis using a one-way ANOVA test, *p < 0.05, **p < 0.01

Fig. 4

The effect of attenuated TGFB signalling on macrophage abundance and location in the mammary gland. Representative F4/80 antibody staining of macrophages around the ductal (a–c) and alveolar (d–f) epithelium. Examples of F4/80-positive epithelial cell-associated macrophages are indicated by arrows, and examples of F4/80-positive stromal macrophages are indicated by arrowheads. The number of F4/80-positive cells within the ductal epithelium and stroma (g) and within the alveolar epithelium and stroma (h) was calculated in the mammary glands from doxycycline-treated single transgenic control Cfms-rtTA (n = 10; a, d) mice, TetO-TbrII (n = 8; b, e) mice and double transgenic Cfms-TbrII (n = 12; c, f) mice. Data are presented as mean + SEM with statistical analysis using a one-way ANOVA test, *p < 0.05, **p < 0.01

Fig. 5

The effect of attenuated TGFB signalling on abundance and location of CCR7-positive cells within the mammary stroma. Representative CCR7 antibody staining in the stroma around the ductal (a–c) and alveolar (d–f) epithelium. Examples of CCR7-positive stromal macrophages are indicated by arrowheads. The number of CCR7-positive (g) cells within the stroma was calculated in the mammary glands from doxycycline-treated single transgenic control Cfms-rtTA (n = 10; a, d) mice, TetO-TbrII (n = 8, b, e) mice and double transgenic Cfms-TbrII (n = 12; c, f) mice. Data are presented as mean + SEM with statistical analysis using a one-way ANOVA test, *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001

Fig. 6

The effect of attenuated TGFB signalling on abundance and location of iNOS-positive cells within the mammary stroma. Representative iNOS antibody staining in the stroma around the ductal (a–c) and alveolar (d–f) epithelium. Examples of iNOS-positive stromal macrophages are indicated by arrowheads. The number of iNOS -positive (g) cells within the stroma was calculated in the mammary glands from doxycycline-treated single transgenic control Cfms-rtTA (n = 10; a, d) mice, TetO-TbrII (n = 8, b, e) mice and double transgenic Cfms-TbrII (n = 12; c, f) mice. Data are presented as mean + SEM with statistical analysis using a one-way ANOVA test, *p < 0.05, **p < 0.01, ***p < 0.005

Fig. 7

The effect of attenuated TGFB signalling in macrophages on mammary gland cancer susceptibility in mice. Mammary tumour latency was monitored following DMBA administration in doxycycline-treated Cfms-rtTA, TetO-TbrII and Cfms-TbrII mice (n = 19–20/gp). Mice were palpated weekly, commencing 1 week after the final DMBA dose, with the zero time point representing mice 12 weeks of age. Tumour latency was determined as the time to the first appearance of palpable mammary tumour. The censored mice (mice were killed because of other tumours or sickness) were represented by a vertical line on the plot. Data are presented as Kaplan-Meier tumour-free survival plot (a). Significant difference observed by log rank analysis (Cfms-rtTA vs. Cfms-TbrII; p < 0.05 and TetO-TbrII vs. Cfms-TbrII; p < 0.05). Examples of F4/80 (b–e) and CCR7 (f, g) staining in tumours classified as glandular adenocarcinoma (b, c), carcinosarcoma (d, e) and solid adenocarcinoma (f, g), in tumour samples from Cfms-rtTA (b, d, f), TetO-TbrII (g) and Cfms-TbrII (c, e) mice

Fig. 8

Relationship between latent TFGB1 and the abundance of CD68-positive macrophages in human breast tissue. Representative latent TGFB1 (a, b) and CD68 (c, d) antibody staining of human non-neoplastic breast tissue. Examples are of high (a) and low (b) staining intensity for latent TGFB1 and the same patient sample stained with CD68 antibody (c and d, respectively). Epithelial-associated CD68-positive macrophages were located between adjacent epithelial cells (arrows), epithelial-aligned CD68-positive macrophages were macrophages that aligned along the epithelium but not invaded into the epithelium (dashed arrows), and stromal CD68-positive macrophages were macrophages within the stroma (arrowheads). The intensity of latent TGFB1 staining and abundance of CD68-positive macrophages in breast tissue was analysed by linear regression (n = 19); latent TGFB1 vs. CD68-positive macrophages in the stroma, p < 0.05 (e); latent TGFB1 vs. CD68-positive macrophages associated with epithelium, p < 0.05 (f)