Cytokine-macrophage regulatory network in mammary gland development and tumourigenesis

Development and function of the mammary gland involves complex and dynamic interactions between epithelial and stromal cells under the influence of hormones and cytokines. Macrophages are a major component of the mammary gland stroma and they are capable of many roles in mammary gland development; importantly, their functions are tightly regulated by signals within the local cytokine microenvironment. The mammary epithelium secrets a number of cytokines, including transforming growth factor beta 1 (TGFB1) and chemokine ligand 2 (CCL2), that might affect the phenotype and function of adjacent stromal macrophages. Furthermore, alterations in cytokine secretion, and macrophage abundance and phenotype have been observed throughout different stages of normal mammary gland development and in tumourigenesis. A number of studies have demonstrated the significance of TGFB1 and CCL2 in regulating macrophages in many other tissues; however, the importance of the function of this cytokine-macrophage regulatory network in mammary gland development and tumourigenesis is yet to be investigated. The studies described in this thesis aimed to investigate the significance of epithelial cell-derived TGFB1 and CCL2 in regulation of macrophages in mammary gland development and mammary cancer susceptibility in the mouse and human mammary gland. Utilising a mouse mammary gland transplant model whereby the mammary gland tissue from Tgfb1 null mutant and wild-type mice were transplanted into TGFB1 replete recipients, we have demonstrated that deficiency in epithelial cell-derived TGFB1 caused a 50% increase of F4/80-positive macrophages invaded into the mammary epithelium, moreover, the number of iNOS-positive (“M1”) and CCR7- positive (“M1”) macrophages was increased by 78% and 200% respectively in the absence of epithelial cell-derived TGFB1. Similarly, immunohistochemical analysis of human non-neoplastic breast tissue revealed that there was a significant inverse relationship between the abundance of latent TGFB1 protein and the abundance of CD68-positive macrophages. We also observed a significant positive relationship between the abundance of latent TGFB1 and the density of stromal-associated CD206- positive (“M2”) macrophages. Further investigation of the role of TGFB-regulated macrophages in mammary gland development and tumourigenesis was undertaken utilising a transgenic (Cfms-rtTA x TetO-TgfbrII) mouse model whereby a dominant negative TGFB receptor is activated in macrophages in the presence of doxycycline, which in turn attenuates TGFB signalling in macrophages in these mice. Whole mount and H&E analysis revealed that impaired TGFB signalling in macrophages caused a 15% and 7% increase in the number of ductal branch points and the percentage of alveolar epithelium respectively in the mammary gland at diestrus. Immunohistochemical analysis using macrophage markers indicated that impaired TGFB signalling in macrophages resulted in a similar alteration in macrophage phenotypes observed in TGFB replete mice transplanted with Tgfb1-/- epithelium. There was a 50% increase in abundance of macrophages invaded into the mammary epithelium, and the number of iNOS-positive (“M1”) macrophages and CCR7-positive (“M1”) stromal macrophages was increased by 110% and 37% respectively. The effect of impaired TGFB signalling in macrophages on mammary gland cancer susceptibility in mice was investigated by challenging the mice with DMBA carcinogen; a significant decrease in mammary tumour incidence and prolonged tumour free survival was observed in mice with impaired TGFB signalling in macrophages compared to controls. The role of epithelial cell-derived CCL2 in regulation of macrophages in mammary gland development and cancer susceptibility was explored in a transgenic mouse model, Mmtv-Ccl2, whereby CCL2 is constitutively expressed by the mammary epithelium under the control of the MMTV promoter. Whole mount and H&E analysis revealed that the number of ductal branch points and the area comprised by alveolar epithelium were increased by 26% and 22% respectively in the presence of abundant epithelial cell-derived CCL2 at proestrus. Immunohistochemical analysis revealed that CCL2 did not affect the proliferation or apoptosis of mammary epithelial cells; however, there was a 40% and 53% increase in macrophage density and collagen deposition respectively around the ductal epithelium of mammary glands of transgenic mice compared to non-transgenic controls. Moreover, quantitative PCR analysis showed that the expression of Lox and Timp3 was increased by 160% and 170% respectively in the mammary glands with constitutive CCL2 expression. In addition, we investigated the effect of constitutive expression of epithelial cell-derived CCL2 on mammary gland cancer susceptibility by challenging the Mmtv-Ccl2 mice with DMBA carcinogen. A significant increase in mammary gland tumour incidence and reduced tumour latency was seen in mice with overabundant CCL2 expression compared to controls. Non-neoplastic breast tissue exhibited variable expression of CCL2 in the epithelium, with protein abundance ranging from low, to moderate and high. However, immunohistochemical analysis of human non-neoplastic breast tissue did not show a significant correlation between the expression of CCL2 and the abundance of macrophages. Interestingly, it was demonstrated that a significant negative relationship was found between the expression of CCL2 and the abundance of stromal-associated iNOS-positive cells in our human breast tissue. Together, these studies suggest that epithelial cell-derived TGFB and CCL2 exert effects on mammary gland development and tumourigenesis through regulation of macrophage functions and phenotypes. This implies that the finely orchestrated cytokine-macrophage regulatory network may be a contributing factor in mammary gland cancer susceptibility. These studies also reveal the possibility of targeting both TGFB and CCL2 signalling as a novel therapeutic approach to breast cancer prevention and/or treatment. However, more research will first be required on the upstream signalling events and underlying mechanisms that affect epithelial cell-derived TGFB and CCL2 macrophage-mediated mammary cancer risk.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Medicine, 2015