Abstract
IntroductionThe functional unit of the mammary gland has been defined as the epithelial cell plus its microenvironment, a hypothesis that predicts changes in epithelial cell function will be accompanied by concurrent changes in mammary stroma. To test this hypothesis, the question was addressed of whether mammary stroma is functionally altered by the anti-oestrogen drug tamoxifen.MethodsForty female rats at 70 days of age were randomised to two groups of 20 and treated with 1.0 mg/kg tamoxifen or vehicle subcutaneously daily for 30 days, followed by a three-day wash out period. Mammary tissue was harvested and effects of tamoxifen on mammary epithelium and stroma determined.ResultsAs expected, tamoxifen suppressed mammary alveolar development and mammary epithelial cell proliferation. Primary mammary fibroblasts isolated from tamoxifen-treated rats displayed a three-fold decrease in motility and incorporated less fibronectin in their substratum in comparison to control fibroblasts; attributes indicative of fibroblast quiescence. Immunohistochemistry analysis of CD68, a macrophage lysosomal marker, demonstrated a reduction in macrophage infiltration in mammary glands of tamoxifen-treated rats. Proteomic analyses by mass spectrometry identified several extracellular matrix (ECM) proteins with expression levels with tamoxifen treatment that were validated by Western blot. Mammary tissue from tamoxifen-treated rats had decreased fibronectin and increased collagen 1 levels. Further, ECM proteolysis was reduced in tamoxifen-treated rats as detected by reductions in fibronectin, laminin 1, laminin 5 and collagen 1 cleavage fragments. Consistent with suppression in ECM proteolysis with tamoxifen treatment, matrix metalloproteinase-2 levels and activity were decreased. Biochemically extracted mammary ECM from tamoxifen-treated rats suppressed in vitro macrophage motility, which was rescued by the addition of proteolysed collagen or fibronectin. Mammary ECM from tamoxifen-treated rats also suppressed breast tumour cell motility, invasion and haptotaxis, reduced organoid size in 3-dimensional culture and blocked tumour promotion in an orthotopic xenograft model; effects which could be partially reversed by the addition of exogenous fibronectin.ConclusionsThese data support the hypothesis that mammary stroma responds to tamoxifen treatment in concert with the epithelium and remodels to a microenvironment inhibitory to tumour cell progression. Reduced fibronectin levels and reduced ECM turnover appear to be hallmarks of the quiescent mammary microenvironment. These data may provide insight into attributes of a mammary microenvironment that facilitate tumour dormancy.
Highlights
The functional unit of the mammary gland has been defined as the epithelial cell plus its microenvironment, a hypothesis that predicts changes in epithelial cell function will be accompanied by concurrent changes in mammary stroma
Extracted mammary extracellular matrix (ECM) from tamoxifen-treated rats suppressed in vitro macrophage motility, which was rescued by the addition of proteolysed collagen or fibronectin
Mammary ECM from tamoxifen-treated rats suppressed breast tumour cell motility, invasion and haptotaxis, reduced organoid size in 3dimensional culture and blocked tumour promotion in an orthotopic xenograft model; effects which could be partially reversed by the addition of exogenous fibronectin
Summary
The functional unit of the mammary gland has been defined as the epithelial cell plus its microenvironment, a hypothesis that predicts changes in epithelial cell function will be accompanied by concurrent changes in mammary stroma. The model of dynamic reciprocity postulates that the microenvironment, in particular the ECM, exerts an influence on gene expression in the mammary epithelial cell and, in turn, gene expression of the epithelial cell influences stromal cells and the composition of the ECM [2,4]. In support of this concept, our laboratory has shown that the composition of rat mammary ECM is dependent on reproductive state, demonstrating that the mammary microenvironment, as with the mammary epithelium, is under endocrine control [5]. Work by others has shown that the mammary ECM protein fibronectin and its specific integrin α5β1 are under hormonal control and in turn mediate hormone response in mammary epithelium, providing further support for the concept of Dynamic Reciprocity in the mammary gland [6,7]
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