title: Tamoxifen induces pleiotrophic changes in mammary stroma resulting in extracellular matrix that suppresses transformed phenotypes.
abstract: BACKGROUND
  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. To test this hypothesis, the question was addressed of whether mammary stroma is functionally altered by the anti-oestrogen drug tamoxifen.
  
  
  METHODS
  Forty 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.
  
  
  RESULTS
  As 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.
  
  
  CONCLUSIONS
  These 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.
authors.affiliation: Department of Medicine, Division of Medical Oncology, University of Colorado Denver, 12801 East 17th Avenue, Aurora, CO 80045, USA. hattar.rhonda@ucdenver.edu
authors.forename: Rhonda
authors.initials: R
authors.lastname: Hattar
authors.forename: Ori
authors.initials: O
authors.lastname: Maller
authors.forename: Shauntae
authors.initials: S
authors.lastname: McDaniel
authors.forename: Kirk C
authors.initials: KC
authors.lastname: Hansen
authors.forename: Karla J
authors.initials: KJ
authors.lastname: Hedman
authors.forename: Traci R
authors.initials: TR
authors.lastname: Lyons
authors.forename: Scott
authors.initials: S
authors.lastname: Lucia
authors.forename: R Storey
authors.initials: RS
authors.lastname: Wilson
authors.forename: Pepper
authors.initials: P
authors.lastname: Schedin
mesh_terms: D000818:Animals; D018931:Antineoplastic Agents, Hormonal / Q000494:pharmacology*; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D018929:Cell Culture Techniques; D002465:Cell Movement / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D008321:Mammary Glands, Animal / Q000187:drug effects*; D051379:Mice; D008819:Mice, Nude; D009361:Neoplasm Invasiveness; D040901:Proteomics; D051381:Rats; D017207:Rats, Sprague-Dawley; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017154:Stromal Cells / Q000187:drug effects* / Q000378:metabolism; D013629:Tamoxifen / Q000494:pharmacology*
chemical_list: D018931:Antineoplastic Agents, Hormonal; D015415:Biomarkers; D005353:Fibronectins; D013629:Tamoxifen