title: The normal breast microenvironment of premenopausal women differentially influences the behavior of breast cancer cells in vitro and in vivo.
abstract: BACKGROUND
  Breast cancer studies frequently focus on the role of the tumor microenvironment in the promotion of cancer; however, the influence of the normal breast microenvironment on cancer cells remains relatively unknown. To investigate the role of the normal breast microenvironment on breast cancer cell tumorigenicity, we examined whether extracellular matrix molecules (ECM) derived from premenopausal African-American (AA) or Caucasian-American (CAU) breast tissue would affect the tumorigenicity of cancer cells in vitro and in vivo. We chose these two populations because of the well documented predisposition of AA women to develop aggressive, highly metastatic breast cancer compared to CAU women.
  
  
  METHODS
  The effects of primary breast fibroblasts on tumorigenicity were analyzed via real-time PCR arrays and mouse xenograft models. Whole breast ECM was isolated, analyzed via zymography, and its effects on breast cancer cell aggressiveness were tested in vitro via soft agar and invasion assays, and in vivo via xenograft models. Breast ECM and hormone metabolites were analyzed via mass spectrometry.
  
  
  RESULTS
  Mouse mammary glands humanized with premenopausal CAU fibroblasts and injected with primary breast cancer cells developed significantly larger tumors compared to AA humanized glands. Examination of 164 ECM molecules and cytokines from CAU-derived fibroblasts demonstrated a differentially regulated set of ECM proteins and increased cytokine expression. Whole breast ECM was isolated; invasion and soft agar assays demonstrated that estrogen receptor (ER)-, progesterone receptor (PR)/PR- cells were significantly more aggressive when in contact with AA ECM, as were ER+/PR+ cells with CAU ECM. Using zymography, protease activity was comparatively upregulated in CAU ECM. In xenograft models, CAU ECM significantly increased the tumorigenicity of ER+/PR+ cells and enhanced metastases. Mass spectrometry analysis of ECM proteins showed that only 1,759 of approximately 8,000 identified were in common. In the AA dataset, proteins associated with breast cancer were primarily related to tumorigenesis/neoplasia, while CAU unique proteins were involved with growth/metastasis. Using a novel mass spectrometry method, 17 biologically active hormones were measured; estradiol, estriol and 2-methoxyestrone were significantly higher in CAU breast tissue.
  
  
  CONCLUSIONS
  This study details normal premenopausal breast tissue composition, delineates potential mechanisms for breast cancer development, and provides data for further investigation into the role of the microenvironment in cancer disparities.
authors.affiliation: Mammary Biology and Tumorigenesis Laboratory, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.
authors.forename: Jodie M
authors.initials: JM
authors.lastname: Fleming
authors.forename: Tyler C
authors.initials: TC
authors.lastname: Miller
authors.forename: Mariam
authors.initials: M
authors.lastname: Quinones
authors.forename: Zhen
authors.initials: Z
authors.lastname: Xiao
authors.forename: Xia
authors.initials: X
authors.lastname: Xu
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Meyer
authors.forename: Erika
authors.initials: E
authors.lastname: Ginsburg
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Veenstra
authors.forename: Barbara K
authors.initials: BK
authors.lastname: Vonderhaar
mesh_terms: D000328:Adult; D001741:Black or African American; D000818:Animals; D001940:Breast / Q000276:immunology* / Q000502:physiology*; D001943:Breast Neoplasms / Q000208:ethnology / Q000276:immunology*; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D005109:Extracellular Matrix / Q000737:chemistry / Q000276:immunology*; D005260:Female; D006801:Humans; D013058:Mass Spectrometry; D051379:Mice; D008875:Middle Aged; D017697:Premenopause; D014183:Transplantation, Heterologous; D014481:United States; D044465:White People; D023041:Xenograft Model Antitumor Assays