Effects of matrix components on aromatase activity in breast stromal cellsin culture

Local estradiol production within breast tissue is maintained by the aromat ase cytochrome P450(arom) complex, which has been localized primarily to th e stromal component of tumors but also has been detected in the breast epit helial cells. Paracrine interactions between stromal and epithelial compone nts of the breast are critical to the sustained growth and progression of b reast tumors. Maintenance of the differentiated state, including hormone an d growth factor responsiveness, requires extracellular matrix proteins as s ubstrata for cells. This research has focused on developing a cell culture system that more closely mimics in vivo interactions in order to dissect ac tual paracrine signaling between these two cell types. Human fibroblasts we re isolated from breast tissue and were maintained in a cell culture system grown on plastic support or on a collagen I support matrix. The collagen I matrix model supports cell maintenance and subsequent differentiation on c ollagen rather than maximal proliferation, therefore allowing for a more ac curate environment for the study of hormonal control and cellular communica tion. Initial experiments compared aromatase activity of patient fibroblast s grown on plastic versus collagen I using the tritiated water release meth od. Constitutive aromatase activity was found to be lower when cells were g rown on a collagen gel for 4-7 days (7.7 fold lower) using DMEM/F12 contain ing 10% dextran coated charcoal stripped serum. However, fibroblasts grown on collagen I appeared to be significantly more responsive to stimulation b y 100 nM dexamethasone (plastic: 6.0 fold induction, collagen: 33.2 fold in duction) when pretreated for 12 h prior to measurement of aromatase activit y. In an effort to examine paracrine interactions between the stromal and e pithelial cells in breast tissue, experiments using conditioned media from fibroblast cultures were performed. Testosterone administration to fibrobla sts results in the production of estradiol into the media in sufficient con centrations to elicit an increase in pS2 expression when the conditioned me dia is administered to MCF-7 cells. The addition of a potent aromatase inhi bitor resulted in a complete suppression of fibroblast-derived estrogens an d showed only a modest increase in pS2 expression. Culturing breast fibrobl asts and epithelial cells on extracellular matrix allows for a more meaning ful examination of the paracrine interactions between these cell types with in the context of an appropriate extracellular environment. This study high lights the need for evaluation of gene expression in cell culture systems t hat accurately reflect the tissue microenvironment. (C) 1999 Elsevier Scien ce Ltd. All rights reserved.