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

Local estradiol production within breast tissue is maintained by the aromatase cytochrome P450arom complex, which has been localized primarily to the stromal component of tumors but also has been detected in the breast epithelial cells. Paracrine interactions between stromal and epithelial components of the breast are critical to the sustained growth and progression of breast tumors. Maintenance of the differentiated state, including hormone and growth factor responsiveness, requires extracellular matrix proteins as substrata for cells. This research has focused on developing a cell culture system that more closely mimics in vivo interactions in order to dissect actual paracrine signaling between these two cell types. Human fibroblasts were 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 collagen rather than maximal proliferation, therefore allowing for a more accurate environment for the study of hormonal control and cellular communication. Initial experiments compared aromatase activity of patient fibroblasts grown on plastic versus collagen I using the tritiated water release method. Constitutive aromatase activity was found to be lower when cells were grown on a collagen gel for 4-7 days (7.7 fold lower) using DMEM/F12 containing 10% dextran coated charcoal stripped serum. However, fibroblasts grown on collagen I appeared to be significantly more responsive to stimulation by 100 nM dexamethasone (plastic: 6.0 fold induction, collagen: 33.2 fold induction) when pretreated for 12 h prior to measurement of aromatase activity. In an effort to examine paracrine interactions between the stromal and epithelial cells in breast tissue, experiments using conditioned media from fibroblast cultures were performed. Testosterone administration to fibroblasts results in the production of estradiol into the media in sufficient concentrations to elicit an increase in pS2 expression when the conditioned media is administered to MCF-7 cells. The addition of a potent aromatase inhibitor resulted in a complete suppression of fibroblast-derived estrogens and showed only a modest increase in pS2 expression. Culturing breast fibroblasts and epithelial cells on extracellular matrix allows for a more meaningful examination of the paracrine interactions between these cell types within the context of an appropriate extracellular environment. This study highlights the need for evaluation of gene expression in cell culture systems that accurately reflect the tissue microenvironment.