Multiple differentiation pathways of rat mammary stromal cells in vitro: acquisition of a fibroblast, adipocyte or endothelial phenotype is dependenton hormonal and extracellular matrix stimulation

It has previously been shown that mammary stromal cells possess the ability to maintain a fibroblastlike phenotype or differentiate in vitro into matu re adipocytes in a hormone-dependent manner. This paper reports that rat ma mmary stromal cells can also differentiate into capillary-like structures i n vitro when cultured on a reconstituted basement membrane (RBM). The diffe rentiation potential of mammary stromal cells was compared with that of hum an umbilical vein endothelial cells (HUVEC) and 3T3-L1 preadipocytes. When cultured on plastic, mammary stromal cells, 3T3-L1 and HUVEC maintained a f ibroblast-like phenotype. Mammary stromal cells and 3T3-L1, but not HUVEC, differentiated into mature adipocytes when cultured in adipogenic medium. W hen plated on reconstituted basement membrane, all three cell types began t o migrate and organize themselves into an interconnected capillary network. By 18-20 h, mammary stromal cells organized into complex, highly branched capillary-like tubules whereas 3T3-L1 cells and HUVEC formed more simple st ructures. Cross-sectional analysis demonstrated the presence of an internal lumen. Mammary stromal cells were unique in their ability to progressively develop into a three-dimensional, highly branched network invading the RBM surface. The network formation was enhanced by the presence of vascular en dothelial growth factor (VEGF) and was inhibited by the anti-angiogenic dru g suramin. Western blotting analysis demonstrated the presence of the endot helial-specific marker flk-1, as well as the presence of the tight-junction -associated protein ZO-1. Mammary stromal cell differentiation into capilla ry structures was not a terminal state, since these cells were still able t o differentiate into adipocytes when ex posed to adipogenic medium. These f indings suggest that mammary stromal cells differentiate into fibroblasts, adipocytes or vascular structures in a hormone- and substatum-dependent man ner, and may explain the dramatic changes in stromal composition during bot h normal mammary gland development and tumorigenesis.