STROMAL INFLUENCES ON BREAST CANCER PROGRESSION

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Transfection of MCF-7 breast carcinoma cells with fibroblast growth factor 4 (FGF-4), an angiogenic growth factor, produces a dramatic change in tumor phenotype when the cells are injected into the mammary fat pads of ovariectomized nude mice. While the parental cells are estrogen- dependent for tumor growth, poorly invasive, and rarely metastatic, the transfectants produce large, progressively growing tumors which are invasive, frequently metastatic and growth-stimulated by tamoxifen, an estrogen antagonist. This in vivo behavior of the transfectants is in contrast to their in vitro behavior, which is not substantially different from the parental MCF-7 cells (specifically, the transfectants are not growth-stimulated by tamoxifen in vitro). Thus, it would seem likely that at least some portion of the change in in vivo phenotype is due to an interaction between the transfected gene product and stromal components of the tumor. Since the transfected gene product is a known angiogenic growth factor, it seems likely that increased angiogenesis in tumors produced by-the transfected cells is at least partially responsible for the change in in vivo phenotype produced by the transfection. A number of FGF receptor (FGFR) genes have been identified which may each encode multiple receptors via alternative mRNA splicing. Evidence is emerging which attributes specific responses or affinity for specific FGF ligands to specific FGFR isoforms. Specific FGFRs in tumors produced by transfected cells may respond to the FGF-4 ligand and/or be up- or down- regulated. Evidence is also accumulating that endothelial cells from specific tissues differ, and that endothelial cells from tumors are different from those in normal tissues. Cultured endothelial cells are quite plastic in their gene expression and behavior depending on their passage and growth conditions. Therefore, attempts to study tumor angiogenesis in vitro must use appropriate endothelial cells under appropriate conditions. This project seeks to relate changes in the process of angiogenesis in tumors produced by parental and FGF-4 transfected MCF-7 cells to the change in in vivo behavior. We will relate microvessel density and total vascularity to tumor size in tumors produced by parental and transfected cells. We will describe the process of angiogenesis in tumors produced by parental and transfected cells by techniques which identify proliferating or immature endothelial cells in tumor sections obtained at very early and later time points after injection of tumor cells. The project will also seek to characterize specific in situ FGFR expression by endothelial and other cells contained within the tumor. We will isolate endothelial cells from tumors produced by parental or FGF-4 transfected MCF-7 cells as well as endothelial cells from normal mammary fat pads. Growth assays in which quiescent cells are stimulated to invade a 3-dimensional matrix will be done utilizing very early passage tumor-derived and normal endothelial cells to identify differences in behavior between the three types of endothelial cells. Tumor-derived and mammary fat pad endothelial cells will be subjected to differential display PCR in a search for genes which are preferentially expressed in tumors produced by FGF-4 transfected MCF-7 cells. Transcription of such genes might be an important downstream event of FGFR stimulation and the protein products of such genes might be important in determining the phenotype of the tumor.