STROMAL INFLUENCES ON BREAST CANCER PROGRESSION

乳腺癌MCF-7细胞转染成纤维细胞生长的研究 因子4（FGF-4），一种血管生成生长因子， 在肿瘤表型中，当细胞被注射到乳房脂肪垫中时， 卵巢切除的裸鼠。母体细胞是雌激素- 依赖于肿瘤生长，侵袭性差，很少转移， 转染子产生大的、进行性生长的肿瘤，这些肿瘤是 侵袭性，经常转移和生长刺激的他莫昔芬， 雌激素拮抗剂转染子的这种体内行为是在 与它们在体外的行为相反， 来自亲本MCF-7细胞的转染子（具体地，转染子不 体外由他莫昔芬刺激生长）。因此，似乎有可能， 体内表型的至少一部分变化是由于 转染的基因产物和基质成分之间的相互作用 肿瘤。由于转染的基因产物是已知的血管生成因子， 生长因子，这似乎可能是增加血管生成的肿瘤 所产生的至少部分负责 通过转染产生的体内表型的变化。一些 已经鉴定了1000个FGF受体（FGFR）基因，其各自可以编码 多个受体通过选择性mRNA剪接。 有证据表明 其归因于对特定FGF配体的特异性反应或亲和力 特异性FGFR亚型。肿瘤细胞产生的特异性FGFR 转染的细胞可以对FGF-4配体产生应答和/或上调或下调FGF-4配体的表达。 监管.越来越多的证据表明， 具体组织不同，肿瘤的内皮细胞 与正常组织不同。培养的内皮细胞 基因表达和行为的可塑性取决于 传代和生长条件。因此，研究肿瘤的尝试 体外血管生成必须使用适当的内皮细胞， 适当的条件。该项目旨在将 由亲本和FGF-4产生的肿瘤中的血管生成过程 转染的MCF-7细胞对体内行为的改变。 我们将 肿瘤中微血管密度和总血管分布与肿瘤大小的关系 由亲本和转染细胞产生。我们将描述这个过程 在由亲本细胞和转染细胞产生的肿瘤中， 这些技术可以识别血管内皮细胞中增殖或未成熟的内皮细胞， 肿瘤切片获得在非常早期和较晚的时间点后， 注射肿瘤细胞。该项目还将寻求描述 内皮细胞和其他细胞的特异性原位FGFR表达 在肿瘤内。我们将从产生的肿瘤中分离出内皮细胞 通过亲本或FGF-4转染的MCF-7细胞以及内皮细胞 从正常的乳房脂肪垫。生长测定，其中静止细胞是 刺激入侵三维矩阵将利用非常 早期传代的肿瘤衍生的和正常的内皮细胞， 三种内皮细胞之间的行为差异。 肿瘤来源的和乳腺脂肪垫内皮细胞将经受 差异显示PCR在寻找基因， 在FGF-4转染的MCF-7细胞产生的肿瘤中表达。 这些基因的转录可能是一个重要的下游事件， FGFR刺激和这些基因的蛋白质产物可能是 这对确定肿瘤的表型很重要。

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.