Normal and Cancer Stem Cells of the Mammary Gland

描述(由申请人提供)：这项研究计划的主要长期目标是确定小鼠正常乳腺组织干细胞(MASC)的特征，并确定MASC如何和/或是否参与癌症的发展。小鼠的这些结果将与人类乳腺肿瘤类型进行比较，以将结果外推到人类系统。一种小鼠转基因模型，在该模型中，增强的绿色荧光蛋白(GFP)在胚胎和成体的几个干/祖细胞群中表达。在成人乳腺组织中，在青春期观察到两种独特的GFP+干细胞类型，与乳腺导管突起相吻合，并在妊娠早期到晚期，在肺泡/小叶结构产生之前观察到同样的情况。青春期的GFP+细胞与末端芽生长端的帽状细胞相对应，形成最初的乳腺导管网络。在怀孕早期，GFP+细胞以肺泡芽的形式出现，直接形成产奶所需的肺泡/小叶。GFP的前瞻性标记提供了一步分离GFP+细胞群体的方法。通过移植生长、使用Cre/loxP技术的谱系追踪和自我更新实验证明，这些GFP+细胞是MASC。本申请中提出的研究将集中于三个具体目标，首先，基于预期的生态位特性来表征MASC的生态位，确定MASC命运的Notch依赖性调控，并通过干细胞消融实验鉴定MASC的功能。其次，MASC的前瞻性鉴定允许直接检验癌症干细胞假说。第二个目标的实验将确定WNT1和ErbB2(也称为neu或HER2)乳腺肿瘤是否含有GFP+肿瘤细胞，GFP+肿瘤细胞包含主要的肿瘤启动活性。此外，所有乳腺组织上皮细胞中的靶细胞将通过细胞分离和直接体外转化来鉴定。肿瘤细胞将在移植到乳房脂肪垫中生长后读出。第三个目标是将GFP+小鼠乳腺肿瘤与基于基因表达的人类乳腺肿瘤分类联系起来。这些GFP+的小鼠乳腺肿瘤中的microRNAs将被鉴定出来，以便进一步分析信号和潜在的治疗用途。与公共卫生相关：乳腺干细胞的前瞻性鉴定和表征使实验得以进行，这在以前是不可能的。因此，该项目的结果将对乳腺干细胞以及它们如何对正常乳腺组织和癌症发展做出贡献产生新的见解。解决癌症干细胞假说将澄清目前靶细胞中的不确定性，而基因表达数据将在小鼠和人类乳腺癌之间画出相似之处。这些研究代表了乳腺癌研究的一个新的、有前途的范例。

DESCRIPTION (provided by applicant): The major long-term objectives of this research proposal are to characterize the normal mammary tissue stem cells (MaSC) in the mouse, and determine how, and/or whether, MaSC participate in cancer development. These results in the mouse will be compared to human breast tumor types for extrapolating results to the human system. A mouse transgenic model in which the enhanced green fluorescent protein (GFP) is expressed in several stem/progenitor cell populations of the embryo and adult. In the adult mammary tissue, two unique GFP+ stem cell types have been observed during puberty, coincident with mammary ductal outgrowth, and again in early to late pregnancy, prior to the generation of alveolar/lobular structures. The GFP+ cells in puberty correspond to the cap cells in the growing end of the terminal end buds, which form the initial mammary ductal network. In early pregnancy the GFP+ cells appear as alveolar buds, which direct formation of alveoli/lobules necessary for milk production. The prospective labeling with GFP offers a one-step isolation of the GFP+ cell populations. These GFP+ cells are MaSC as demonstrated by transplantation outgrowth, lineage tracing using Cre/LoxP technology, and self- renewal experiments. The research proposed in this application will focus on three specific Aims, which will first, characterize the MaSC niche based on expected niche properties, determine Notch-dependent regulation of MaSC fate, and identify MaSC functions by stem cell ablation experiments. Secondly, the prospective identification of MaSC allows the direct test of the cancer stem cell hypothesis. Experiments in the second Aim will determine whether Wnt1 and ErbB2 (also called neu or HER2) mammary tumors contain GFP+ tumor cells, which contain the primary tumor-initiating activity. In addition, the target cell, within all mammary tissue epithelial cells, will be identified by cell isolation and direct in vitro transformation. Tumor cell readout will follow transplantation into mammary fat pads for outgrowth. The third Aim, will tie together GFP+ murine mammary tumors with gene expression-based human mammary tumor classifications. MicroRNAs in these GFP+ murine mammary tumors will be identified for further analysis of signaling and potential therapeutic use. PUBLIC HEALTH RELEVANCE: The prospective identification and characterization of mammary stem cells has enabled experiments to be performed, which were not previously possible. Therefore, results from this project will generate new insights into mammary stem cells and how they may contribute to normal mammary tissue and to cancer development. Addressing the cancer stem cell hypothesis will clarify present uncertainties in the target cells for which therapies should be directed, while gene expression data will draw parallels between mouse and human mammary cancers. These studies represent a new and promising paradigm for breast cancer research.