Normal and Cancer Stem Cells of the Mammary Gland

描述（由申请人提供）：本研究计划的主要长期目标是表征小鼠正常乳腺组织干细胞（MaSC），并确定MaSC如何和/或是否参与癌症发展。这些小鼠的结果将与人类乳腺肿瘤类型进行比较，以推断出人类系统的结果。增强的绿色荧光蛋白（GFP）在胚胎和成体的几个干细胞/祖细胞群中表达的小鼠转基因模型。在成人乳腺组织中，两种独特的GFP+干细胞类型在青春期被观察到，与乳腺导管的生长一致，在怀孕早期到晚期，在肺泡/小叶结构的产生之前。青春期的GFP+细胞对应于终末芽生长端的帽细胞，形成最初的乳腺导管网络。在妊娠早期，GFP+细胞以肺泡芽的形式出现，它指导了产奶所必需的肺泡/小叶的形成。GFP的前瞻性标记提供了GFP+细胞群的一步分离。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。本申请提出的研究将集中在三个具体目标上，首先，根据预期的生态位特性表征MaSC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确定MaSC功能。其次，MaSC的前瞻性鉴定可以直接验证癌症干细胞假说。第二阶段的实验将确定乳腺肿瘤中Wnt1和ErbB2（也称为neu或HER2）是否含有含有原发肿瘤启动活性的GFP+肿瘤细胞。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。肿瘤细胞的读数将跟随移植到乳腺脂肪垫中生长。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结合起来。这些GFP+小鼠乳腺肿瘤中的microrna将被鉴定用于进一步的信号分析和潜在的治疗应用。公共卫生相关性：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及其如何促进正常乳腺组织和癌症发展产生新的见解。解决癌症干细胞假说将澄清目前靶细胞的不确定性，而基因表达数据将得出小鼠和人类乳腺癌之间的相似之处。这些研究代表了一种新的、有希望的乳腺癌研究范式。

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.