Molecular and functional analysis of mammary stem cells throughout development

描述（由申请人提供）：乳腺癌是女性癌症死亡的第二大原因。亚型根据组织病理学外观和基因表达进行分类。基础亚型预后较差，部分原因是缺乏治疗策略。基底肿瘤具有独特的特征，如高度的基因组不稳定性、肿瘤内异质性和与乳腺干细胞相似的基因表达谱。这些特征可能是由正常干细胞的异常繁殖引起的。另外，在这种亚型中常见的突变可能促进茎样状态。为了研究这些可能性，需要对纯干细胞群体进行表征。乳腺中的干细胞活性在胚胎发育晚期可以检测到，在出生前达到峰值，出生前迅速下降，并在成年后保持较低水平。胎儿乳腺干细胞（fMaSCs）是通过表达与成人乳腺干细胞（aMaSCs）相同的标记物分离出来的，并且在移植后同样能够再生腺体。有趣的是，富含fMaSCs而非aMaSCs的群体的基因表达谱与基底肿瘤的基因表达谱显示出惊人的重叠。当fMaSCs以基础肿瘤相关突变为目标并移植到受体小鼠中时，肿瘤在五周内出现。携带相同突变的aMaSCs移植的小鼠在6个月后仍未发生乳腺肿瘤。这些数据表明fMaSCs极易发生肿瘤，可能是基底样肿瘤的来源。Lgr5在fMaSCs的活性高峰高度表达。Lgr5+胎儿细胞是体外检测到的fMaSCs最富集的群体。Lgr5+细胞将在整个发育过程中进行分析，以测量干细胞的潜力。Lgr5+胚胎和成体细胞的命运将被跟踪，以测试成体龟头中胎儿样干细胞的持久性。Lgr5+细胞的综合基因表达谱将在整个发育过程中生成，以鉴定与干细胞状态相关的基因。在高度富集的Lgr5+ fMaSCs群体中，将进行单细胞RNA测序以完善这些特征。这将允许确定亚群和标记，为其未来的分离和功能分析。细胞类型和突变对基因组不稳定性和肿瘤内异质性的相对贡献尚不清楚。当以这种亚型中常见的突变为靶标时，处于不同发育阶段的干细胞产生基底样肿瘤的能力将被测试。肿瘤将分析基因组不稳定性、肿瘤内异质性和基因表达。通过确定干细胞状态的分子相关性并测试发育如何影响肿瘤表型，胎儿乳腺干细胞是基础肿瘤来源的假设将得到验证。该分析将揭示正常发育与肿瘤发生和发展之间的联系，为乳腺癌治疗策略提供见解。

DESCRIPTION (provided by applicant): Breast cancer is the second leading cause of cancer death in women. Subtypes are classified by histopathological appearance and gene expression. The basal subtype has a poor prognosis due in part to lack of therapeutic strategies. Basal tumors have unique characteristics such as high degrees of genomic instability, intra-tumor heterogeneity, and gene expression profiles similar to those of mammary stem cells. These characteristics may arise through the aberrant propagation of normal stem cells. Alternatively, mutations commonly found in this subtype may promote a stem-like state. To investigate these possibilities, pure populations of stem cells need to be characterized. Stem cell activity in the mammary gland becomes detectable in late embryonic development, peaks right before birth, diminishes rapidly after birth, and remains low in the adult. Fetal mammary stem cells (fMaSCs) are isolated by expression of the same markers as adult mammary stem cells (aMaSCs), and are similarly able to regenerate glands upon transplantation. Interestingly, gene expression profiles of populations enriched for fMaSCs, but not aMaSCs, show striking overlap with those of basal tumors. When fMaSCs were targeted with basal tumor-associated mutations and transplanted into a recipient mouse, a tumor arose within five weeks. Mice transplanted with aMaSCs bearing the same mutation have yet to develop mammary tumors after six months. These data suggest that fMaSCs are highly prone to tumorigenesis and may be a source of basal-like tumors. Lgr5 is highly expressed in fMaSCs at the peak of their activity. Lgr5+ fetal cells represent the most enriched population of fMaSCs measured in vitro. Lgr5+ cells will be analyzed throughout development to measure stem cell potential. The fate of Lgr5+ fetal and adult cells will be followed to test for the persistence of fetal-like stem cells in the adult glan. Comprehensive gene expression profiles from Lgr5+ cells will be generated throughout development to identify genes associated with a stem cell state. In the highly enriched population of Lgr5+ fMaSCs, single cell RNA sequencing will be conducted to refine these signatures. This will allow the identification of subpopulations and markers for their prospective isolation and functional analysis. The relative contributions of cell type and mutation to genomic instability and intra-tumor heterogeneity are poorly understood. The capacity of stem cells at various stages of development to give rise to basal-like tumors when targeted with mutations commonly found in this subtype will be tested. Tumors will be analyzed for genomic instability, intra-tumor heterogeneity, and gene expression. By defining the molecular correlates of a stem cell state and testing how development influences tumor phenotype, the hypothesis that fetal mammary stem cells are a source of basal tumors will be tested. This analysis will reveal links between normal development and tumor initiation and progression, providing insight into strategies for breast cancer treatment.