Molecular and functional analysis of mammary stem cells throughout development

描述(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表现和基因表达情况进行分类。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点，如高度的基因组不稳定性，肿瘤内的异质性，以及类似于乳腺干细胞的基因表达谱。这些特征可能是通过正常干细胞的异常繁殖而产生的。或者，在这个亚型中常见的突变可能促进干状状态。为了研究这些可能性，需要确定干细胞的纯群体的特征。乳腺中的干细胞活动在胚胎发育后期就可以检测到，出生前达到顶峰，出生后迅速下降，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)相同的标记而分离出来的，并且在移植后能够类似地再生腺体。有趣的是，富含fMaSCs而不是aMaSCs的人群的基因表达谱与基底肿瘤的基因表达谱有显著重叠。当fMaSCs以基础肿瘤相关突变为靶点并移植到受体小鼠体内时，在五周内就出现了肿瘤。移植了携带相同突变的aMaSCs的小鼠在六个月后仍未患上乳腺肿瘤。这些数据表明，fMaSCs非常容易发生肿瘤，可能是基底样瘤的来源之一。LGR5在fMaSCs活性高峰期高表达。Lgr5+胎儿细胞代表了体外检测到的最丰富的fMaSCs群体。在整个发育过程中，将对Lgr5+细胞进行分析，以测量干细胞潜力。将跟踪Lgr5+胎儿和成体细胞的命运，以测试成体龟头中胎儿样干细胞的持久性。在整个发育过程中，将生成来自Lgr5+细胞的全面基因表达谱，以识别与干细胞状态相关的基因。在高度丰富的Lgr5+fMaSCs群体中，将进行单细胞RNA测序来提炼这些特征。这将允许识别亚群和标记，以便进行前瞻性分离和功能分析。细胞类型和突变对基因组不稳定和肿瘤内异质性的相对贡献还知之甚少。当以这一亚型中常见的突变为靶点时，处于不同发育阶段的干细胞产生基底样瘤的能力将得到测试。将对肿瘤进行基因组不稳定性、肿瘤内异质性和基因表达的分析。通过定义干细胞状态的分子相关性并测试发育如何影响肿瘤表型，将检验胎儿乳腺干细胞是基础肿瘤来源的假设。这项分析将揭示正常发育与肿瘤发生和发展之间的联系，为乳腺癌的治疗策略提供洞察。

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.