Tracking the evolution of breast cancer through single cell analyses of premalignant breast tissues from women at high risk for cancer development

摘要： 建议研究的主要目标是识别和表征早期癌前病变。 携带与乳腺癌高风险相关的基因改变的女性的乳房组织最终 制定检测和预防乳腺癌发展的策略。为了实现这一目标，我们有 优化了三种分析单个乳腺细胞(MECs)的技术：(1)CyTOF质量细胞术 并行跟踪&gt；30细胞谱系和增殖标记，(2)单细胞RNA测序以鉴定 富含突变载体的细胞群体的表达程序；(3)多链循环免疫荧光 成像(CyCIF)在原位同时成像&gt；50个标志物。这些技术使人们有可能检测到 少数细胞群体的差异将被整体群体分析所掩盖。到目前为止，我们有 对30多名患有野生型或突变型BRCA1或BRCA2的妇女的乳腺组织进行了细胞学分析，并 从BRCA1的乳腺组织中鉴定出不同的、以前未被识别的细胞亚群 和/或BRCA2携带者。这些丰富的亚群可能代表直接在通往 恶性肿瘤或间接导致这些高危妇女罹患癌症。我们已经鉴定出核糖核酸 与这些丰富的亚群相关的特征，包括用于分离它们的表面标记 以研究这两种可能性，并在乳腺肿瘤内追踪它们。签名 与其中一个丰富的种群相关联，也提供了关于它们积累基础的线索 作为防止其积累的潜在战略。使用CyCIF，我们已经能够识别富集物 乳腺组织中原位细胞的亚群，并追踪它们与异常组织学之间的关系。我们有 还开发了保持所有主要MEC谱系以及富含BRCA1/2的有机培养物 并且能够在免疫受损的小鼠体内重建腺体结构。我们相信 这些工具为跟踪人类癌症的发展提供了前所未有的机会。在建议的 在研究中，我们将调查BRCA1/2/MUT丰富的亚群是否以及如何对 突变携带者的肿瘤发生。我们还将调查这些人口丰富的基础，并 DNA损伤对其浓缩的贡献。后期研究将重点放在制定战略上 干扰肿瘤进展，重要的是开发新的诊断策略来告知 预防性干预。此外，我们还将检查携带其他乳房突变的女性的组织。 癌症易感基因以确定是否在其他高危个体中检测到类似的亚群。 最后，我们将研究这些细胞代表散发性乳腺肿瘤起源细胞的可能性 在更广泛的人群中出现。

Abstract: The overarching objective of the proposed studies is to identify and characterize early premalignant changes in breast tissues from women that carry genetic alterations associated with a high risk of breast cancer to ultimately develop strategies to detect and prevent the development of breast cancer. To accomplish this goal, we have optimized three technologies to profile single breast mammary cells (MECs): (1) CyTOF mass cytometry to allow tracking in parallel of >30 cell lineage and proliferation markers, (2) single cell RNA sequencing to identify expression programs of cell populations enriched in mutation-carriers, and (3) multi-plex cyclic immunofluorescence imaging (CyCIF) to simultaneous image >50 markers in situ. These technologies make it possible to detect differences in small populations of cells that would be masked by bulk population analyses. To date, we have profiled breast tissues from over 30 women with wild-type or mutant BRCA1 or BRCA2 by CyTOF and have identified distinct, previously unrecognized subpopulations of cells that are enriched in breast tissues from BRCA1 and/or BRCA2 carriers. These enriched subpopulations may represent cells that are either directly on the path to malignancy or indirectly contribute to the development of cancer in these high-risk women. We have identified RNA signatures associated with these enriched subpopulations, which include surface markers to isolate them from breast tissue to investigate both these possibilities and to track them within breast tumors. The signatures associated with one of the enriched populations have provided clues as to the basis for their accumulation, as well as potential strategies to prevent their accumulation. Using CyCIF, we have been able to identify enriched subpopulations of cells in situ within breast tissues and track their association with aberrant histologies. We have also developed organoid cultures that maintain all of the major MEC lineages as well as the BRCA1/2-enriched populations, and that are able to reconstitute glandular structures in immunocompromised mice. We believe that these tools provide an unprecedented opportunity to track the development of human cancer. In the proposed studies, we will investigate whether and how the BRCA1/2+/mut-enriched subpopulations contribute to tumorigenesis in mutation carriers. We will also investigate the basis for the enrichment of these populations and the contribution of DNA damage to their enrichment. Later stage studies will focus on the development of strategies to interfere with tumor progression, and importantly to develop novel diagnostic strategies to inform on the timing of prophylactic interventions. In addition, we will examine tissues from women who carry mutations in other breast cancer predisposition genes to establish whether similar subpopulations are detected in other high-risk individuals. And lastly, we will examine the possibility that these cells represent cells-of-origin of sporadic breast tumors that arise more broadly in the population.