Harnessing Single Cell Technology to Define Self-Renewal in Normal and Malignant Stem Cells

! 项目摘要 大多数癌症的主要临床问题是复发。患者通常会对 化疗，并可以去多年没有任何疾病的迹象，但一个子集的患者将不可避免地重新- 患上癌症，最终预后很差。复发是因为我们目前的化疗 不能可靠地和完全地消除肿瘤增殖细胞，也称为癌症干细胞。 这些细胞在肿瘤细胞群中是独特的，因为它们可以自我更新，这意味着它们可以在肿瘤细胞群中自我更新。 可以无限地补充肿瘤细胞群，类似于正常组织干细胞在肿瘤中的作用。 组织和器官的维护。阻止肿瘤增殖细胞的自我更新将导致它们 终末分化，从而阻断其形成复发的能力。不幸的是，肿瘤的自我更新 繁殖细胞的方法还不清楚，这妨碍了合理的药物设计。一个主要问题是， 研究这些细胞是它们的稀有性;它们通常在总肿瘤细胞中每105-107个细胞中包含1个 在人类癌症和小鼠模型中，体外培养改变了它们的自我更新能力。 研究人员必须依赖于基于某些细胞表面标志物的FACS富集，但这种偏见 并排除自我更新细胞的一些亚群。 这个项目的目标是通过定义自我更新在生物医学领域产生重大影响 在肿瘤增殖细胞中以完全无偏的方式。我们将使用白血病增殖细胞作为 模型，并确定这些细胞与正常造血干细胞，也可以自我， 更新，以及它们与其他不能自我更新的白血病细胞有何不同。根据这些数据， 我们将找到检测患者体内白血病增殖细胞的方法，并确定可以抑制其增殖的药物。 自我更新能力。最初，我们将使用一组高度自我更新的急性淋巴细胞白血病， 从斑马鱼模型中分离的正常造血干细胞，这将使我们能够使用单细胞 RNA测序鉴定自我更新白血病增殖的独特基因表达谱 不需要FACS富集。我们将把我们的发现转化为人类细胞， 生物标志物面板，可以检测患者样本中自我更新细胞的频率，并告诉我们 化疗是否成功地消除了它们。我们还将描述干细胞龛在 具有高和低自我更新率的白血病，以确定生态位如何调节自我更新率。 最后，我们将在斑马鱼中使用移植方法，以及新的斑马鱼模型，其中， 白血病增殖细胞被荧光标记，用于高通量的体内药物筛选， 损害自我更新的化合物。总之，该项目将提供一个公正的基因组和功能， 肿瘤增殖细胞的分析，使我们能够回答有关肿瘤生物学的基本问题。 这种重要的肿瘤细胞类型

! Project Summary A major clinical issue in most cancers is relapse. Patients often respond very well to chemotherapy, and can go years without any sign of disease, but a subset of patients will invariably re- develop their cancer with a poor final prognosis. Relapse occurs because our current chemotherapies are unable to reliably and completely eliminate tumor propagating cells, also known as cancer stem cells. These cells are unique among the tumor cell population in that they can self-renew, meaning that they can replenish a tumor cell population indefinitely, similar to the role of the normal tissue stem cells in tissue and organ maintenance. Preventing self-renewal in tumor propagating cells would cause them to terminally differentiate, thereby blocking their ability to form relapse. Unfortunately, self-renewal of tumor propagating cells is not well understood, precluding rational drug design. A major issue in regards to studying these cells is their rarity; they often comprise 1 in every 105-107 cells within the total tumor cell population in human cancers and mouse models, and culture ex vivo alters their self-renewal capability. Researchers necessarily rely on FACS enrichment based on certain cell surface markers, but this biases towards the cells expressing the markers and excludes some subsets of self-renewing cells. The goal of this project is to have a major impact in the biomedical field by defining self-renewal in tumor propagating cells in a completely unbiased manner. We will use leukemia propagating cells as a model and determine how these cells differ from normal hematopoeitic stem cells, which can also self- renew, and how are they unique from other leukemic cells that cannot self-renew. Based on these data, we will find ways to detect leukemia propagating cells in patients, and identify drugs that can inhibit their self-renewal ability. Initially, we will use a panel of high self-renewing acute lymphoblastic leukemias and normal hematopoietic stem cells isolated from zebrafish models, which will allow us to use single cell RNA sequencing to identify the unique gene expression profile of self-renewing leukemia propagating cells without the need for FACS enrichment. We will translate our findings to human cells to build a biomarker panel that can detect the frequency of self-renewing cells in patient samples, and tell us whether chemotherapy has successfully eliminated them. We will also characterize the stem cell niche in leukemias with high and low self-renewal rates, to identify how the niche is regulating self-renewal rate. Finally, we will use transplantation approaches in zebrafish, as well as new zebrafish models in which the leukemia propagating cells are fluorescently labeled, for high-throughput, in vivo drug screens to identify compounds that impair self-renewal. In total, this project will provide an unbiased genomic and functional analysis of tumor propagating cells, allowing us to answer fundamental questions about the biology of this important tumor cell type.