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

好了！ 项目摘要 大多数癌症的一个主要临床问题是复发。患者通常对药物反应良好 化疗，可以多年来没有任何疾病的迹象，但一部分患者总是会重新- 患上癌症，最终预后很差。复发的发生是因为我们目前的化疗 无法可靠地完全消除肿瘤增殖细胞，也就是众所周知的癌症干细胞。 这些细胞在肿瘤细胞群中是独一无二的，因为它们可以自我更新，这意味着它们 可以无限补充肿瘤细胞群，类似于正常组织中干细胞的作用 组织和器官的维护。阻止肿瘤增殖细胞的自我更新会导致它们 最终分化，从而阻止他们形成复发的能力。不幸的是，肿瘤的自我更新 对细胞的繁殖还没有很好的了解，这排除了合理的药物设计。关于……的一个主要问题 研究这些细胞是罕见的；它们通常占总肿瘤细胞中每105-107个细胞中就有一个 人类癌症和小鼠模型中的种群和体外培养改变了它们的自我更新能力。 研究人员必然依赖于基于某些细胞表面标记的FACS浓缩，但这是有偏见的 而不包括一些自我更新细胞的子集。 该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 以一种完全公正的方式在肿瘤细胞中繁殖。我们将使用白血病增殖细胞作为 建立模型并确定这些细胞与正常的造血干细胞有何不同，正常的造血干细胞也可以自我 更新，以及它们如何区别于其他不能自我更新的白血病细胞。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，并识别能够抑制其增殖的药物 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性白血病和 从斑马鱼模型中分离出正常的造血干细胞，这将使我们能够使用单细胞 RNA测序技术鉴定自发性白血病增殖期独特的基因表达谱 细胞，而不需要FACS浓缩。我们将把我们的发现转化到人类细胞中，以建立一个 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的利基 白血病具有高和低的自我更新率，以确定利基是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，以及在新的斑马鱼模型中 白血病增殖细胞被荧光标记，用于高通量体内药物筛选以识别 有损自我更新的化合物。总而言之，这个项目将提供一个公正的基因组和功能 对肿瘤增殖细胞的分析，使我们能够回答关于 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.