Single Cell Analysis of Sphingosine Kinase Activity in Human Leukemia Stem Cells

描述（由申请人提供）：急性髓性白血病（AML）是最致命和最常见的成人白血病。在美国，每年有超过1万人被诊断出患有急性髓性白血病。虽然大多数患者在最初的化疗治疗后癌症得到缓解，但大约80%的患者随后复发。不到30%的AML患者在复发后存活超过一年。AML的高复发率归因于被称为白血病干细胞的一小部分细胞。LSCs对多种化疗药物具有高度耐药性，并能在初始化疗中存活。它们能够积极地恢复白血病细胞群。开发一种靶向和杀死LSCs的方法将极大地造福AML患者。然而，由于可以从患者身上分离出的LSC数量很少，研究LSC生物学的进展受到限制。这项研究将开发出能够在单细胞水平上进行调查的技术；LSCs鞘氨酸激酶（SphK）活性的变化。SphK是一种致癌激酶，已知可增加白血病细胞对化疗药物的耐药性。该提案的中心假设是SphK在LSCs中被上调，这将使其成为药物治疗的一个有希望的靶点。SphK的活性将通过装载荧光报告细胞来测量，荧光报告细胞可以被活性SphK修饰。单细胞毛细管电泳（CE）将用于分离每个细胞中修饰的报告基因和未修饰的报告基因。CE是一种非常敏感的技术，使其成为单个细胞生物分析的理想选择。然而，单细胞CE的一个主要限制是低通量（每天5-35个细胞）。该项目的目标是开发一个自动化的单细胞CE平台，该平台将大大提高吞吐量，每天可以分析数百到数千个细胞。这种自动化CE仪器将用于测量ML患者LSCs中的SphK活性。这项研究将首次在个体患者体内研究人类LSCs的生物信号通路。表征LSCs中的SphK活性有望有助于开发特异性靶向LSCs的治疗方法，从而预防AML复发。

DESCRIPTION (provided by applicant): Acute myelogenous leukemia (AML) is the deadliest and most common form of adult leukemia. Each year, over 10,000 people in the United States are diagnosed with AML. While most patients experience a remission of the cancer after initial chemotherapy treatments, approximately 80% subsequently relapse. Fewer than 30% of AML patients survive past one year after relapsing. The high rate of AML relapse has been attributed to a small fraction of cells termed leukemia stem cells. LSCs are highly resistant to multiple chemotherapeutic drugs, and survive initial chemotherapy treatments. They are capable of aggressively restoring the leukemia cell population. Developing a method to target and kill LSCs would benefit AML patients tremendously. However, progress in studying LSC biology has been limited because of the low numbers that can be isolated from patients. This research will develop technology that can investigate, on the single cell level; the sphingosine kinase (SphK) activity of LSCs. SphK is an oncogenic kinase that is known to increase resistance to chemotherapeutic drugs in leukemia cells. The central hypothesis of this proposal is that SphK is up regulated in LSCs, which would make it a promising target for drug therapy. SphK activity will be measured by loading cells with a fluorescent reporter, which can be modified by active SphK. Single-cell capillary electrophoresis (CE) will be used to separate the modified from the unmodified reporter in each cell. CE is an extremely sensitive technique, making it ideal for the biological analysis of individual cells. However, a major limitation of single-cell CE is low throughput (5-35 cells per day). The aim of this project is to develop an automated single-cell CE platform that will dramatically improve throughput, enabling hundreds to thousands of cells to be analyzed per day. This automated CE instrument will then be used to measure SphK activity in LSCs from ML patients. This study will be the first to investigate a biological signalng pathway in human LSCs within individual patients. Characterizing SphK activity in LSCs is expected to contribute to the development of therapies that specifically target LSCs and therefore prevent AML relapse.