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

描述（由申请人提供）：急性骨髓性白血病（AML）是最致命和最常见的成人白血病。每年，美国有超过10，000人被诊断患有AML。虽然大多数患者在最初的化疗治疗后经历了癌症的缓解，但约80%随后复发。不到30%的AML患者在复发后存活超过一年。AML的高复发率归因于一小部分称为白血病干细胞的细胞。LSC对多种化疗药物具有高度抗性，并在初始化疗治疗中存活。它们能够积极地恢复白血病细胞群。 开发一种靶向和杀死LSC的方法将使AML患者受益匪浅。然而，LSC生物学研究的进展受到限制，因为可以从患者中分离出的数量很少。这项研究将开发可以在单细胞水平上研究LSC的鞘氨醇激酶（SphK）活性的技术。SphK是一种致癌激酶，已知其增加白血病细胞对化疗药物的抗性。该提议的中心假设是SphK在LSC中上调，这将使其成为药物治疗的有希望的靶点。SphK活性将通过用荧光报告物加载细胞来测量，所述荧光报告物可以被活性SphK修饰。单细胞毛细管电泳（CE）将用于分离每个细胞中的修饰报告基因和未修饰报告基因。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的主要限制是低通量（每天5-35个细胞）。该项目的目的是开发一个自动化的单细胞CE平台，该平台将大大提高通量，每天可以分析数百至数千个细胞。然后，这种自动化CE仪器将用于测量ML患者LSC中的SphK活性。 这项研究将是第一个研究个体患者体内人类LSC中的生物信号通路的研究。表征LSC中的SphK活性有望有助于开发特异性靶向LSC的疗法，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.