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

人白血病干细胞中鞘氨醇激酶活性的单细胞分析

• 批准号: 8395236
• 负责人: Alexandra Jazz Dickinson
• 金额: $ 3.23万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8395236
• 关键词: Acute Myelocytic Leukemia; Address; Adult Acute Myeloblastic Leukemia; Area; Biological; Biological Assay; Biological Process; Blood; Blood capillaries; Cancer Hospital; Cancer Remission; Capillary Electrophoresis; Cell Count; Cell Fraction; Cell Line; Cell Proliferation; Cells; Charge; Cytolysis; Detection; Diagnosis; Fluorescence; Goals; Grant; Health; Hematologic Neoplasms; Heterogeneity; Human; Individual; Institutional Review Boards; LIF gene; Lasers; Malignant Neoplasms; Manuals; Measures; Membrane Transport Proteins; Methods; Mitotic; Mole the mammal; North Carolina; Oncogenic; P-Glycoproteins; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Phosphotransferases; Population; Proliferating; Protocols documentation; Relapse; Reporter; Research; Resistance; Sampling; Second Messenger Systems; Signal Pathway; Signal Transduction; Specificity; Sphingosine; Survival Rate; System; Techniques; Technology; Time; Tissue Banking; Tissue Banks; Translating; United States; Up-Regulation; adult leukemia; capillary; cell growth; cell type; chemotherapy; experience; improved; inorganic phosphate; instrument; instrumentation; interest; killings; leukemia; leukemic stem cell; migration; peripheral blood; prevent; second messenger; single cell analysis; sphingosine kinase; stem cell biology; stem cell population; therapy development; tool

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. PUBLIC HEALTH RELEVANCE: The activity of an oncogenic kinase will be characterized in leukemia stem cells (LSCs) from patients with acute myeloid leukemia (AML). The goal is to identify a kinase pathway that can be targeted to decrease LSC resistance to chemotherapeutics, and therefore prevent AML relapse.

描述（由申请人提供）： 急性髓性白血病 (AML) 是最致命且最常见的成人白血病。美国每年有超过 10,000 人被诊断患有 AML。虽然大多数患者在初次化疗后癌症得到缓解，但大约 80% 的患者随后会复发。不到 30% 的 AML 患者在复发后能存活超过一年。 AML 的高复发率归因于一小部分被称为白血病干细胞的细胞。 LSC 对多种化疗药物具有高度耐药性，并且能够在初始化疗后存活。它们能够积极恢复白血病细胞群。 开发一种靶向并杀死 LSC 的方法将使 AML 患者受益匪浅。然而，由于可以从患者体内分离的LSC数量很少，研究LSC生物学的进展受到限制。这项研究将开发可以在单细胞水平上进行研究的技术； LSC 的鞘氨醇激酶 (SphK) 活性。 SphK 是一种致癌激酶，已知会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 LSC 中表达上调，这将使其成为药物治疗的有希望的靶点。 SphK 活性将通过在细胞上装载荧光报告基因来测量，荧光报告基因可以通过活性 SphK 进行修饰。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与未修饰的报告基因。 CE 是一种极其灵敏的技术，非常适合单个细胞的生物分析。然而，单细胞 CE 的一个主要限制是通量低（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毛细管电泳平台，该平台将显着提高通量，每天可以分析数百至数千个细胞。然后，该自动化 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 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专门针对 LSC 的疗法，从而预防 AML 复发。 公共健康相关性：将在急性髓性白血病 (AML) 患者的白血病干细胞 (LSC) 中表征致癌激酶的活性。目标是确定一种激酶途径，可降低 LSC 对化疗药物的耐药性，从而预防 AML 复发。