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: 8573551
• 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/8573551
• 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; public health relevance; 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.

描述（由申请人提供）：急性骨髓性白血病（AML）是成人白血病的最致命，最常见的形式。每年，美国有10,000多人被诊断出患有AML。尽管大多数患者在初次化疗后经历了癌症的缓解，但随后大约80％的复发。复发后一年中，不到30％的AML患者存活。 AML复发的高率归因于一小部分称为白血病干细胞的细胞。 LSC对多种化学治疗药物具有高度耐药性，并且可以在初始化疗治疗中生存。他们能够积极恢复白血病细胞群体。 开发靶向和杀死LSC的方法将极大地使AML患者受益。但是，研究LSC生物学方面的进展受到限制，因为可以从患者中分离出来的数量较低。这项研究将开发可以在单细胞水平上调查的技术。 LSC的鞘氨醇激酶（SPHK）活性。 SPHK是一种致癌激酶，已知可以增加白血病细胞中化学治疗药物的抗性。该提案的中心假设是SPHK在LSC中受到了调节，这将使它成为药物治疗的有希望的靶标。 SPHK活性将通过用荧光报告基因加载细胞来测量，该细胞可以通过Active SPHK修改。单细胞毛细管电泳（CE）将用于将修饰的修饰者与每个单元中未修饰的报告基因分开。 CE是一种非常敏感的技术，使其非常适合单个细胞的生物学分析。但是，单细胞CE的主要局限性是低吞吐量（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，该平台将大大改善吞吐量，每天可以分析数百到数千个细胞。然后，该自动化CE仪器将用于测量ML患者LSC的SPHK活性。 这项研究将是第一个研究个别患者中人类LSC的生物信号途径的研究。 LSC中SPHK活性的表征有望有助于特异性针对LSC并因此防止AML复发的疗法的发展。