Isolation and Characterization of Melanoma Tumor Stem Cells

黑色素瘤肿瘤干细胞的分离和表征

基本信息

批准号：
7743838
负责人：
ALEXANDER D BOIKO
金额：
$ 5.38万
依托单位：
STANFORD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-12-01 至 2010-11-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): The goal of this proposal is to isolate and characterize melanoma tumor stem cells (MTSCs) from the patients diagnosed with various stages of this disease. Tumor stem cells (TSCs) are often insensitive to growth arresting signals and have infinite proliferative capacity allowing them to re-establish the entire tumor cell population eliminated after traditional medical regiments even with more aggressive phenotype. Thus, to treat cancer efficiently TSCs have to be identified and their properties analyzed to design the therapies that specifically target and eradicate all of them. Specifically, our studies will address the existence of MTSCs at each stage of melanoma development and analyze the role of stem cell self-renewing pathways in maintaining tumorigenic potential of MTSCs. We will achieve our goal by completing following specific aims: 1) To identify and purify melanoma tumor stem cells (MTSCs) from human melanoma samples; 2) To analyze MTSCs for activation of signaling pathways involved in self-renewal and maintenance of normal stem cells. Our research design is based on the advanced techniques for stem cell sorting and identification of stem cell related markers developed in our laboratory. We plan to pursue aim 1 in the following steps: i) to determine candidate cell surface markers suitable for enrichment of MTSCs using combinatorial mAb staining and fluorescent activated cell sorting (FACS); ii) to determine tumor stem cell identity of fractionated melanoma cells in-vitro by performing tumor sphere formation and proliferation assays; iii) to determine tumor stem celt identity and purity of candidate MTSCs in-vivo by performing tumor xenograft and serial transplantation assays in immunocompromised mice. To understand molecular mechanisms that might be involved in emergence and maintenance of MTSCs we will analyze these cells for activation of signaling pathways involved in self-renewal and maintenance of normal stem cells. This aim will be pursued by utilizing lentiviral reporter assays combined with RNAi mediated mechanism of gene suppression to asses the role of candidate transcription factors in tumorigenic potential of MTSCs. Our studies will contribute to the new understanding of mechanisms underlying the origins of melanoma and its progression. Completion of our aims will identify new cellular and molecular targets for drug and immune melanoma therapies. Isolated MTSCs can be transplanted into immunodeficient mice and such tumor bearing mice will become a very useful model for preclinical testing of new melanoma treatments and diagnostics. These procedures can be designed to specifically irradicatge all of MTSCs and thus improve patient survival rate and functional outcome of tumor therapy.

描述（由申请人提供）：该提案的目标是从诊断为该疾病不同阶段的患者中分离和表征黑色素瘤肿瘤干细胞（MTSC）。肿瘤干细胞（TSC）通常对生长抑制信号不敏感，并且具有无限的增殖能力，使它们能够重建传统医疗治疗后消除的整个肿瘤细胞群，甚至具有更具侵袭性的表型。因此，为了有效治疗癌症，必须识别 TSC 并分析其特性，以设计专门针对和根除所有 TSC 的疗法。具体来说，我们的研究将解决黑色素瘤发展各个阶段的 MTSC 的存在，并分析干细胞自我更新途径在维持 MTSC 致瘤潜力中的作用。我们将通过完成以下具体目标来实现我们的目标：1）从人类黑色素瘤样本中鉴定和纯化黑色素瘤肿瘤干细胞（MTSC）； 2) 分析 MTSC 激活参与正常干细胞自我更新和维持的信号通路。我们的研究设计基于我们实验室开发的干细胞分选和干细胞相关标记物鉴定的先进技术。我们计划通过以下步骤实现目标 1：i) 使用组合 mAb 染色和荧光激活细胞分选 (FACS) 确定适合富集 MTSC 的候选细胞表面标记； ii) 通过进行肿瘤球形成和增殖测定来确定体外分离的黑色素瘤细胞的肿瘤干细胞身份； iii) 通过在免疫功能低下的小鼠中进行肿瘤异种移植和连续移植测定，确定体内候选 MTSC 的肿瘤干细胞身份和纯度。为了了解可能涉及 MTSC 出现和维持的分子机制，我们将分析这些细胞激活参与正常干细胞自我更新和维持的信号通路。该目标将通过利用慢病毒报告基因检测结合 RNAi 介导的基因抑制机制来评估候选转录因子在 MTSC 致瘤潜力中的作用。我们的研究将有助于对黑色素瘤起源及其进展机制的新认识。完成我们的目标将为药物和免疫黑色素瘤治疗确定新的细胞和分子靶点。分离的 MTSC 可以移植到免疫缺陷小鼠体内，这种荷瘤小鼠将成为新黑色素瘤治疗和诊断的临床前测试非常有用的模型。这些程序可以设计用于专门照射所有 MTSC，从而提高患者的生存率和肿瘤治疗的功能结果。