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）：该提案的目的是与诊断出患有该疾病的各个阶段的患者分离和表征黑色素瘤肿瘤干细胞（MTSC）。肿瘤干细胞（TSC）通常对生长引起的信号不敏感，并且具有无限的增殖能力，从而使他们能够在传统的医疗方案后重新建立整个肿瘤细胞种群，即使具有更具侵略性的表型。因此，必须确定有效治疗癌症，并且必须分析其特性，以设计专门针对和消除所有这些的疗法。具体而言，我们的研究将探讨在黑色素瘤发育的每个阶段的MTSC存在，并分析干细胞自我更新途径在维持MTSC的致瘤潜力中的作用。我们将通过以下特定目的来完成目标：1）从人黑色素瘤样品中识别和净化黑色素瘤肿瘤干细胞（MTSC）； 2）分析MTSC以激活与正常干细胞的自我更新和维持有关的信号通路。我们的研究设计基于用于干细胞分选和鉴定干细胞相关标记的先进技术。我们计划在以下步骤中追求AIM 1：i）确定适合使用组合MAB染色和荧光活化细胞分选（FACS）的候选细胞表面标记； ii）通过执行肿瘤球体形成和增殖测定法，确定分离的黑色素瘤细胞的肿瘤干细胞认同； iii）通过在免疫功能低下的小鼠中执行肿瘤异种移植和连续移植测定，确定候选MTSC的肿瘤celt认同和纯度。为了了解可能涉及MTSC的出现和维持的分子机制，我们将分析这些细胞，以激活与正常干细胞的自我更新和维持有关的信号传导途径。将通过利用慢病毒报告基因抑制机制来实现此目标，以评估候选转录因子在MTSC的致瘤潜力中的作用。我们的研究将有助于对黑色素瘤起源及其进展的基础机制的新理解。我们目标的完成将确定用于药物和免疫黑色素瘤疗法的新细胞和分子靶标。可以将分离的MTSC移植到免疫缺陷的小鼠中，这种肿瘤轴承小鼠将成为一种非常有用的模型，用于临床前测试新的黑色素瘤治疗和诊断。这些程序可以设计为专门辐照所有MTSC，从而提高患者的存活率和肿瘤治疗的功能结果。