Isolation and Characterization of Melanoma Tumor Stem Cells

黑色素瘤肿瘤干细胞的分离和表征

• 批准号: 7329397
• 负责人: ALEXANDER D BOIKO
• 金额: $ 4.96万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 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
• 来源: https://reporter.nih.gov/project-details/7329397
• 关键词: Address; Asses; Biological Assay; Cell Separation; Cell surface; Cells; Development; Diagnosis; Diagnostic; Disease; Event; Genes; Goals; Growth; Human; Immune; Immunocompromised Host; Immunodeficient Mouse; In Vitro; Laboratories; Maintenance; Malignant Neoplasms; Mediating; Medical; Melanoma Cell; Modeling; Molecular; Molecular Target; Mus; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Population; Preclinical Testing; Procedures; Property; RNA Interference; Reporter; Research Design; Role; Sampling; Signal Pathway; Signal Transduction; Staging; Staining method; Stains; Stem cells; Survival Rate; Techniques; Transplantation; Tumor Stem Cells; base; cancer cell; combinatorial; design; functional outcomes; improved; in vivo; melanocyte; melanoma; neoplastic cell; self-renewal; stem; therapy design; transcription factor; tumor; tumor xenograft; tumorigenic

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)：本提案的目标是从诊断为本病不同阶段的患者中分离和鉴定黑色素瘤肿瘤干细胞(MTSCs)。肿瘤干细胞(TSCs)通常对生长抑制信号不敏感，具有无限的增殖能力，使其能够重建传统药物治疗后被消灭的整个肿瘤细胞群，甚至具有更具侵袭性的表型。因此，为了有效地治疗癌症，必须识别TSCs并分析其特性，以设计专门针对并根除所有TSCs的治疗方法。具体地说，我们的研究将探讨黑色素瘤发展的每个阶段中MTSCs的存在，并分析干细胞自我更新途径在维持MTSCs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)从人黑色素瘤样本中鉴定和纯化黑色素瘤肿瘤干细胞(MTSCs)；2)分析MTSCs激活与自我更新和维持正常干细胞有关的信号通路。我们的研究设计是基于我们实验室开发的干细胞分选和干细胞相关标记鉴定的先进技术。我们计划在以下步骤中实现目标1：i)使用组合单抗染色和荧光激活细胞分选(FACS)来确定适合于富集MTSCs的候选细胞表面标志；ii)通过进行肿瘤球体形成和增殖试验来确定分离的黑色素瘤细胞在体外的肿瘤干细胞特性；iii)通过在免疫低下小鼠中进行肿瘤移植和系列移植试验来确定候选MTSCs在体内的肿瘤干细胞特性和纯度。为了了解可能参与MTSCs产生和维持的分子机制，我们将分析这些细胞是否激活了参与正常干细胞自我更新和维持的信号通路。这一目标将通过慢病毒报告实验结合RNAi介导的基因抑制机制来实现，以评估候选转录因子在MTSCs致瘤潜力中的作用。我们的研究将有助于重新理解黑色素瘤的起源和进展的潜在机制。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MTSCs可以移植到免疫缺陷小鼠体内，这样的荷瘤小鼠将成为临床前测试新的黑色素瘤治疗和诊断的非常有用的模型。这些程序可以被设计成专门照射所有的MTSCs，从而提高患者的存活率和肿瘤治疗的功能结果。