Isolation and Characterization of Melanoma Tumor Stem Cells

描述（由申请人提供）：本提案的目标是从诊断患有该疾病的各个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（TSCs）通常对生长停滞信号不敏感，并且具有无限的增殖能力，允许它们重建在传统医疗团之后消除的整个肿瘤细胞群体，甚至具有更具侵袭性的表型。因此，为了有效地治疗癌症，必须鉴定TSC并分析其特性，以设计特异性靶向和根除所有TSC的疗法。具体而言，我们的研究将解决在黑色素瘤发展的每个阶段MTSC的存在，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。我们将通过完成以下具体目标来实现我们的目标：1）从人黑色素瘤样品中鉴定和纯化黑色素瘤肿瘤干细胞（MTSC）; 2）分析MTSC中参与自我更新和维持正常干细胞的信号通路的激活。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志物鉴定的先进技术。我们计划在以下步骤中追求目标1：i）使用组合mAb染色和荧光激活细胞分选（FACS）确定适合于富集MTSC的候选细胞表面标志物; ii）通过进行肿瘤球形成和增殖测定来确定体外分级分离的黑素瘤细胞的肿瘤干细胞身份; iii）通过在免疫受损小鼠中进行肿瘤异种移植和系列移植测定来体内确定候选MTSC的肿瘤干细胞身份和纯度。为了了解可能参与MTSC的出现和维持的分子机制，我们将分析这些细胞参与正常干细胞自我更新和维持的信号通路的激活。这一目标将通过利用慢病毒报告基因测定结合RNAi介导的基因抑制机制来评估候选转录因子在MTSC致瘤潜力中的作用来实现。我们的研究将有助于对黑色素瘤的起源及其进展机制的新认识。我们目标的完成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中，并且这种荷瘤小鼠将成为用于新的黑素瘤治疗和诊断的临床前测试的非常有用的模型。这些程序可以被设计为特异性地照射所有的MTSC，从而提高患者的生存率和肿瘤治疗的功能结果。

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.