Isolation and Characterization of Tumor Stem Cells from Melanoma Patients

描述(由申请人提供)：在缺乏有效的治疗方案的情况下，黑色素瘤在美国迅速上升，这突显了深入了解这种疾病的发病机制的紧迫性和重要性。在这个项目中，我们认为黑色素瘤是由CD271阳性的肿瘤启动细胞亚群驱动的。其他癌症中的肿瘤干细胞(TSCs)被证明对大多数传统的化疗和放射治疗具有抵抗力，具有自我更新和有效补充传统药物治疗后消除的整个肿瘤细胞群的能力。因此，要设计出更成功的癌症治疗方法，必须分离和研究各自的TSCs的特性。这项建议的主要目的是从广谱的黑色素瘤中分离高度浓缩的黑色素瘤TSCs，并鉴定它们的分子特征。我们将通过设计和完成以下实验任务来实现这一目标： 1.通过CD271鉴定和前瞻性分离黑色素瘤TSC，并通过先进的荧光激活细胞分选和体内肿瘤移植试验表达更多的细胞表面标志。 2.通过使用微阵列和慢病毒报告技术进行MTSC的全球基因表达分析，鉴定CD271+MTSC功能根源的遗传和/或表观遗传学改变；确认特定基因候选在MTSC表型中的关键作用 3.通过分析MTSC的侵袭特性和CD271在人皮肤重建小鼠模型中的作用，分析MTSC在与人类自然发生的癌症密切相关的环境中的高级致瘤特性。

DESCRIPTION (provided by applicant): The rapid rise of melanoma, in the United States, in the absence of effective therapeutic regimens underscores the urgency and importance of obtaining a deeper knowledge about pathogenesis of this disease. In this project we propose that melanoma is driven by subpopulation of CD271 positive tumor initiating cells. Tumor stem cells (TSCs) in other cancers were shown to be resistant to most conventional chemo and radio therapies, to have the ability to self-renew and efficiently replenish the entire tumor cell population eliminated after traditional medical regimens. Thus to design more successful cancer therapy one must isolate and study the properties of respective TSCs. The primary goal of this proposal is to putatively isolate highly enriched melanoma TSCs from broad spectrum of melanomas and to characterize their molecular profile. We will reach this goal by designing and accomplishing following experimental tasks: 1. Identify and prospectively isolate melanoma TSC by CD271 and additional cell surface markers expression using advanced Fluorescent Activated Cell Sorting and in-vivo tumor transplantation assays. 2. Characterize genetic and/or epigenetic alterations that lie at the root of CD271+ MTSC function by performing global gene expression analysis of MTSCs using microarray and lentiviral reporter technologies; confirm critical role of specific gene candidates in MTSC phenotype 3. Analyze MTSC's advanced tumorigenic properties in the environment that closely reflects natural occurrence of this cancer in humans by characterizing invasive characteristics of MTSCs and a functional role of CD271 in human skin reconstruct mouse model.