Isolation and Characterization of Tumor Stem Cells from Melanoma Patients

描述（由申请人提供）：在缺乏有效治疗方案的情况下，黑色素瘤在美国的迅速上升，强调了获得有关该疾病发病机制的更深入知识的紧迫性和重要性。在这个项目中，我们提出黑色素瘤是由CD 271阳性肿瘤起始细胞亚群驱动的。其他癌症中的肿瘤干细胞（TSCs）对大多数常规化疗和放疗具有抗性，能够自我更新并有效补充传统药物治疗后消除的整个肿瘤细胞群。因此，为了设计更成功的癌症治疗，必须分离和研究各自的TSC的特性。该提议的主要目标是从广谱黑色素瘤中纯化分离高度富集的黑色素瘤TSC并表征其分子谱。我们将通过设计和完成以下实验任务来实现这一目标： 1.使用先进的荧光激活细胞分选和体内肿瘤移植试验，通过CD 271和其他细胞表面标志物表达识别并前瞻性分离黑色素瘤TSC。 2.通过使用微阵列和慢病毒报告基因技术对MTSC进行全局基因表达分析，表征位于CD 271 + MTSC功能根源的遗传和/或表观遗传改变;确认特定候选基因在MTSC表型中的关键作用 3.通过描述MTSC的侵袭性特征和CD 271在人类皮肤重建小鼠模型中的功能作用，分析MTSC在环境中的晚期致瘤性，该环境密切反映了这种癌症在人类中的自然发生。 公共卫生相关性：传统的抗癌疗法是基于缩小大块肿瘤大小的筛查方法开发的。虽然它们在减小肿瘤的大小方面是有效的，但它们可能会丢失肿瘤干细胞或癌症的“根源”;因此，肿瘤干细胞可以在原发部位重建整个肿瘤或迁移到远端器官。黑色素瘤中TSC的鉴定表征将在用靶向疗法治疗这种疾病方面开辟全新的维度，所述靶向疗法是专门针对这些细胞设计的，并且与先前已知的方案组合将消除肿瘤的所有细胞组分。

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. PUBLIC HEALTH RELEVANCE: Conventional anti-cancer therapies are developed based on screening method that shrink the size of bulk tumors. While they are effective in reducing the size of the tumor, they may be missing the TSCs or the "roots" of cancer; as a result TSCs can either reconstitute an entire tumor at the primary site or migrate to distal organs. Identification characterization of TSCs in melanomas will open entirely new dimension in treating this disease with targeted therapies that are designed specifically against these cells and in combination with previously known regimens will eliminate all cellular components of the tumor.