Isolating Circulating Tumor Cells

 描述(由申请人提供)：我们建议开发一种从乳腺癌患者的外周血中分离和筛选CTC的有效、无标记的方法。对于这个提议的R21项目，我们将专注于乳腺癌，但我们的技术通常可以应用于其他癌症，如结肠癌、前列腺癌、肺癌和黑色素瘤。我们的方法将包括根据大小分离候选CTC，然后以无标记的方式同时筛选所分离的细胞的多个表面标志，例如EpCAM、AXL、CD44、CD24、ALDH1和CD45(以识别污染的白细胞)，从而识别上皮样CTC、EMT CTC或干细胞样CTC。细胞将根据表型进行分类，并随时可用于进一步的下游分析/分子表征。我们将使用健康的人类捐献者血液对我们的平台进行参数化和优化，使用来自乳腺癌细胞系MCF-7、SKBR3(HER2+细胞系)和MDA-MB-231(三阴性的间叶性乳腺癌细胞系)的细胞，以及通过EMT主控调控因子LBX1的异位表达诱导MCF-10A细胞进入EMT。我们将筛选无标记的分离细胞的上皮标记EpCAM、间充质标记CD44和CD24、干细胞标记ALDH1、EMT标记Ax1和白细胞标记CD45(以识别和去除受污染的白细胞)。我们将使用乳腺癌患者的血液对我们的平台进行基准测试，并将我们的结果(CTCs数量/患者抽血)与CellSearch进行比较。我们建议的技术的主要创新方面包括：1)全血中CTCs的无标记浓缩和筛选；以及2)CTCs的多标记筛选和随后的亚群分类。在单一平台上进行无标签多标记筛选和将CTC分类为子群体是我们的方法所独有的，并解决了生物医学研究和医学界尚未满足的需求。研究人员可以对CTCs进行二次分析或分子诊断，而不必担心细胞没有受到标记的影响。临床医生可能会潜在地将亚群与预后和疾病进展相关联。皮莉迪亚·L·孙(Pi Lydia L.Sohn，Assoc.机械工程系教授。加州大学伯克利分校将领导这个NIH R21项目。劳伦斯伯克利国家实验室的癌症生物学家Mark LaBarge博士将就该项目的生物学方面提供指导，加州大学戴维斯综合癌症中心内科科学家兼第一阶段项目联席主任李天宏将就样本选择、实验设计、数据分析和临床相关性提供指导

 DESCRIPTION (provided by applicant): We propose to develop an efficient, label-free method of isolating and screening CTCs from peripheral blood of breast-cancer patients. For this proposed R21 project, we will focus on breast cancer, but our technique could be generally applied to other cancers such as colon, prostate, lung, and melanoma. Our method will involve isolating candidate CTCs based on size and then screening in a label-free manner the isolated cells for multiple surface markers simultaneously, e.g. EpCAM, Axl, CD44, CD24, ALDH1, and CD45 (to identify contaminating leukocytes), thereby identifying CTCs which are epithelial, EMT, or stem-cell like. Cells would be sorted based on phenotype and readily available for further downstream analysis/molecular characterization. We will parameterize and optimize our platform using healthy human donor blood spiked with cells from breast-cancer cell lines, MCF-7, SKBR3 (a HER2+ cell line), and MDA-MB- 231 (a "triple-negative" mesenchymal breast cancer cell line), and with MCF-10A cells induced into EMT via the ectopic expression of the EMT master regulator LBX1. We will screen label-free isolated cells for the epithelial marker, EpCAM, mesenchymal markers CD44 and CD24, stem-cell marker ALDH1, EMT marker Axl, and leukocyte marker CD45 (to identify and remove contaminating leukocytes). We will benchmark our platform using breast-cancer patient blood and compare our results (number of CTCs/patient blood draw) with CellSearch. The key innovative aspects of our proposed technology include: 1) the label-free enrichment and screening of CTCs from whole blood; and 2) multi-marking screening and subsequent sorting of CTCs into sub-populations. Performing label-free multi-marker screening and sorting CTCs into sub-populations on a single platform are unique to our method, alone, and address unmet needs of both biomedical research and medical communities. Researchers could perform secondary analysis or molecular diagnostics of CTCs without worry that the cells have not been affected by labeling. Clinicians could potentially correlate sub- populations with prognostic outcome and disease progression. PI Lydia L. Sohn, Assoc. Prof. of Mechanical Eng. at the University of California, Berkeley will lead this NIH R21 project. Drs. Mark LaBarge, an expert cancer biologist at Lawrence Berkeley National Laboratory, who will provide guidance on the biological aspects of the project, and Tianhong Li, physician scientist and co-Director of the Phase I Program at UC Davis Comprehensive Cancer Center, who will provide guidance on sample choice, experimental design, data analysis, and clinical relevance, will advise her