Isolating Circulating Tumor Cells

 描述（由申请人提供）：我们提出开发一种从乳腺癌患者的外周血中分离和筛选CTC的有效的、无标记的方法。对于这个拟议的R21项目，我们将专注于乳腺癌，但我们的技术可以普遍应用于其他癌症，如结肠癌，前列腺癌，肺癌和黑色素瘤。我们的方法将涉及基于大小分离候选CTC，然后以无标记的方式同时筛选分离的细胞的多种表面标志物，例如EpCAM、Axl、CD 44、CD 24、ALDH 1和CD 45（以鉴定污染的白细胞），从而鉴定上皮、EMT或干细胞样的CTC。将基于表型对细胞进行分选，并易于用于进一步的下游分析/分子表征。我们将使用健康的人类供体血液来参数化和优化我们的平台，这些血液掺有来自乳腺癌细胞系MCF-7、SKBR 3（一种HER 2+细胞系）和MDA-MB- 231（一种“三阴性”间充质乳腺癌细胞系）的细胞，并且通过EMT主调节因子LBX 1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志物EpCAM、间充质标志物CD 44和CD 24、干细胞标志物ALDH 1、EMT标志物Axl和白细胞标志物CD 45（以鉴定和去除污染的白细胞）。我们将使用乳腺癌患者血液对我们的平台进行基准测试，并将我们的结果（CTC/患者抽血的数量）与CellSearch进行比较。我们提出的技术的关键创新方面包括：1）从全血中无标记富集和筛选CTC;以及2）多标记筛选和随后将CTC分选成亚群。在单一平台上进行无标记的多标记筛选和将CTC分选为亚群是我们的方法所独有的，并且解决了生物医学研究和医学界未满足的需求。研究人员可以对CTC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。临床医生可能将亚群与预后结果和疾病进展联系起来。莉迪亚湖加州大学伯克利分校机械工程副教授Sohn将领导NIH R21项目。Mark LaBarge博士是劳伦斯伯克利国家实验室的癌症生物学家专家，他将为该项目的生物学方面提供指导，Tianhong Li博士是加州大学戴维斯分校综合癌症中心I期项目的医生科学家和联合主任，他将为样本选择，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