Sen Moen, Wang Ling, Yu Liping, Carpenter Erica L
Division of Hematology and Oncology, Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
BD Technologies and Innovation, Research Triangle Park, NC, USA.
Methods Mol Biol. 2019;2032:213-226. doi: 10.1007/978-1-4939-9650-6_13.
Noninvasive isolation of circulating tumor cells (CTCs) from patient blood samples allows for interrogation of valuable molecular and phenotypic information useful for disease diagnosis and monitoring response to therapy. However, CTCs are extremely rare relative to red and white blood cells (R/WBC), thus making CTC isolation from unmanipulated whole blood very time-consuming. Moreover, single CTC analysis often requires hand-picking, a step that can result in more CTC loss and compromised cell integrity. Here we describe an automated flow cytometry-based approach for isolation and analysis of single, viable CTCs that combines gentle RBC lysis and magnetic, no-wash negative-depletion of WBCs, followed by a highly adaptable sorting protocol for rare cells of interest. Multiparametric flow-cytometric panels allow probing of numerous extracellular markers for immunophenotyping, while whole transcriptome analysis contributes to molecular characterization of individual CTCs. Index sorting links single CTC proteogenomics information.
从患者血液样本中无创分离循环肿瘤细胞（CTC），有助于获取对疾病诊断和治疗反应监测有价值的分子和表型信息。然而，相对于红细胞和白细胞（R/WBC），CTC极其罕见，因此从未经处理的全血中分离CTC非常耗时。此外，单个CTC分析通常需要手工挑选，这一步骤可能导致更多的CTC损失并损害细胞完整性。在此，我们描述了一种基于自动流式细胞术的方法，用于分离和分析单个活的CTC，该方法结合了温和的红细胞裂解以及白细胞的磁性免洗阴性去除，随后是针对稀有目标细胞的高度适应性分选方案。多参数流式细胞术面板允许探测众多细胞外标志物进行免疫表型分析，而全转录组分析有助于单个CTC的分子特征鉴定。索引分选将单个CTC的蛋白质基因组学信息联系起来。
