Division of Drug Discovery and Development, Shizuoka Cancer Center Research Institute, Shizuoka, Japan.
Cytometry A. 2014 Mar;85(3):206-13. doi: 10.1002/cyto.a.22422. Epub 2013 Dec 10.
The presence and number of circulating tumor cells (CTCs) in the blood of patients with solid tumors are predictive of their clinical outcomes. To date, the CellSearch system is the only US Food and Drug Administration-approved CTC enumeration system for advanced breast, prostate, and colon cancers. However, sensitivity issues due to epithelial cellular adhesion molecule (EpCAM)-based enrichment and limited capability for subsequent molecular analysis must be addressed before CTCs can be used as predictive markers in the clinical setting. We have developed a multicolor CTC detection system using cross-contamination-free flow cytometry, which permits the enumeration and characterization of CTCs for multiple molecular analyses. Tumor cell lines with different expression levels of EpCAM were spiked into peripheral blood obtained from healthy donors. Spike-in samples were negatively enriched using anti-CD45-coated magnetic beads to remove white blood cells, and this was followed by fixation and labeling with CD45-Alexa Fluor 700, EpCAM-phycoerythrin, cytokeratin (CK)-fluorescein isothiocyanate antibodies, and/or 7-aminoactinomycin D for nuclei staining. Excellent detection (slope = 0.760-0.888) and a linear performance (R(2) = 0.994-0.998) were noted between the observed and expected numbers of tumor cells, independent of EpCAM expression. The detection rate was markedly higher than that obtained using the CellSearch system, suggesting the superior sensitivity of our system in detecting EpCAM- tumor cells. Additionally, the incorporation of an epithelial-mesenchymal transition (EMT) marker allowed us to detect EpCAM-/CK- cells and EMT-induced tumor cells. Taken together, our multicolor CTC detection system may be highly efficient in detecting previously unrecognized populations of CTCs.
循环肿瘤细胞（CTC）在实体瘤患者血液中的存在和数量可预测其临床结局。迄今为止，CellSearch 系统是唯一获得美国食品和药物管理局批准的用于晚期乳腺癌、前列腺癌和结肠癌的 CTC 计数系统。然而，由于基于上皮细胞黏附分子（EpCAM）的富集以及后续分子分析能力有限，在 CTC 可作为临床预测标志物之前，必须解决敏感性问题。我们使用无污染的流式细胞术开发了一种多色 CTC 检测系统，该系统可用于对 CTC 进行计数和特征分析，进行多种分子分析。将具有不同 EpCAM 表达水平的肿瘤细胞系掺入来自健康供体的外周血中。使用抗 CD45 包被的磁性珠对掺入的样本进行负性富集，以去除白细胞，然后进行固定和标记，使用 CD45-Alexa Fluor 700、EpCAM-藻红蛋白、细胞角蛋白（CK）-异硫氰酸荧光素抗体和/或 7-氨基放线菌素 D 进行核染色。在独立于 EpCAM 表达的情况下，观察到与预期肿瘤细胞数量之间存在极好的检测（斜率=0.760-0.888）和线性性能（R(2)=0.994-0.998）。检测率明显高于 CellSearch 系统获得的检测率，表明我们的系统在检测 EpCAM-肿瘤细胞方面具有更高的灵敏度。此外，上皮-间质转化（EMT）标志物的加入使我们能够检测到 EpCAM-/CK-细胞和 EMT 诱导的肿瘤细胞。总之，我们的多色 CTC 检测系统在检测先前未被识别的 CTC 群体方面可能非常有效。
