Zhou Fangbin, Zhou Yaying, Yang Ming, Wen Jinli, Dong Jun, Tan Wenyong
Department of Oncology, The Second Clinical Medical College, Shenzhen People's Hospital, Jinan University, Shenzhen, People's Republic of China.
Integrated Chinese and Western Medicine Postdoctoral Research Station, Jinan University, Guangzhou, People's Republic of China.
Cancer Manag Res. 2018 Mar 8;10:447-464. doi: 10.2147/CMAR.S157837. eCollection 2018.
Circulating endothelial cells (CECs) and their subpopulations could be potential novel biomarkers for various malignancies. However, reliable enumerable methods are warranted to further improve their clinical utility. This study aimed to optimize a flow cytometric method (FCM) assay for CECs and subpopulations in peripheral blood for patients with solid cancers.
An FCM assay was used to detect and identify CECs. A panel of 60 blood samples, including 44 metastatic cancer patients and 16 healthy controls, were used in this study. Some key issues of CEC enumeration, including sample material and anticoagulant selection, optimal titration of antibodies, lysis/wash procedures of blood sample preparation, conditions of sample storage, sufficient cell events to enhance the signal, fluorescence-minus-one controls instead of isotype controls to reduce background noise, optimal selection of cell surface markers, and evaluating the reproducibility of our method, were integrated and investigated. Wilcoxon and Mann-Whitney tests were used to determine statistically significant differences.
In this validation study, we refined a five-color FCM method to detect CECs and their subpopulations in peripheral blood of patients with solid tumors. Several key technical issues regarding preanalytical elements, FCM data acquisition, and analysis were addressed. Furthermore, we clinically validated the utility of our method. The baseline levels of mature CECs, endothelial progenitor cells, and activated CECs were higher in cancer patients than healthy subjects (<0.01). However, there was no significant difference in resting CEC levels between healthy subjects and cancer patients (=0.193).
We integrated and comprehensively addressed significant technical issues found in previously published assays and validated the reproducibility and sensitivity of our proposed method. Future work is required to explore the potential of our optimized method in clinical oncologic applications.
循环内皮细胞（CECs）及其亚群可能是多种恶性肿瘤潜在的新型生物标志物。然而，需要可靠的计数方法来进一步提高其临床应用价值。本研究旨在优化一种流式细胞术（FCM）检测方法，用于检测实体癌患者外周血中的CECs及其亚群。
采用FCM检测法检测和鉴定CECs。本研究使用了一组60份血样，包括44例转移性癌症患者和16例健康对照。综合研究了CEC计数的一些关键问题，包括样本材料和抗凝剂的选择、抗体的最佳滴定、血样制备的裂解/洗涤程序、样本储存条件、增强信号所需的足够细胞事件、用荧光减一对照代替同型对照以降低背景噪声、细胞表面标志物的最佳选择以及评估我们方法的可重复性。采用Wilcoxon和Mann-Whitney检验确定统计学上的显著差异。
在本验证研究中，我们改进了一种五色FCM方法，用于检测实体瘤患者外周血中的CECs及其亚群。解决了有关分析前因素、FCM数据采集和分析的几个关键技术问题。此外，我们在临床上验证了我们方法的实用性。癌症患者中成熟CECs、内皮祖细胞和活化CECs的基线水平高于健康受试者（<0.01）。然而，健康受试者和癌症患者之间静息CEC水平无显著差异（=0.193）。
我们整合并全面解决了先前发表的检测方法中发现的重大技术问题，并验证了我们所提出方法的可重复性和敏感性。未来需要开展工作，探索我们优化方法在临床肿瘤学应用中的潜力。
