Yin Jian, Wang Yi, Yin Hanlu, Chen Wenping, Jin Guangfu, Ma Hongxia, Dai Juncheng, Chen Jiaping, Jiang Yue, Wang Hui, Liu Zhian, Hu Zhibin, Shen Hongbing
Department of Epidemiology & Biostatistics, School of Public Health, Nanjing Medical University, Nanjing, China; Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Cancer Center, School of Public Health, Nanjing Medical University, Nanjing, China.
Department of Respiratory Diseases, Nanjing Chest Hospital, Nanjing, China.
PLoS One. 2015 Aug 28;10(8):e0137076. doi: 10.1371/journal.pone.0137076. eCollection 2015.
It has been considered that the detection methods for circulating tumor cells (CTCs) based on epithelial cell adhesion molecule (EpCAM) underestimate the number of CTCs and may miss a metastatic subpopulation with cancer stem cell (CSC) properties. Therefore, we investigated EpCAM-positive and -negative CTCs in non-small cell lung cancer (NSCLC) patients at different stages, assessed the clinical value of these CTCs and explored their capacity in the following CSC model.
CTCs were enriched by the depletion of leukocytes with bi-antibodies using a magnetic bead separation technique and then identified by the expression of EpCAM and cytokeratin 7 and 8 using multi-parameter flow cytometry. We determined the distribution of CTCs classified by the expression of EpCAM in 46 NSCLC patients with stages I to IV, assessed the diagnostic value of these CTCs by longitudinal monitoring in 4 index patients during adjuvant therapy and characterized the stemness of these CTCs by the expression of CXCR4 and CD133 in 10 patients.
EpCAM-negative (E-) CTCs were detected to be significantly higher than EpCAM-positive (E+) CTCs in stage IV (p = 0.003). The patients with the percentage of E-CTCs more than 95% (r > 95%) were detected to be significantly increased from 13.3% in stage I-II to 61.1% in stage IV (p = 0.006). Kaplan-Meier analysis indicated that the patients with r > 95% had significantly shorter survival time than those with r ≤ 0.95 (p = 0.041). Longitudinal monitoring of CTCs indicated that the patients with a high percentage of E-CTCs in the blood were not responsive to either chemotherapy or targeted therapy. Further characterization of CTCs revealed that a stem-like subpopulation of CXCR4+CD133+ CTCs were detected to be significantly more prevalent in E-CTCs than that in E+CTCs (p = 0.005).
The enrichment of CTCs by the depletion of leukocytes with bi-antibodies is a valuable method for estimating the number of CTCs, which can be potentially applied in predicting the prognosis, monitoring the therapeutic effect of NSCLC patients and further analyzing the biology of CTCs.
基于上皮细胞粘附分子（EpCAM）的循环肿瘤细胞（CTC）检测方法被认为低估了CTC数量，可能会遗漏具有癌症干细胞（CSC）特性的转移亚群。因此，我们研究了不同分期非小细胞肺癌（NSCLC）患者中EpCAM阳性和阴性CTC，评估了这些CTC的临床价值，并在后续的CSC模型中探索了它们的能力。
使用磁珠分离技术通过双抗体去除白细胞来富集CTC，然后使用多参数流式细胞术通过EpCAM、细胞角蛋白7和8的表达来鉴定。我们确定了46例I至IV期NSCLC患者中按EpCAM表达分类的CTC分布，通过对4例指标患者在辅助治疗期间的纵向监测评估了这些CTC的诊断价值，并通过10例患者中CXCR4和CD133的表达来表征这些CTC的干性。
在IV期检测到EpCAM阴性（E-）CTC显著高于EpCAM阳性（E+）CTC（p = 0.003）。E-CTC百分比超过95%（r > 95%）的患者从I-II期的13.3%显著增加到IV期的61.1%（p = 0.006）。Kaplan-Meier分析表明，r > 95%的患者生存时间明显短于r≤0.95的患者（p = 0.041）。CTC的纵向监测表明，血液中E-CTC百分比高的患者对化疗或靶向治疗均无反应。对CTC的进一步表征显示，CXCR4+CD133+ CTC的干细胞样亚群在E-CTC中比在E+CTC中明显更普遍（p = 0.005）。
通过双抗体去除白细胞来富集CTC是一种估计CTC数量的有价值方法，可潜在应用于预测NSCLC患者的预后、监测治疗效果以及进一步分析CTC的生物学特性。
