Khoo Bee Luan, Warkiani Majid Ebrahimi, Tan Daniel Shao-Weng, Bhagat Ali Asgar S, Irwin Darryl, Lau Dawn Pingxi, Lim Alvin S T, Lim Kiat Hon, Krisna Sai Sakktee, Lim Wan-Teck, Yap Yoon Sim, Lee Soo Chin, Soo Ross A, Han Jongyoon, Lim Chwee Teck
Mechanobiology Institute, National University of Singapore, Singapore, Singapore.
BioSystems and Micromechanics (BioSyM) IRG, Singapore-MIT Alliance for Research and Technology (SMART) Centre, Singapore, Singapore.
PLoS One. 2014 Jul 7;9(7):e99409. doi: 10.1371/journal.pone.0099409. eCollection 2014.
Circulating tumor cells (CTCs) are cancer cells that can be isolated via liquid biopsy from blood and can be phenotypically and genetically characterized to provide critical information for guiding cancer treatment. Current analysis of CTCs is hindered by the throughput, selectivity and specificity of devices or assays used in CTC detection and isolation.
METHODOLOGY/PRINCIPAL FINDINGS: Here, we enriched and characterized putative CTCs from blood samples of patients with both advanced stage metastatic breast and lung cancers using a novel multiplexed spiral microfluidic chip. This system detected putative CTCs under high sensitivity (100%, n = 56) (Breast cancer samples: 12-1275 CTCs/ml; Lung cancer samples: 10-1535 CTCs/ml) rapidly from clinically relevant blood volumes (7.5 ml under 5 min). Blood samples were completely separated into plasma, CTCs and PBMCs components and each fraction were characterized with immunophenotyping (Pan-cytokeratin/CD45, CD44/CD24, EpCAM), fluorescence in-situ hybridization (FISH) (EML4-ALK) or targeted somatic mutation analysis. We used an ultra-sensitive mass spectrometry based system to highlight the presence of an EGFR-activating mutation in both isolated CTCs and plasma cell-free DNA (cf-DNA), and demonstrate concordance with the original tumor-biopsy samples.
CONCLUSIONS/SIGNIFICANCE: We have clinically validated our multiplexed microfluidic chip for the ultra high-throughput, low-cost and label-free enrichment of CTCs. Retrieved cells were unlabeled and viable, enabling potential propagation and real-time downstream analysis using next generation sequencing (NGS) or proteomic analysis.
循环肿瘤细胞（CTCs）是可通过液体活检从血液中分离出来的癌细胞，可对其进行表型和基因特征分析，以提供指导癌症治疗的关键信息。目前对CTCs的分析受到CTCs检测和分离所用设备或检测方法的通量、选择性和特异性的阻碍。
方法/主要发现：在此，我们使用一种新型的多重螺旋微流控芯片，从晚期转移性乳腺癌和肺癌患者的血液样本中富集并鉴定假定的CTCs。该系统在高灵敏度下（100%，n = 56）（乳腺癌样本：12 - 1275个CTCs/ml；肺癌样本：10 - 1535个CTCs/ml）能从临床相关血量（5分钟内7.5 ml）中快速检测到假定的CTCs。血液样本被完全分离为血浆、CTCs和外周血单个核细胞（PBMCs）成分，每个组分通过免疫表型分析（全细胞角蛋白/CD45、CD44/CD24、上皮细胞黏附分子（EpCAM））、荧光原位杂交（FISH）（棘皮动物微管相关蛋白4 - 间变性淋巴瘤激酶（EML4-ALK））或靶向体细胞突变分析进行特征鉴定。我们使用基于超灵敏质谱的系统来突出在分离的CTCs和血浆游离DNA（cf-DNA）中表皮生长因子受体（EGFR）激活突变的存在，并证明与原始肿瘤活检样本一致。
结论/意义：我们已在临床上验证了我们用于CTCs超高通量、低成本和无标记富集的多重微流控芯片。回收的细胞未标记且有活力，能够进行潜在的增殖，并使用下一代测序（NGS）或蛋白质组分析进行实时下游分析。
