Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu, Taiwan.
Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan.
Lab Chip. 2024 Jan 17;24(2):375-382. doi: 10.1039/d3lc00862b.
Cholangiocarcinoma (CCA) is an aggressive cancer that originates from the epithelial cells lining the bile ducts. Due to its location deep within the body and nonspecific symptoms in the early stages, it is often diagnosed at the advanced stage, thus leading to worse prognosis. Circulating tumor cells within liquid biopsies ( blood) have been considered as promising biomarkers for CCA diagnosis, though current methods for profiling them are not satisfactory in terms of sensitivity and specificity. Herein we developed a new cancer cell probing and immuno-tracking assay known as "CAPTURE", which was performed on an integrated microfluidic system (IMS) to automate CCA diagnosis from bile with a sample amount of only 1 mL. The assay utilized magnetic beads surface-coated with two affinity reagents, a nucleic acid aptamer (HN16) and a glycosaminoglycan (SCH 45-mix), for capturing cancer cells in bile; the "gold standard" anti-epithelial cell adhesion molecule was used as a comparison. In a single-blind test of 54 CCA-positive (+) and 102 CCA-negative (-) clinical samples, sensitivities and specificities of 96 and 80%, respectively, were documented with the CAPTURE assay on-bench. An IMS composed of a centrifugal module for sample pretreatment and a CAPTURE module for cell capture and staining was integrated with a new "vertical integration module" for detecting cancer cells from bile without human intervention. Furthermore, a novel micro-tier structure within the centrifugal module was designed to block passage of gallbladder stones with diameters >1 mm, thereby preventing their interference during the subsequent CAPTURE assay. Improved sensitivity and specificity (100 & 83%, respectively) by using three affinity reagents were achieved on the IMS when using 26 clinical bile samples, confirming its clinical bio-applicability for CCA diagnosis. This approach could be therefore used for early-stage CCA diagnostics, ideally enabling effective treatment, as well as reducing potential for relapse.
胆管癌（CCA）是一种起源于胆管上皮细胞的侵袭性癌症。由于其位置深在体内，早期症状不特异，因此常常在晚期诊断，导致预后较差。液体活检（血液）中的循环肿瘤细胞已被认为是 CCA 诊断的有前途的生物标志物，但目前用于分析这些细胞的方法在灵敏度和特异性方面并不令人满意。在此，我们开发了一种新的癌细胞探测和免疫追踪检测方法，称为“CAPTURE”，该方法在集成微流控系统（IMS）上进行，只需 1mL 样本量即可自动从胆汁中诊断 CCA。该检测方法利用表面涂有两种亲和试剂的磁性珠，即核酸适体（HN16）和糖胺聚糖（SCH 混合），用于捕获胆汁中的癌细胞；将抗上皮细胞黏附分子用作比较。在对 54 份 CCA 阳性（+）和 102 份 CCA 阴性（-）临床样本的单盲测试中，CAPTURE 检测方法在实验台上的灵敏度和特异性分别为 96%和 80%。一个由用于样品预处理的离心模块和用于细胞捕获和染色的 CAPTURE 模块组成的 IMS 与一个新的“垂直集成模块”集成，用于在无需人为干预的情况下从胆汁中检测癌细胞。此外，在离心模块中设计了一种新颖的微梯结构，用于阻挡直径>1mm 的胆囊结石通过，从而防止它们在随后的 CAPTURE 检测中干扰。在使用 26 份临床胆汁样本时，通过使用三种亲和试剂，在 IMS 上实现了更高的灵敏度和特异性（分别为 100%和 83%），证实了其用于 CCA 诊断的临床生物适用性。这种方法可用于早期 CCA 诊断，理想情况下可实现有效治疗，并降低复发的可能性。
