Institute of Molecular Medicine, Department of Laboratory Medicine, Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine, Renji Hospital, School of Medicine and School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai, China.
Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility (SSRF), CAS Key Laboratory of Interfacial Physics and Technology, Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai, China.
Nat Protoc. 2020 Jul;15(7):2163-2185. doi: 10.1038/s41596-020-0326-4. Epub 2020 Jun 22.
Circulating tumor cells (CTCs) enable noninvasive liquid biopsy and identification of cancer. Various approaches exist for the capture and release of CTCs, including microfluidic methods and those involving magnetic beads or nanostructured solid interfaces. However, the concomitant cell damage and fragmentation that often occur during capture make it difficult to extensively characterize and analyze living CTCs. Here, we describe an aptamer-trigger-clamped hybridization chain reaction (atcHCR) method for the capture of CTCs by porous 3D DNA hydrogels. The 3D environment of the DNA networks minimizes cell damage, and the CTCs can subsequently be released for live-cell analysis. In this protocol, initiator DNAs with aptamer-toehold biblocks specifically bind to the epithelial cell adhesion molecule (EpCAM) on the surface of CTCs, which triggers the atcHCR and the formation of a DNA hydrogel. The DNA hydrogel cloaks the CTCs, facilitating quantification with minimal cell damage. This method can be used to quantitively identify as few as 10 MCF-7 cells in a 2-µL blood sample. Decloaking of tumor cells via gentle chemical stimulus (ATP) is used to release living tumor cells for subsequent cell culture and live-cell analysis. We also describe how to use the protocol to encapsulate and release cells of cancer cell lines, which can be used in preliminary experiments to model CTCs. The whole protocol takes ~2.5 d to complete, including downstream cell culture and analysis.
循环肿瘤细胞 (CTCs) 可实现非侵入性的液体活检和癌症鉴定。目前已有多种方法可用于捕获和释放 CTCs，包括微流控方法和涉及磁性珠或纳米结构固体界面的方法。然而，在捕获过程中经常发生的细胞损伤和碎片化，使得难以广泛地对活 CTCs 进行特征分析和研究。在这里，我们描述了一种适体触发夹式杂交链式反应 (atcHCR) 方法，用于通过多孔 3D DNA 水凝胶捕获 CTCs。DNA 网络的 3D 环境可最大限度地减少细胞损伤，随后可释放 CTCs 进行活细胞分析。在本方案中，具有适体-toehold 双嵌段的起始 DNA 特异性结合 CTCs 表面的上皮细胞黏附分子 (EpCAM)，从而触发 atcHCR 和 DNA 水凝胶的形成。DNA 水凝胶包裹 CTCs，在最小化细胞损伤的情况下进行定量。该方法可用于在 2µL 血液样本中定量鉴定低至 10 个 MCF-7 细胞。通过温和的化学刺激 (ATP) 使肿瘤细胞脱衣，从而释放活的肿瘤细胞，用于随后的细胞培养和活细胞分析。我们还介绍了如何使用该方案包封和释放癌细胞系的细胞，这些细胞可用于初步实验以模拟 CTCs。整个方案大约需要 2.5 天才能完成，包括下游细胞培养和分析。
