Abstract
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.
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Data availability
The main data supporting the examples of this protocol are available within the article and its Supplementary Information files. Extra data are available from the corresponding author upon reasonable request. The source data underlying Figs. 3e,f, 6d,e and 7c–f and Supplementary Figs. 2, 3, 5, 6b, 8a–c and 9 are provided as source data files.
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Acknowledgements
This work was financially supported by the Ministry of Science and Technology of China (2016YFA0201200), the National Natural Science Foundation of China (21904086, 21804088, 21804091), the Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (20171913), the “Shuguang Program” supported by the Shanghai Education Development Foundation, and the Shanghai Municipal Education Commission (18SG16).
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Contributions
X. Zuo, Q.L. and C.F. supervised the projects; D.Y., M.L., T.Z., P.S., L.S., H.W., X.M., X. Zuo and C.F. designed and conducted the experiments; F.W., X. Zhang, J.S., Z.G., L.W. and Q.L. analyzed the data; and Q.L., X. Zuo and C.F. wrote the manuscript.
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Related links
Key reference using this protocol
Song, P. et al. Nano Lett. 17, 5193–5198 (2017): https://doi.org/10.1021/acs.nanolett.7b01006
Key data used in this protocol
Song, P. et al. Nano Lett. 17, 5193–5198 (2017): https://doi.org/10.1021/acs.nanolett.7b01006
Supplementary information
Supplementary Information
Supplementary Figures 1–9.
Supplementary Video 1
E. coli movement in PBS
Supplementary Video 2
E. coli movement in DNA hydrogel
Supplementary Data 1
Statistical source data for Supplementary Figure 2
Supplementary Data 2
Statistical source data for Supplementary Figure 3
Supplementary Data 3
Statistical source data for Supplementary Figure 5
Supplementary Data 4
Statistical source data for Supplementary Figure 6
Supplementary Data 5
Statistical source data for Supplementary Figure 8
Supplementary Data 6
Statistical source data for Supplementary Figure 9
Source data
Source Data Fig. 3
Statistical source data
Source Data Fig. 4
Unprocessed gels
Source Data Fig. 6
Statistical source data
Source Data Fig. 7
Statistical source data
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Ye, D., Li, M., Zhai, T. et al. Encapsulation and release of living tumor cells using hydrogels with the hybridization chain reaction. Nat Protoc 15, 2163–2185 (2020). https://doi.org/10.1038/s41596-020-0326-4
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DOI: https://doi.org/10.1038/s41596-020-0326-4
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