Encapsulation and release of living tumor cells using hydrogels with the hybridization chain reaction

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. An aptamer-trigger-clamped hybridization chain reaction (atcHCR) captures tumor cells in a protective, porous, 3D DNA hydrogel. Subsequent addition of ATP facilitates release of the tumor cells for use in downstream assays.

[1]  G. Church,et al.  Large-scale de novo DNA synthesis: technologies and applications , 2014, Nature Methods.

[2]  Jiye Shi,et al.  Hybridization chain reaction amplification of microRNA detection with a tetrahedral DNA nanostructure-based electrochemical biosensor. , 2014, Analytical chemistry.

[3]  C. Fan,et al.  DNA Nanostructure-Based Engineering of the Biosensing Interface for Biomolecular Detection , 2017 .

[4]  J. Chao,et al.  A Surface‐Confined Proton‐Driven DNA Pump Using a Dynamic 3D DNA Scaffold , 2016, Advanced materials.

[5]  H. Pei,et al.  Electrochemical single nucleotide polymorphisms genotyping on surface immobilized three-dimensional branched DNA nanostructure , 2011 .

[6]  Itamar Willner,et al.  Switchable bifunctional stimuli-triggered poly-N-isopropylacrylamide/DNA hydrogels. , 2014, Angewandte Chemie.

[7]  Shuang Hou,et al.  Programming Thermoresponsiveness of NanoVelcro Substrates Enables Effective Purification of Circulating Tumor Cells in Lung Cancer Patients , 2014, ACS nano.

[8]  Xiaochun Xu,et al.  Specific Capture and Release of Circulating Tumor Cells Using Aptamer‐Modified Nanosubstrates , 2013, Advanced materials.

[9]  D. Seliktar.  Designing Cell-Compatible Hydrogels for Biomedical Applications , 2012, Science.

[10]  Jiye Shi,et al.  Programmable engineering of a biosensing interface with tetrahedral DNA nanostructures for ultrasensitive DNA detection. , 2015, Angewandte Chemie.

[11]  K. Anseth,et al.  Hydrogel Cell Cultures , 2007, Science.

[12]  K. Isselbacher,et al.  Isolation of circulating tumor cells using a microvortex-generating herringbone-chip , 2010, Proceedings of the National Academy of Sciences.

[13]  Xiaolei Zuo,et al.  Ultrasensitive electrochemical detection of prostate-specific antigen by using antibodies anchored on a DNA nanostructural scaffold. , 2014, Analytical chemistry.

[14]  S. Digumarthy,et al.  Isolation of rare circulating tumour cells in cancer patients by microchip technology , 2007, Nature.

[15]  Itamar Willner,et al.  pH‐Stimulated DNA Hydrogels Exhibiting Shape‐Memory Properties , 2015, Advanced materials.

[16]  Tao Zhang,et al.  Self‐Assembled DNA Hydrogels with Designable Thermal and Enzymatic Responsiveness , 2011, Advanced materials.

[17]  H. Tseng,et al.  Capture and Stimulated Release of Circulating Tumor Cells on Polymer‐Grafted Silicon Nanostructures , 2013, Advanced materials.

[18]  Xiaoling Zhang,et al.  An aptamer cross-linked hydrogel as a colorimetric platform for visual detection. , 2010, Angewandte Chemie.

[19]  Zhi Zhu,et al.  Au@Pt nanoparticle encapsulated target-responsive hydrogel with volumetric bar-chart chip readout for quantitative point-of-care testing. , 2014, Angewandte Chemie.

[20]  Dongsheng Liu,et al.  A Triggered DNA Hydrogel Cover to Envelop and Release Single Cells , 2013, Advanced materials.

[21]  Mehmet Toner,et al.  Inertial Focusing for Tumor Antigen–Dependent and –Independent Sorting of Rare Circulating Tumor Cells , 2013, Science Translational Medicine.

[22]  Cuichen Wu,et al.  Self-assembly of DNA Nanohydrogels with Controllable Size and Stimuli-Responsive Property for Targeted Gene Regulation Therapy , 2015, Journal of the American Chemical Society.

[23]  Robert M. Dirks,et al.  Triggered amplification by hybridization chain reaction. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[24]  Xiaolei Zuo,et al.  Target-responsive, DNA nanostructure-based E-DNA sensor for microRNA analysis. , 2014, Analytical chemistry.

[25]  Lei Jiang,et al.  Programmable Fractal Nanostructured Interfaces for Specific Recognition and Electrochemical Release of Cancer Cells , 2013, Advanced materials.

[26]  Jie Chao,et al.  Clamped Hybridization Chain Reactions for the Self-Assembly of Patterned DNA Hydrogels. , 2017, Angewandte Chemie.

[27]  Xiaolei Zuo,et al.  Valence-Engineering of Quantum Dots Using Programmable DNA Scaffolds. , 2017, Angewandte Chemie.

[28]  Dongsheng Liu,et al.  Rapid formation of a supramolecular polypeptide-DNA hydrogel for in situ three-dimensional multilayer bioprinting. , 2015, Angewandte Chemie.

[29]  David J. Mooney,et al.  Designing hydrogels for controlled drug delivery. , 2016, Nature reviews. Materials.

[30]  W. Duan,et al.  Selection of DNA aptamers against epithelial cell adhesion molecule for cancer cell imaging and circulating tumor cell capture. , 2013, Analytical chemistry.

[31]  Jie Chao,et al.  Multivalent capture and detection of cancer cells with DNA nanostructured biosensors and multibranched hybridization chain reaction amplification. , 2014, Analytical chemistry.

[32]  Kristi S Anseth,et al.  Materials science. Hydrogel cell cultures. , 2007, Science.

[33]  Weian Zhao,et al.  Bioinspired multivalent DNA network for capture and release of cells , 2012, Proceedings of the National Academy of Sciences.

[34]  Hao Yan,et al.  A DNA Nanostructure‐based Biomolecular Probe Carrier Platform for Electrochemical Biosensing , 2010, Advanced materials.

[35]  Juwen Shen,et al.  Valency-Controlled Framework Nucleic Acid Signal Amplifiers. , 2018, Angewandte Chemie.

[36]  Alan Bensky,et al.  Technologies and applications , 2019, Short-range Wireless Communication.

[37]  Jie Chao,et al.  DNA Hydrogel with Aptamer-Toehold-Based Recognition, Cloaking, and Decloaking of Circulating Tumor Cells for Live Cell Analysis. , 2017, Nano letters.

[38]  Daniel F. Hayes,et al.  Sensitive capture of circulating tumour cells by functionalised graphene oxide nanosheets , 2013, Nature nanotechnology.