DNA Hydrogel with Aptamer-Toehold-Based Recognition, Cloaking

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DNA hydrogel with aptamer-toehold based recognition, cloaking and decloaking of circulating tumor cells for live cell analysis Ping Song, Dekai Ye, Xiaolei Zuo, Jiang Li, Jianbang Wang, Huajie Liu, Michael Taeyoung Hwang, Jie Chao, Shao Su, Lihua Wang, Jiye Shi, Lianhui Wang, Wei Huang, Ratnesh Lal, and Chunhai Fan Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b01006 • Publication Date (Web): 03 Aug 2017 Downloaded from http://pubs.acs.org on August 3, 2017

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DNA hydrogel with aptamer-toehold based recognition, cloaking and decloaking of circulating tumor cells for live cell analysis Ping Song1,2,†, Dekai Ye2,†, Xiaolei Zuo1,2,*, Jiang Li2, Jianbang Wang2, Huajie Liu2, Michael T. Hwang3, Jie Chao4, Shao Su4, Lihua Wang2, Jiye Shi5, Lianhui Wang4, Wei Huang4, Ratnesh Lal3, Chunhai Fan2 1

Institute of Molecular Medicine, Renji Hospital, School of Medicine and School of Chemistry

and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200127, China 2

Division of Physical Biology and Bioimaging Center, Shanghai Synchrotron Radiation Facility,

Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800, China 3

Materials Science and Engineering Program, Department of Bioengineering, Department of

Mechanical and Aerospace Engineering, Institute of Engineering in Medicine, University of California, San Diego, La Jolla, CA 92093 4

Key Laboratory for Organic Electronics and Information Displays (KLOEID), Institute of

Advanced Materials (IAM) and School of Materials Science and Engineering, Nanjing University of Posts and Telecommunications, Nanjing 210046, China 5

Kellogg College, University of Oxford, Oxford, OX2 6PN, United Kingdom

ACS Paragon Plus Environment

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Table of Contents


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ABSTRACT Circulating tumor cells (CTCs) contain molecular information of the primary tumor and can be used for predictive cancer diagnostics. Capturing rare live CTCs and their quantification in whole blood remain technically challenging. Here we report an aptamer-trigger clamped hybridization chain reaction (atcHCR) method for in-situ identification and subsequent cloaking/de-cloaking of CTCs by porous DNA hydrogels. These de-cloaked CTCs were then used for live cell analysis. In our design, a DNA staple strand with aptamer-toehold bi-blocks specifically recognizes epithelial cell adhesion molecule (EpCAM) on the CTC surface that triggers subsequent atcHCR via toehold-initiated branch migration. Porous DNA hydrogel based-cloaking of single/cluster of CTCs allows capturing of living CTCs directly with minimal cell damage. The ability to identify a low number of CTCs in whole blood by DNA hydrogel cloaking would allow high sensitivity and specificity for diagnosis in clinically-relevant settings. More significantly, de-cloaking of CTCs using controlled and defined chemical stimuli can release living CTCs without damages for subsequent culture and live cell analysis. We expect this liquid biopsy tool to open new powerful and effective routes for cancer diagnostics and therapeutics.

KEYWORDS Circulating tumor cell; DNA hydrogel; CTC capture; CTC release; live cell analysis


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Circulating tumor cells (CTCs) are viable tumor-derived cells actively circulating in peripheral blood and contain molecular information about the primary tumor. Selective isolation of these CTCs without complex invasive tumor biopsy would allow predictive diagnosis of primary tumor with major impact on clinical diagnosis and therapy1-4. Microfluidic methods to isolate CTCs relies upon the difference in the size of the tumor cells vs normal cells5, 6. The specificity of CTCs isolation is often enhanced by EpCAM-based affinity methods; most cancer cells overexpress EpCAM1, 2, 7-9. Various nanostructured solid interfaces have been used to increase the sensitivity of the detection; cancer cells often show enhanced topological interactions with nanostructured interfaces

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. Although these methods have considerable promises, they suffer

from the cell damage or fragmentation caused by the fluidic turbulence and shear force 2, 4. Although the detection and isolation of CTCs provide a useful tool for diagnosis, the release and downstream analysis of live CTCs would provide more valuable information about the molecular signature and functional properties

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. Cell capturing devices or nanostructured interfaces

typically fail to release the captured CTCs without damage and thus limits any live-cell analysis 2, 4

; cell viability is often constrained by fluidic shear force, enzymatic treatment, electrochemical

repulsion and temperature oscillation associated with these devices. Hydrogel has emerged as a promising matrix for 3-dimensional (3-D) cell culture via the gel-sol phase transition

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,and

has the potential to minimize the loss of cell viability and molecular integrity. DNA hydrogels are 3-D DNA networks that are extensively hydrated and provide elastic, semiwet and 3-D environments. These characteristics are suitable for communications between cells and extracellular matrix with wide applications in drug delivery and tissue engineering 24-32. The optical transparency of DNA hydrogel allows standard staining, labeling and microscopy for live cell study. More importantly, responsive DNA hydrogel can be rationally designed as smart


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sensors and devices to characterize proteins, small molecules and ions, by changes in the morphology, color, and size in response to external stimuli33,

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. Here, we report the liquid

cloaking CTCs via aptamer triggered in-situ DNA gelation for live cell analysis.

Figure 1. DNA gelation-based cloaking and decloaking of CTCs. (a) At the initial solution phase, the MCF-7 cells were dispersed in solution. The aptamer-initiator bi-blocks were able to specifically bind to the EpCAM on cell surface which could then trigger the atcHCR reaction to assemble DNA hydrogel. The ATP was used to destroy the DNA hydrogel with designed ATPresponsive region in DNA hydrogel that cloaked on cell surface. (b) DNA hairpins H1 and H2


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were trapped in metastable state. Without DNA initiator, H1 and H2 could not hybridize. With DNA initiator, the H1 was opened and triggered the subsequent hybridization chain reaction. (c) Confocal images of aptamer-initiator bi-blocks (red) colocalized with DiO stained lipid on cell membrane (green). (d) 3D stack of MCF-7 cells cloaked in DNA hydrogel with FDA staining in green which show multilayered cells in hydrogel. Stack height: 40 µm. (e) By adding the ATP, the MCF-7 cells were released and dispersed in solution.

We first studied the phase transition of DNA hydrogel on CTCs. The phase transition of DNA hydrogel can be programmed by adding the DNA initiator as a trigger or ATP as a stimulus, respectively (Figure 1). The well dispersed MCF-7 cells in solution with aptamer-initiator biblocks binding on the cell surface could trigger the clamped hybridization chain reaction (HCR) and induce phase transition from solution to hydrogel. The in-situ DNA gelation via clamped HCR was used to cloak the CTCs. Further, in order to achieve the phase transition from hydrogel to solution, we designed a stimulus-responsive DNA hydrogel. We incorporated an ATP aptamer in the clamped HCR (aptamer-triggered clamped HCR, atcHCR) to de-cloak the DNA hydrogel on cell surface. We identified the specific aptamer-initiator bi-blocks binding on the cell surface of CTC. A specific ani-EpCAM aptamer

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was used to anchor the DNA initiator on cell surface via the

formation of aptamer-initiator bi-blocks (Figure 1a). To identify the specific binding of aptamerinitiator bi-blocks with EpCAM on the cell surface of MCF-7, we performed colocalization experiments with a two-color confocal microscopy. We labeled the aptamer-initiator bi-blocks with a red-emitting fluorophore (cyanine3, cy3) and stained the cell membrane with a greenemitting fluorophore (3’-dioctadecyloxacarbocyanine perchlorate, DiO). We


colocalized

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fluorescence from cy3 (red) and DiO (green) (Figure 1c and Figure 2a-e) which indicates the binding of aptamer-initiator bi-blocks and EpCAM on the cell surface. The successful binding of aptamer-initiator bi-blocks on cell surface initiated DNA gelation. As a control experiment, we used a random DNA sequence instead of the aptamer and observed no red fluorescence emission on the cell surface indicating the specific binding between aptamer and EpCAM (Figure S1a). In addition, we examined the binding of the aptamer with another EpCAM-negative cell line (HEK293). No obvious fluorescence signal was observed on the cell surface as well (Figure S1b). With the specific aptamer-initiator bi-blocks binding with EpCAM, the atcHCR triggered the insitu DNA gelation on the cell surface (Figure 1). In order to characterize the DNA hydrogel and the entrapped cells, we used optical microscopy and environmental scanning electron microscope (ESEM) to image the in-situ assembly of DNA hydrogel (Figure 1d, Figure 2f and 2g). DNA hydrogel formation appears to be triggered by MCF-7 cells dispersed in solution or adhered on cell culture dish (Figure S2). The 3-D stack of cells shows the multilayered spatial topology of cells in DNA hydrogel (Figure 1d and Video S1, S2). ESEM images reveal that the MCF-7 cells were cloaked by DNA hydrogel; cells were encapsulated in DNA hydrogel by initiating the gelling process (Figure 2f). We characterized the freeze-dried DNA hydrogel by scanning electron microscope (SEM) and observed porous microstructures - a typical hydrogel structure previously reported for other DNA hydrogels 35, 36-38 (Figure 2g).


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Figure 2. CTC cloaking imaged by optical microscopy and SEM. (a) Schematics of CTC cloaking and the characterization by 3D confocal microscopy. (b-e) Confocal images of aptamerinitiator bi-blocks (red) colocalized with DiO stained lipid on cell membrane (green), indicating the aptamer-initiator bi-blocks was attached on the cell surface. Scale bar: 10 µm. (f) ESEM characterization of MCF-7 cells that cloaked with DNA hydrogel. Scale bar: 20 µm. (g) SEM image showed porous structures of freeze-dried DNA hydrogel. Scale bar: 20 µm.


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Next, we designed the ATP-responsive DNA hydrogel to be de-cloaked from the cell surface (Figure 1). In the absence of ATP, the aptamer adopted an unfolded state that ensured the DNA gelation (Video S3). By adding ATP, the conformational change of the aptamer from the unfolded state to a tertiary state forced the collapse of DNA hydrogel, resulting in the decloaking of the entrapped MCF-7 cells (Figure 1e, Video S4). In-situ DNA gelation occurred by atcHCR in which metastable DNA hairpins hybridized as chain reaction when they encountered the DNA initiator. The atcHCR consisted of two different designs of DNA hairpins (H1 and H2) that were kinetically trapped in a metastable state (Figure 1b and S3). The potential energies of H1 and H2 were stored in a short loop region protected by long stems. Based on the computational simulation with NUPACK, the free energies of the secondary structures were estimated as -55.31 and -20.67 kcal / mol for H1 and H2, respectively. The metastability of H1 and H2 prevented the system from spontaneous hybridization of H1 and H2. In the presence of a DNA initiator, the free energy to drive the hybridization reaction is reduced significantly. This is presumably due to the sum of the enthalpic benefit to form stacked base-pairs between the initiator, H1 and H2, and the entropic benefit to open the hairpins (Figure 1b). A chain reaction of alternating hybridization of H1 and H2 driven by the free energy led to the formation of double stranded DNA helices. H1 was designed to form a dimer to bridge the double helices and H2 was designed to form a flexible single stranded region in the HCR products to facilitate the formation of 3-dimentional (3D) DNA network (Figure 1b and S3, S4). The average size of HCR products is inversely related to the initiator concentration, the feature that can be used for quantitative sensing (the inherent characteristics of the originally invented HCR). When we used a relatively high concentration of reactants, we were able to fabricate transparent DNA hydrogel (Figure S4).


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We used bacteria as a living indicator for real-time monitoring of the kinetics of DNA gelation (Figure 3a). A typical bacterium movement was recorded in real-time in solution and in DNA hydrogel, respectively. (Figure 3c). With the hydrogel formation, the speed of bacterial movement decreased drastically. During ~2.5 h of gelling process, the speed decreased to only ~7% of the initial speed (Figure 3d, 3e and Video S6). This could be due to the hydrogel cloaking on the cancer cell surface (Figure 3b). In the solution phase, the bacteria moved freely in random trajectories and in relatively high speed during the same time course of 2.5 h (Figure 3a and Video S5).

Figure 3. In-operando monitoring of CTC cloaking kinetics using a bacterial indicator. (a) The motion of bacteria in solution appears random. The movement trajectories of three representative bacteria are highlighted with yellow, red and green line, respectively. Scale bar: 25 µm. (b) The movement of bacteria was limited in the process of hydrogel formation. The trajectories of


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movement of two representative bacteria are highlighted with red and green line, respectively. Scale bar: 25 µm. (c) Representative time-lapse images for bacteria moving in solution and DNA hydrogel. Scale bar: 10 µm. (d) Velocity proﬁle for a single bacterium track over time in solution (yellow) and the DNA hydrogel (grey). The speed of bacterial motion decreased rapidly during the process of hydrogel assembly (red line); whereas, the speed of bacterial motion in solution was almost unchanged (green line). Hence, the bacterial motion reveals the kinetics of DNA gelation. (e) Velocity distribution of bacteria moving in solution (grey) and DNA hydrogel (red), respectively. We further examined the living state of the cloaked CTCs as well as the released CTCs after decloaking. The cell viability of MCF-7 in the DNA hydrogel was examined by FDA (fluorescein diacetate) staining (Figure 1d and Video S1). We observed the green fluorescence emission from all MCF-7 cells in our study, which indicates that the cell membrane remained intact and the cells were viable in the DNA hydrogel. During the DNA gelation, the cell morphology remained the same as that at the initial state (Figure S5). Significantly, the MCF-7 cells released after decloaking could also be re-cultured. The differentiation and proliferation ability were retained after the gelation process (Figure 4a-c). To evaluate the molecular composition of the cells, we examined the messenger RNA (mRNA) expression of cytokeratin 19 (CK19) and epidermal growth factor receptor (EGFR) by using gel electrophoresis and real-time polymerase chain reaction (RT-PCR) (Figure 4d, 4e, 4f, S6). There appears to be negligible difference in the expression level of EGFR and CK19 before and after the DNA gelation, which indicates that the phase transition of the DNA hydrogel did not affect the molecular properties of cells.


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Figure 4. The release, downstream culture and mRNA analysis of live CTCs. (a) Phase transition from DNA hydrogel to solution was achieved by adding ATP; while the conformational change of aptamers broke the bridge of 3-D DNA networks and transited DNA hydrogel into solution. (b) The released cells were subsequently cultured; the cell morphology retained characteristic morphology of original MCF-7 cancer cells. (c) A microscopic image of released cells after being cultured for one day. The released cells retained the proliferation ability after the cloaking and de-cloaking process. (d) Scheme of RT-PCR expression profiling of mRNA. (e and f) Comparison of Ct values of RT-PCR for CK19 mRNA and EGFR mRNA of MCF-7 and released MCF-7. R: Relative fluorescence.


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Based on our results, we used the DNA gelation via liquid cloaking on cells to quantify the isolated and identified CTCs. We used gold nanoparticles (AuNPs) as indicators through the encapsulation ability of DNA hydrogel. We could visualize the hydrogel formation by adding AuNPs in the solution before gelation. The color at this state was red due to the plasmon resonance adsorption of AuNPs (Figure S7). After the gelation, the AuNPs were encapsulated in the hydrogel with red color, which were not dispersed by adding buffer solution (Figure 5a). Without the formation of hydrogel, the AuNPs were well dispersed (Figure 5a). Then, when we used 50000 MCF-7 cells to initiate the DNA gelation, the AuNPs were not dispersed by adding buffer. This observation indicates the successful assembly of the hydrogel. We monitored the plasmonic adsorption of AuNPs in the supernatant although the detection signal was negligible (Figure 5b). As a control experiment, without MCF-7 cell, the AuNPs were not encapsulated due to the lack of DNA gelation. We observed significant plasmonic adsorption of AuNPs in the solution (Figure 5b). Interestingly, the ability to encapsulate AuNPs of DNA hydrogel is significantly related to the number of MCF-7 cells (Figure 5b). With the reduced number of cancer cells, the ability to encapsulate AuNPs decreased monotonically, this correlation provided a solid foundation for quantitative detection of the cancer cells. We used various numbers of cancer cells to initiate the DNA hydrogel assembly and obtained an excellent correlation between the cell number and plasmonic adsorption of AuNPs (Figure 5b). As few as 5 cancer cells could be detected. More significantly, we transferred our method from the buffer solution to blood samples by spiking MCF-7 cells into blood. Interestingly, the intrinsic blood cells in blood samples were able to be encapsulated in the DNA hydrogel and were not be dispersed by adding the buffer. We thus achieved a sensitive detection of cancer cells with a detection limit of as low as 10 cancer cells (Figure 5c, 5d and Figure S8).


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Figure 5. CTC quantification with DNA gelation-based CTC cloaking. (a) AuNPs were used as an indicator to visualize the hydrogel assembly. (b) The AuNPs encapsulation ability of DNA hydrogel was related to the cell numbers that initiated the hydrogel assembly. The cancer cells were quantitatively detected by monitoring the plasmonic adsorption of AuNPs that were released into the buffer solution. The plasmonic adsorption was increased along with the decrease of cell number (inset). Five MCF-7 cells can be sensitively detected. (c and d) The method can be used to detect the MCF-7 cells in blood samples. The intrinsic blood cells in the blood sample were used as indicator. The blood cells encapsulation ability of DNA hydrogel was related to the cell numbers that initiated the hydrogel assembly. The 8-bit grey values for blood samples with various CTC numbers were obtained from histogram averages in Adobe Photoshop (c). The intensity change was increased with the increase of CTC number in blood samples (d). Abs: Absorbance.


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Exploiting the differences in the interaction of cell and 3-D DNA network should allow selective release of encapsulated non-specific cell population during the hydrogel swelling while retaining the cancer cells and thus enriching the cancer cells in the hydrogel (Figure 6). To demonstrate this, we mixed MCF-7 cells (EpCAM positive cell) and L1210 cells (EpCAM negative, nonspecific cells) as a model system to observe the enrichment of MCF-7 cells. The formation of hydrogel was initiated by the specific binding of aptamer-initiator bi-blocks on EpCAM of MCF7 cells (Figure 6a). After interaction with the buffer solution, the swelling of hydrogel initiated the selective release of L1210 cells due to the lack of specific interaction between L1210 cells and 3D DNA network (Figure 6b). The specific interaction of MCF-7 cells and 3D DNA network retained the MCF-7 cells in hydrogel and realized the enrichment of the MCF-7 cell population (Figure 6c-f).

Figure 6. Cancer cell enrichment based on biased non-specific cell release via hydrogel swelling. a) The MCF-7 cells initiate the hydrogel formation, which encapsulate the MCF-7 Cells and non-


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specific cells (L1210 cells) in hydrogel. The dashed line indicates the border of hydrogel. b) In the process of hydrogel swelling, the lack of specific interaction between DNA networks and L1210 cells allows biased release of L1210 cell population encapsulated in hydrogel, and thus the cancer cells can be enriched in the hydrogel. The dashed line indicates the border of hydrogel and buffer solution. c) Schematics shows the enrichment of cancer cells based on hydrogel swelling. The specific interaction between MCF-7 cell and DNA network retains the MCF-7 cells in hydrogel, while, L1210 cells are selectively released during the hydrogel swelling. d) The representative fluorescent imaging result shows the MCF-7 cells (stained green) are mixed with L1210 cells (stained red). e) The L1210 cells are released during the hydrogel swelling. f) The frequency of the released L1210 cells containing no MCF-7 cells is ~16-fold higher than that of the mixture of MCF-7 and L1210 cells, indicating the released cells from the hydrogel are non-specific cells (L1210). Scale bar: 100 µm. In summary, we have achieved DNA gelation-based liquid cloaking of CTCs for live-cell analysis. The 3D DNA network structure of DNA hydrogel can provide a biocompatible, gentle environment and condition for live CTCs capturing. Other approaches to capture CTCs including microfluidic devices are based on microstructures, trapping arrays and microfilters. However, the fluidic turbulence, shear force and other harsh conditions for living cells during the capturing process usually induce nonphysiological stress and often damage the CTCs and thus limiting the detection accuracy and the further analysis of CTCs 2. The recognition elements are not limited to EpCAM on cell membrane, other proteins that express on cancer cell surface could be used as recognition elements in principle, which allows us to generalize our method to capture and quantify EpCAM negative cancer cells39-41.


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More importantly, our DNA hydrogel based CTC capturing provides a simple and gentle cellrelease approach by using the ATP-responsive DNA hydrogel. The DNA hydrogel can be gently denatured and hence destroyed by ATP within 10 minutes. The captured cells can then be easily released. While cell separation devices with complex surface topologies (e.g., microposts, herringbone structures) typically fail to gently release the captured cells4. The binding affinity of cell capturing via dendrimer-mediated multivalent binding and rolling circle amplification associated multivalent capturing are also reported. However, the high binding affinity would limit the release of the non-damaged captured cells for subsequent cell culture and whole cell analysis. Finally, our DNA hydrogel based CTC capturing provides an approach for capturing live CTCs. Our FDA staining results indicated the excellent cell viability of captured cells in DNA hydrogel. The RT-PCR analysis of mRNA expression suggested minimal effect (or damage) on captured cells. These studies show a great potential of our method to gather faithful information of primary tumor from captured CTCs.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Experimental Section, Figures S1−S8 (PDF) AUTHOR INFORMATION


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Corresponding Author *Email: [email protected] Author Contributions †

P. S. and D. Y. contributed equally to this work.

ACKNOWLEDGMENT This work was financially supported NSFC (grant numbers 21422508, 31470960, 21390414), Ministry

of

Science

and

Technology

of

China

(2016YFA0201200, 2013CB933802, 2013CB932803), the Chinese Academy of Sciences (QYZDJ-SSW-SLH031), and National Institute on Aging National Institutes of Health (Grant AG028709). We also thank Grace Jang for the helpful discussion.

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