Capture and Release of Cancer Cells by Combining On-Chip

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Capture and release of cancer cells by combining onchip purification and off-chip enzymatic treatment Xiaolei Yu, Bingrui Wang, Nangang Zhang, Changqing Yin, Hao Chen, Lingling Zhang, Bo Cai, Zhaobo He, Lang Rao, Wei Liu, Fubing Wang, Shi-Shang Guo, and Xingzhong Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b06791 • Publication Date (Web): 13 Oct 2015 Downloaded from http://pubs.acs.org on October 20, 2015

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ACS Applied Materials & Interfaces

Capture and release of cancer cells by combining onchip purification and off-chip enzymatic treatment ⊥

†

‖

Xiaolei Yu, Bingrui Wang, Nangang Zhang, Changqing Yin,§ Hao Chen, § Lingling Zhang, †

†

†

†

†

†,

Bo Cai, Zhaobo He, Lang Rao, Wei Liu, Fu-Bing Wang, §,* Shi-Shang Guo * and XingZhong Zhao

†

†

Key Laboratory of Artificial Micro- and Nano-Structures of Ministry of Education, School of

Physics and Technology, Wuhan University, Wuhan, 430072, P. R. China §

Department of Laboratory Medicine & Center for Gene Diagnosis, Zhongnan Hospital of

Wuhan University, Wuhan University, Wuhan, 430072, P. R. China ‖

Advanced Micro-nano Textile Innovation Research Center, Hubei Collaborative Innovation

Center for Key Technologies in Textiles, Wuhan Textile University, Wuhan, 430073, P. R. China ⊥

College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, 430070,

P. R. China

Corresponding Authors: *Email: [email protected] *Email: [email protected]

ACS Paragon Plus Environment

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ABSTRACT As “liquid biopsies”, circulating tumor cells (CTCs) have been thought to hold significant insights for cancer diagnosis and treatment. Despite the advances of microfluidic techniques improve the capture of CTCs to a certain extent, recovering the captured CTCs with enhanced purity at the same time remains a challenge. Here, by combining on-chip purification and offchip enzymatic treatment, we demonstrate a two-stage strategy to enhance the purity of captured cancer cells from blood samples. The on-chip purification introduces stirring flow to increase the capture sensitivity and decrease non-specifically bounded cells. The off-chip enzymatic treatment enables the cancer cells to be released from the attached magnetic beads, further improving the purity and enabling next re-culture. For the proof-of-concept study, spiked cancer cells are successfully obtained from unprocessed whole blood with high recovery rate (~68%) and purity (~61%), facilitating next RNA expression analysis.

KEYWORDS: circulating tumor cells, hepatocellular carcinoma, aptamers, release, microfluidics


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Introduction Circulating tumor cells (CTCs) shed off the edges of a tumor and travel in the bloodstream or lymphatic system.1 As “liquid biopsies”, CTCs have been thought to hold significant medical insights for cancer diagnosis and treatment.2 Specifically, counting the number of CTCs in peripheral blood can track the severity of a cancer and monitor the efficacy of a surgery;3 In addition, the molecular analysis of CTCs can reveal the heterogeneities and gene mutations,4 thereby providing opportunities to realize personalized therapy. To efficiently obtain CTCs from peripheral blood, various approaches have been developed from macro-scale methods to microchip-based techniques.5 Compared with macro-scale approaches, microfluidic devices offer multiple advantages for CTC isolation6,7 such as the enhanced interactions between the CTCs and the functionalized surface,8,9 and the dynamic flows to prohibit non-specific binding.10,11 However, other than capture and enumeration,12 recovering the captured CTCs with high purity is equivalently important because the impurities (peripheral blood monocytes (PBMCs) or other blood components) along with the CTCs will inevitably disturb the downstream characterization such as RNA/genome analysis.13-16 Nanostructured materials with stimuliresponsive coating proved to be highly efficient at capture and release of CTCs,17-19 but the release mechanism is to degrade the whole sacrificial layer20,21 by temperature,22,23 UV light24 or laser-dissection25,26, resulting in not only the impurities released from the substrate as well but also the poor reusability of the device. Surface acoustic waves (SAW) has been integrated into microfluidics as label-free, contact-free cell manipulation and separation approach, yet without the affinity agents, the specificity and purity will be a critical issue to be addressed.27-29 Immunomagnetic beads (MBs) can realize non-invasive recovery of CTCs30,31 and have been utilized in the commercial CellSearch system.32 However, the capture purity of CellSearch


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system is known to be less than 1% even the capture efficiency reaches ~80%.33,34 Moreover, the MBs still attach on the cell surface after isolation, causing cytotoxicity in subsequent cultivation process.35-37 Recently, nucleic acid aptamers have been screened for targeting numerous cancers and utilized for efficient capture of cancer cells.38-40 Like antibodies, nucleic acid aptamers can fold into unique secondary or tertiary structures to recognize surface receptors on targeted cells with comparable affinities.38 On the other hand, the aptamers can be conveniently decomposed by enzymatic treatment to realize cell-release without breaking the integrity of the substrate.41-43 Herein, by combining on-chip purification and off-chip enzymatic treatment, we demonstrate a two-stage platform to enhance the purity of captured CTCs from blood samples, as schemed in Figure 1. The microfluidic chip (PN-chip) consists of a polydimethylsiloxane (PDMS) herringbone structure and a substrate of Ni micropillar arrays. The PDMS herringbone structure worked as a chaotic mixer to induce transverse flows,44 which enhanced the cell-surface interaction and decreased non-specific bindings compared with the flat device without the herringbone structure. By subsequent enzymatic treatment of the cells washed away from the microfluidic device, the aptamers were cleaved from the MBs, making the cells further release from the MBs. Such off-chip enzymatic treatment enhanced the purity again and enabled the reculture of the release cells by removal of the cytotoxicity of MBs. Finally, cancer cells were spiked in un-processed whole blood with low abundance as simulated CTCs to prove the function of our platform. As a result, both the recovery rate and purity were >60 %, and RT-PCR analysis was successfully conducted toward the purified cancer cells.

MATERIALS AND METHODS


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General materials. Paraformaldehyde (PFA), Triton X-100, Bovine serum albumin (BSA), Fluorescein diacetate (FDA), Propidium iodide (PI), 1-ethyl-3-[3-dimethylamino-propyl] carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), Normal goat serum and 4, 6Diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma-Aldrich. Streptavidin (SA) and Tween-20 were bought from Invitrogen. Phycoerythrin (PE) conjugated anti-cytokeratin antibody and Fluorescein (FITC) conjugated anti-human CD45 antibody were purchased from BD Biosciences. Exonuclease (Purity > 99%), used for digestion of aptamer, was bought from TAKARA. PBS (Hyclone, 1x), Dulbecco’s modified eagle medium (DMEM, Hyclone, high glucose) were purchased from Thermo Scientific. Deionized water was generated from a MILLI-Q system (Millipore, USA). The biotinylated aptamers (LY-1: biotin-5’ ATC CAG AGT GAC GCA GCA TTG GGT GTT AGG CTG GTC TTA ATC GGG TCG GGT TGC GTG GAC ACG GTG GCT TAGT-3’) were harvested from Professor Fubing Wang’s Lab in Zhongnan Hospital of Wuhan University.45 Cells and blood samples. The hepatocellular carcinoma (HCC) cell lines HCCLM9 and MHCC97-L were harvested from Zhongnan Hospital of Wuhan University. Whole blood samples from healthy donors were obtained from the Department of Clinical Laboratory, Zhongnan Hospital of Wuhan University according to Institutional Review Board (IRB) protocol. All blood specimens were collected into anti-coagulant tubes (EDTA-K2, 2 mL, violet cap) (Wuhan Zhiyuan Medical Technology Co. Ltd., China). Numerical simulation. Finite element simulations were performed by using commerciallyavailable software COMSOL Multiphysics. Navier-Stokes equations were used for the study, and in order to simplify the calculation we only chose a repetitive unit of herringbone structures to do the simulation. A flow rate of 1 mL h-1 was applied on the inlet and no slip condition was


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set on the wall boundary. To show the effect of enhanced mixing by herringbone structures compared with flat channel, the velocity magnitude at the surface perpendicular to the flow direction was plotted inside the microchannel. On-chip purification. Before the cells were introduced into the micropillar device, the micropillar device was washed with PBS and then 200 µL of the prepared LY-1@MBs was injected into the microchannel using syringe pump (Longer, China). Once the whole microchannel was filled with solutions, two NdFeB permanent magnets were put at the two sides of the PDMS, resulting in the LY-1@MBs immobilized on the micropillars. For device optimization, HCCLM9 cancer cells were prestained by FDA (100 µg mL-1 in acetone) and dilute to a concentration of 104 mL-1 in PBS as test samples. Then 1 mL test sample was introduced into the device and the captured cells were observed and counted under an inverted microscope (IX-81, Olympus, Japan). Cell-capture from Cell mixtures or blood samples were performed in a similar way described above except spiking various numbers (102~103) of HCCLM9 cells in 1 mL cell suspension containing 106 MHCC97-L or 1 mL whole blood, respectively. For a low number cell spiking (0~100), HCCLM9 cells were diluted in DMEM starting at an initial concentration of 104 mL-1. 10 µL of the concentrated cell suspension was transferred to a low-attachment 96-well plate. The transferred cells were counted under the microscope, then immediately pipetted into a 1 mL of whole blood. After removing the cells from the 96-well plate, we counted the remaining cells at the same position. By subtracting these cells left behind from the original spot, the total number of cells spiked into blood was estimated. During the cell-capture assays, the cells that got rid of the micropillar device were collected at the outlet and defined as escaped cells. The captured simulated CTCs (HCCLM9 cells spiked in whole blood) were then fixed with paraformaldehyde (4 % in PBS) for 15 min, successively


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refilled with Triton X-100 (0.2 % in PBS) for cell permeation for 10 min and blocked with BSA (3 % in PBS). Followed by staining of anti-CK, anti-CD45 and DAPI, the captured cells were counted and imaged under the inverted microscope. Off-chip enzymatic treatment. After the cell-capture, the permanent magnets were removed and the microchannel was rinsed with PBS. The captured cells were then washed away from the micropillars and collected at the outlet. To further release HCCLM9 cells from the attached MBs, the collected suspension was injected in a 96-well plate and treated with exonuclease (10 µL and 10 µL exonuclease buffer in 80 µL DMEM) at 37 °C for 15 min in a standard incubator (Thermo Scientific, USA). After the enzymatic treatment and magnetic separation, the released HCCLM9 cells were re-suspended in DMEM in a 96-well plate and cultivated at 37 °C under a humidified 5 % CO2 atmosphere. The culture medium was replaced once a day. RT-PCR analysis. RT-PCR analysis was conducted toward captured HCCLM9 cells and escaped HCCLM9 cells (100 cells). Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA). Total RNA was purified by chloroform, precipitated with isopropyl alcohol, washed with 75 % ethanol and redissolved in 0.1 % diethylpyrocarbonate (DEPC)-treated ddH2O and stored at -80 °C. The cDNA was prepared by the first-strand cDNA synthesis kit according to the protocol provided by themanufacturer (Toyobo, Japan). A 423-bp PCR product of CK-19 was amplified using primers, 5´-CTG AGT GAC ATG CGA AGC CAA TA-3´ (sense primer) and 5´-ATC TTC CTG TCC CTC GAG CA-3´ (antisense primer). The primers for amplification of the human GAPDH: forward (5’-ACC ACA GTC CAT GCC ATC AC-3’) and reverse (5’TCC ACC ACC CTG TTG CTG TA-3’). PCR reactions were carried out in a DNA thermal cycler (ABI 2700, USA) in the presence of sense and antisense primer (final concentration 500


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nM). The amplified PCR products were analyzed by electrophoresis in 1 % agarose gel and visualized with ethidium bromide staining.

RESULTS AND DISCUSSION Fabrication of the PN-chip. The PN-chip we present here takes advantages of both

magnetic separation and micromixing technique. The ITO glass substrate has ~5100 electroplated nickel micropillars with diameter of ~50 µm, which form a magnetic filter to trap the past MBs under external magnetic field.46 The PDMS herringbone structure works as a chaotic mixer to induce transverse flow in the microchannel, which will further enhance the interaction between the target cells and functionalized surfaces (Figure S1).44 LY-1 are singlestranded oligonucleotides generated by Cell-SELEX process through a positive selection using HCCLM9 high metastatic HCC cell line and a negative selection using MHCC97-L low metastatic HCC cell line.45 Different HCC cell lines HCCLM9 and MHCC97-L, along with leukocytes (WBC) and Peripheral blood monocytes (PBMC) were used to test the binding specificity of LY-1 aptamers by flow cytometry (Figure S2). LY-1 had highly binding capacity to HCCLM9 and low binding capacity to MHCC97-L, but little binding to WBC or PBMC, implying the specific recognition of LY-1 aptamers to high metastatic HCCLM9 cell line. To functionalize the micropillars, LY-1 aptamers were firstly conjugated to the graphite oxide (GO)-coated Fe3O4 MBs, to give LY-1@MBs, and then the aptamer-conjugated MBs were trapped onto the micropillar arrays (Figure S3). The dynamic light scattering measurement showed no obvious difference in hydrodynamic size before or after aptamer conjugation. The zeta potential measurement, however, indicated that before conjugation the zeta potential of MBs was -13.8 mV, while after conjugation the zeta potential of LY-1@MBs became -17.4 mV, which could be attributed to the negative charges carried by nucleic acid aptamers (Figure S3c).40 The


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high-packed LY-1@MBs immobilized on the micropillars enabled the effective capture of cancer cells (Figure S3d). On-chip purification of HCC cells from cell mixture. To validate the capture performance of PN-chip and to optimize the experimental conditions, 103 HCCLM9 cells were labeled with a fluorescent dye (FDA, green fluorescence) and mixed with 106 MHCC97-L cells, which were prestained with DAPI (blue fluorescence), in 1 mL PBS as test samples. After injecting the cell mixture into the PN-chip, the captured cells were imaged and counted under a fluorescent microscopy. Another device only had flat mainchannel and conventional off-chip magnetic separations were used as controls. The simulation results (Figure 2a) demonstrated the impact of PDMS chaotic mixer. Under the laminar, uniaxial flow conditions presented in the flat device, cells followed streamlines and displayed minimal diffusion across flow channels. This lack of mixing resulted in a limited number of interactions between the cells and the micropillars, especially with high channel heights. Upon the integration of chaotic mixer, stirring flows were introduced, ensuring effective contacts of cells with the bottom substrate. Consistent with the simulation, the PN-chip proved highly efficient at capturing HCCLM9 cells, whereas significantly less capture was observed with the flat chamber device (Figure 2b). Different flow rates (from 0.4 mL h-1 to 1.6 mL h-1) were tried and 0.8 mL h-1 was found as the optimal one with final capture efficiency ~91% (Figure 2c). Compared with on-chip purification, the capture efficiency of off-chip magnetic separation is ~70%. Besides to flow rate, another two parameters: the heights of the microchannel and the micropillar also influence the capture performance to a certain extent because the alternation of either one will change the flow pattern inside the PN-chip and affect the cell-micropillar interaction. For our PN-chip, 80 µm of channel height (55 µm for mainchannel and 25 µm for


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herringbone) and 24 µm of micropillar height were found as the optimal parameters (Figure S4). Although more desirable, higher pillar heights may introduce more defects and undermine the overall robustness. Moreover, the higher channel height would offset the mixing effect brought by the herringbone structures and lead to less interaction between the flowing cells and micropillars. Unlike conventional off-chip magnetic separations which were always processed in a centrifuge tube,49-50 the effective functionalized surface created by the micropillar arrays and the enhanced cell-micropillar interactions induced by the herringbone structure not only synergistically contribute to the enhanced capture performance but also provide a dynamic condition to prohibit non-specific binding. The purity of off-chip separation is very poor (50%, the final purity is poor (~8.5%). Moreover, we also directly injected exonuclease into the microchannel and conduct the enzymatic treatment on-chip. Unexpectedly, the recovery effect for on-chip enzymatic treatment is 18.4%, even less than off-chip magnetic separation. This phenomenon can be explained by that the high-packed density of aptamerconjugated MBs on the micropillars make the enzymatic treatment incomplete. Compared with off-chip separation or on-chip enzymatic treatment, combing on-chip purification and off-chip enzymatic treatment can reach better performance toward artificial blood samples with recovery rate of 68.4% and purity of 60.8%. Obviously, compared with the assays of spiked cells in PBS or cell-mixture, the great amount of blood cells (WBC or RBC) and other blood components (free-DNA or RNA) interfered the effective interactions between the simulated CTCs and the micropillars or aptamers. To demonstrate the feasibility of downstream analysis, we extracted RNA from the captured HCCLM9 cells and escaped HCCLM9 cells (cells not to be caught but collected at the outlet),


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respectively. The RT-PCR result (Figure 4d) showed that the captured HCCLM9 cells expressed more mRNA expression of CK-19. Because the overexpression of CK-19 correlated to the higher metastatic potential of HCC cells,48 the relatively higher CK-19 expression of captured HCCLM9 cells than escaped HCCLM9 cells probably suggested the LY-1 aptamers tended to recognize the high metastatic subpopulation within the whole HCCLM9 cell lines. Although nucleic acid aptamers are selected by using cell lines and may have poor performance for conducting real blood samples from cancer patients. In this work we emphasize more on the combination of on-chip purification and off-chip enzymatic treatment, and their synergistic effect to get more purified cancer cells from blood samples. On the other hand, for patients’ blood, DNA sequences can be also conjugated to the antibodies (i.e. anti-EpCAM) as capture probe and cleaved by enzymatic treatment to realize cell-release.43 Additionally, the dimensions of our PNchip are able to be expanded to process larger volumes of blood while maintaining the proper flow rate at the same time.51

CONCLUSIONS In conclusion, a two-stage purification strategy is demonstrated for enhancing the purity of

captured cancer cells in blood samples. The PN-chip introduces stirring flow to continuous magnetic separation, increasing the capture sensitivity and decreasing non-specifically bounded cells. The subsequent off-chip enzymatic treatment enables the cancer cells further released from the attached MBs, improving the purity and facilitating next re-culture. For the proof-of-concept study, spiked cancer cells in whole blood were successfully recovered with enhanced purity for next RNA expression analysis by the synergistic effect of our two-stage purification approach.


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Notes The authors declare no competing financial interest.

Author contributions X. Y., B. W. and N. Z. contributed equally to this work

Supporting Information Information of the fabrication of PN-chip, the construction and characterization of aptamerconjugated MBs and Figure S1-S7. This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgement

We thank Prof. Quan Yuan for her helpful advices. This work was supported by the National Natural Science Foundation of China (Nos. 51132001, 51272184, 81371897 and 81572860), the Research Fund for the Doctoral Program of Higher Education of China (No. 20130141110059), the Natural Science Foundation of Hubei Province (No. 2013CFA027), and the Independent Research Fund Program of Wuhan University (No. 2042014kf0241).


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Figure captions

Figure 1. Conceptual illustration of the enhanced purity of captured HCC cells by the synergistic effect of on-chip purification and subsequent off-chip enzymatic treatment.


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Figure 2. On-chip purification of HCCLM9 cells in cell mixture. (a) Simulation of the flow patterns in the chaotic channel or flat channel. (b) Representative fluorescent images of the captured performance by using PN-chip or flat-channel chip, respectively. The 1 mL cell mixture contains 103 HCCLM9 cells and 106 MHCC97-L cells. (c) Comparison of capture efficiencies of HCCLM9 cells at various flow rates (0.4 mL h-1, 0.8 mL h-1, 1.2 mL h-1 and 1.6 mL h-1) in PN chip or flat-channel chip. Off-chip magnetic separation is also conducted as control group. (d) Comparison of purity by using different devices. The error bars represent a mean ± standard deviation from three repeats.


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Figure 3. Off-chip enzymatic treatment of captured HCCLM9 cells. (a) Top: Schematic of the mechanism on the enzymatic release of HCCLM9 cells from the aptamer-conjugated MBs. Bottom: representative images of a HCCLM9 cell before and after enzymatic treatment, respectively. (b) The counts of recovered HCCLM9 cells towards different numbers of initial spiked cells. (c) Representative dark-field images of cell-mixture before (left) and after (right) enzymatic treatment. Most HCCLM9 cells (green) were further released from the suspension. “1000” and “100” are the initial spiked number of cells. (d) Enhanced purity of released HCCLM9 cells by enzymatic treatment. The error bars represent a mean ± standard deviation from three repeats.


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Figure 4. Validation of the PN-chip using HCCLM9 cells spiked in whole blood. (a) Immunostaining of simulated-CTC and WBC captured by a micropillar on the PN-chip. The dash lines indicate the boundary of the micropillar. (b) Released CTC and WBC after enzymatic treatment. The CTC was further detached from the MBs while the WBC was still wrapped by the MBs, as the arrow indicated. (c) Recovery rate and purity for low-abundance spiked 0~100 HCCLM9 cells in whole blood by different experimental conditions. (d) RT-PCR results (from left to right): lane 1: GAPDH of captured HCCLM9, lane 2: GAPDH of escaped HCCLM9, lane 3: mRNA expression of CK-19 of captured HCCLM9, lane 4: mRNA expression of CK-19 of escaped HCCLM9, lane 5: Maker.


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Table Of Contents


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Capture and Release of Cancer Cells by Combining On-Chip

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