Highly sensitive detection of cancer cells based ... - Wiley Online Library

1504 Soyi Chung Minkyung Cho Jae Hwan Jung Tae Seok Seo Department of Chemical and Biomolecular Engineering (BK21 Program), Korea Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea

Received January 2, 2014 Revised February 7, 2014 Accepted February 8, 2014

Electrophoresis 2014, 35, 1504–1508

Short Communication

Highly sensitive detection of cancer cells based on the DNA barcode assay and microcapillary electrophoretic analysis Considering rarity of circulating tumor cells (CTCs) in human blood, the development of highly sensitive detection techniques for cancer cells is crucial for prediction, diagnosis, and prognosis of cancers. In this study, we propose an advanced cellular detection method by combining a biobarcode assay and microcapillary electrophoresis (␮CE) technology. While the DNA biobarcode assay can provide ultrasensitive and multiplex detection platforms, the ␮CE chip can analyze barcode DNAs with high speed and accuracy according to the DNA size. We designed the barcode DNA size as 20 bp for indicating the expression of epithelial cell adhesion molecules (EpCAM) biomarkers and 30 bp for assigning CDX2 expression which is specific for colorectal cancer cells with addition to two bracket ladders (15 and 45 bp). Using MCF-7 (breast cancer) and SW620 (colorectal cancer) as models, we conducted a biobarcode assay and analyzed the resultant biobarcode DNA on the ␮CE chip. We could detect the 20 bp CE peak in the electropherogram even with ten MCF-7 and SW620 cells in a volume of 200 ␮L, thereby demonstrating the highly sensitive detection of cancer cells. We furthermore identified the type of colorectal cancer by observing two positive peaks (20 bp for EpCAM and 30 bp for CDX2) in the ␮CE analysis. Keywords: Biobarcode assay / Breast cancer / CE / Circulating tumor cell / Colorectal cancer DOI 10.1002/elps.201400001

Additional supporting information may be found in the online version of this article at the publisher’s web-site

Recent efforts on the cancer diagnostics have been dedicated to detecting circulating tumor cells (CTCs) which are the cells detached from the edges of a tumor and circulate in the bloodstream. The advantages of simple blood draw instead of painful bone marrow aspiration and real-time cancer monitoring to predict prognosis of cancers endow CTC as a nextgenerating cancer diagnostic tool. However, the bottleneck of CTC analysis lies in the scarcity of CTC, since only one CTC approximately exists among one billion blood cells [1]. Thus, to identify the CTCs in the limited human blood, a highly sensitive analytical technique for cancer cell is required.

Correspondence: Professor Tae Seok Seo, Department of Chemical and Biomolecular Engineering (BK21 Program), Korea Advanced Institute of Science and Technology (KAIST), 291 Daehakro, Yuseong-gu, Daejeon 305-701, Korea E-mail: [email protected] Fax: +82-42-350-3933

Abbreviations: ␮CE, microcapillary electrophoresis; CTC, circulating tumor cell; EpCAM, epithelial cell adhesion molecule; MMP, magnetic microparticle; PMP, polystyrene microparticle C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

To address this issue, two main approaches have been developed so far: size-based filtration and affinity selection [2,3]. Size-based filtration methods use the porous cylindrical polycarbonate membrane whose diameter of pores is 8 ␮m. While the erythrocytes and leukocytes are passing through the membrane, the large CTCs are filtered and isolated for further downstream analysis. This technique is simple, label-free, and prevents morphology damage of cells, but the size of leukocytes is partially overlapped with that of CTCs or the relatively small sized CTCs can be penetrated. Thus, the capture yield and purity of the isolated CTCs should be compromised [4–6]. Affinity selection basically relies on specific antibody– antigen interaction to separate CTCs [7–11]. Epithelial cell adhesion molecule (EpCAM) on the CTCs can be recognized by anti-EpCAM antibody, and anti-EpCAM antibody coated magnetic particles or microposts were employed for capturing and identifying CTCs [12, 13]. Nagrath et al. reported the affinity based CTCs isolation in cancer patients by using the micropost array integrated microdevice with 99% yield and 50% purity. Despite fine manipulation capability of cell

Colour Online: See the article online to view Figs. 1–3 in colour.

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Figure 1. (A) Schematic illustration of detecting cancer cells by combining the biobarcode assay and ␮CE analysis. (B) Microscopic image of attachment of anti-EpCAM MMPs on the MCF-7 cell. Scale bar: 10 ␮m. (C) Bright field and fluorescence images of the (anti-EpCAM MMP)–(MCF-7)–(anti-EpCAM & 20 bp PMPs) complexes. Scale bar: 10 ␮m.

sorting rare-cell detection, the low dimension of a microfluidic channel has inherent limitations in terms of high-throughput treatment of processing a large volume of samples. Regarding the highly sensitive cellular detection methods, we adopted the advantages of the biobarcode assay techniques [14–19]. The DNA biobarcode assay which uses two types of particle probes (magnetic microparticles (MMPs) with recognition elements for the target of interest and gold nanoparticles with a second recognition agent and barcode DNAs) has demonstrated its ability for detecting target proteins or nucleic acids with ultrahigh sensitivity. Since the barcode DNAs on a single nanoparticle are abundant enough to be detected, even a single analyte can be potentially analyzed. Moreover, the ease of tuning the size of barcode DNAs allows us to detect multiple targets simultaneously. As for the detection methods for the released barcode DNAs, the CE on a microchip is superior in terms of precision, simplicity, rapidity, and quantitative analysis [20]. Size-dependent elution time of peaks in the electropherogram enables us to recognize the target DNA with ease, and the single base resolution separation on a chip makes it possible to analyze the multiplex DNA molecules [21]. Due to these advantages, the microCE (␮CE) based genetic analysis is widely applied for DNA sequencing [22, 23], short tandem repeat genotyping [24, 25], and single nucleotide polymorphism analysis [26, 27]. In this

C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

study, we developed an advanced cancer cell detection method based on the biobarcode assay and the ␮CE. Combining the advantages of both techniques, we could detect the cancer cells with high sensitivity and speed. In addition, the multiplexing ability of the proposed techniques could differentiate colorectal cancer cells from breast cancer cells. Figure 1A illustrates the overall scheme for cancer detection using the DNA biobarcode assay and the ␮CE on a chip. First, we synthesized the particle probes: anti-EpCAM monoclonal antibody labeled MMPs (anti-EpCAM MMPs, 1 ␮m diameter), anti-EpCAM polyclonal antibody and 20 bp barcode DNA labeled polystyrene microparticles (anti-EpCAM & 20 bp PMPs, 1 ␮m diameter), anti-CDX2 polyclonal antibody and 30 bp barcode DNA labeled PMPs (anti-CDX2 & 30 bp PMPs), 15 bp and 45 bp bracket ladder DNA labeled MMPs (15 bp MMPs and 45 bp MMPs, 2.8 ␮m diameter). See the detailed information on the particle probe synthesis in the Supporting Information. MCF-7 (breast cancer cell line) and SW620 (colorectal cancer cell line) were used for cancer cell detection. Fifty microliters of anti-EpCAM MMPs, 20 ␮L of 15 bp MMPs, and 20 ␮L of 45 bp MMPs were washed with 1 mL of 0.01 M PBS twice, and mixed with a cell solution. After making the total volume of 200 ␮L with a PBS buffer, the mixture was incubated at room temperaR Sample Mixer in orture for 30 min using a Dynabeads der to attach the MMPs on the cells through EpCAM and


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anti-EpCAM interaction. Figure 1B shows anti-EpCAM MMPs on the surface of cancer cells. A significant number of the MMPs exist on the cells which enable cell purification by an external magnet without loss of the target cells. After a magnetic separation and washing step, (anti-EpCAM MMP)–(MCF-7) complexes were resuspended with a solution containing 50 ␮L of anti-EpCAM & 20 bp PMPs. The mixture solution was incubated for 30 min, and then vigorous washing was followed to ensure the complete removal of the excess particle probes. As a result, (anti-EpCAM MMP)– (MCF-7)–(anti-EpCAM & 20 bp PMPs) sandwich complexes were produced. The MMP and PMP attachment on the MCF-7 cells was verified by observing the fluorescence signal which was derived from the FAM labeled barcode DNAs on the PMPs. Figure 1C shows a bright field microscope image (left) and fluorescence image (right) of the (anti-EpCAM MMP)– (MCF-7)–(anti-EpCAM & 20 bp PMPs) complexes. Depending on the injected particle probes and cell lines, the following sandwich structured immunocomplexes ((anti-EpCAM MMP)–(MCF-7)–(anti-EpCAM & 20 bp PMPs), (anti-EpCAM MMP)–(SW620)–(anti-EpCAM & 20 bp PMPs), and (antiEpCAM MMP)–(SW620)–(anti-CDX2 & 30 bp PMPs)) could be generated. Those complexes were resuspended with 20 ␮L R by Life Techof Hi-DiTM Formamide (Applied Biosystems nologies) and denatured at 70°C for 2 min to separate a FAM labeled single stranded DNA (20 bp or 30 bp) from the doublestranded barcode DNA along with 15 and 45 bp bracket ladder DNAs which were coming from 15 and 45 bp MMPs. The released FAM labeled single stranded DNA was collected during the magnetic purification and analyzed on the ␮CE chip. The ␮CE glass chip was fabricated according to the previous reports [28]. Figure 2A shows the simple cross-designed CE chip (width: 220 ␮m and depth: 40 ␮m) and the CE operation process. Prior to the CE operation, the microchannels were first cleaned using piranha (3:1 H2 SO4 /H2 O2 ) solution for 15 min. After extensive H2 O washing, the channels were pretreated with a 50% dynamic coating (DEH-100, The Gel Co., San Francisco, CA, USA) in methanol for 15 min to suppress electro-osmotic flow. A 5% w/v linear polyacrylamide (LPA) with 6 M urea in 1 × Tris TAPS EDTA (TTE) was injected as a separation sieving matrix, from the anode hole. The separation channels (effective length: 6 cm) were heated at 70°C with a silicon rubber heater (SR020312, Hanil Electric Heat Engineering Co., Korea) for denaturing conditions. The recovered barcode DNA solution (8 ␮L) was loaded in the sample reservoir and added into the injection channel (1 cm) under electric field strength of 1000 V/cm for 30 s. Then, 900 V was applied in the sample and the waste reservoir, and 2100 V and 0 V were supplied in the anode and cathode reservoir, respectively, for 10 s as a backbiasing to prevent bleeding of the DNA sample into the separation channel. The DNA sample (1.37 nL) in the intersection part was isolated and separated along the separation channel under an electric field strength of 257 V/cm and detected by a laser-induced confocal fluorescence microscope (C1si, Nikon, Japan) near the anode reservoir. Since the appearance of the 20 bp barcode DNA C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


Figure 2. (A) The design of the ␮CE chip and the CE operation process. (B) Electropherograms of the bracket ladders and the barcode DNAs. (Top panel) Bracket ladders of 15 and 45 bp DNAs with barcode DNAs of 20 and 30 bp. (Middle panel) Bracket ladders of 15 and 45 bp DNAs with barcode DNAs of 20 bp. (Bottom panel) Bracket ladders of 15 and 45 bp DNAs with barcode DNAs of 30 bp.



indicates the expression of the EpCAM, the CE peak corresponding to 20 bp suggests that the targets are cancer cells (either MCF-7 or SW620 in this study). The presence of both 20 and 30 bp peaks implies that the targets are cancer cells as well as colorectal cancer type, since the 30 bp barcode DNA is matched with the anti-CDX2 antibody which specifically captures the CDX2 epitopes of colorectal cancer cells (SW620 in this study). The sequence information is shown in Supporting Information Table 1, and their melting temperature is in the range from 56 to 59°C. Figure 2B shows the electropherogram of four FAMlabeled barcode DNAs. From left to right, the peak of 15, 20, 30, and 45 bp appeared in a consecutive order. However, the absolute elution time of each peak could be variable according to the CE operation, polymer gel matrix, and temperature (as shown in Fig. 2B from top to bottom). Thus, to clarify the peak assignment regardless of the external influences, we added the bracket ladders (15 and 45 bp) and calculated the relative elution time of 20 and 30 bp barcode DNAs with respect to the bracket ladders. From the top panel of Fig. 2B, we obtained the elution time ratio of 20 bp DNA (0.164) by dividing the time difference (4.18 s) between the elution time of 20 and 15 bp by the time difference (25.489 s) between the elution time of 45 and 15 bp. In the same way, the elution time ratio of 30 bp DNA was calculated as 0.552. Such a relative elution time ratio was valid in the middle and bottom CE data in Fig. 2B, although the absolute elution time of bracket ladders was changed. Detailed information for the relative elution time ratio was presented in Table 2 (Supporting Information). Figure 3 shows the results of the breast and colorectal tumor cell detection according to the scheme of Fig. 1A. Figure 3A shows that MCF-7 cancer cells could be detected in the range of 10 to 105 cells. The signal of a barcode DNA obtained from the input cell number of 10 was clearly distinguishable from the background level with a S/N of 3. As shown in the inset of Fig. 3A, the peak height was augmented in proportion to the logarithm of the input cell number with a R2 value of 0.996. Since the CE analysis can provide quantitative information, we could determine the cancer cell number by using the CE calibration curve. We also demonstrated a highly sensitive detection of SW620 cancer cells with a low limit of detection of 10 cells (Fig. 3B). As the input cell number increased from 10 to 105 , the peak intensities were gradually augmented. The overall absolute peak height in Fig. 3B was less than that of Fig. 3A, which might be due to the different EpCAM and anti-EpCAM binding affinity depending on the cell type. The addition of the bracket ladders (15 and 45 bp) enables us to assign the 20 bp barcode DNA with high accuracy. Finally, we performed multiplex barcode assay using anti-EpCAM MMP, anti-EpCAM & 20 bp PMPs, and antiCDX2 & 30 bp PMPs with SW620 cells. As shown in Fig. 3C, the 20 (the relative elution time ratio of 0.156) and 30 bp (the relative elution time ratio of 0.522) DNA peaks were displayed between the bracket ladders. These results imply that the target was a cancer cell due to the formation of ((antiEpCAM MMP)–(SW620)–(anti-EpCAM & 20 bp PMPs)) as C 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Figure 3. (A) LOD study for detecting MCF-7 breast cancer cells. The input cell number ranged from 10 (back) to 105 (front), and even 10 cells generated a significant barcode DNA peak in the electropherogram. Inset: the correlation of the peak intensity versus the logarithm of the input cell number. (B) LOD study for detecting SW620 colorectal cancer cells. The input cell number ranged from 10 (back) to 105 (front), and even 10 cells generated a significant barcode DNA peak in the electropherogram. The bracket ladders could clearly assign the eluted peak. (C) Multiplex analysis for EpCAM (20 bp) and CDX2 (30 bp) epitopes revealed that the targets were colorectal cancer cells.


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well as a colorectal cancer cell due to the formation of ((antiEpCAM MMP)–(SW620)–(anti-CDX2 & 30 bp PMPs)). Thus, our methodology could execute multiplex biomarker analysis at the same time, providing more detailed and enriched information to diagnose the cancers with high accuracy and reliability. In conclusion, we have developed an advanced diagnostic platform for detecting cancer cells by combining DNA biobarcode assay and ␮CE analysis. The whole process could be complete in 1.5 h, and a low LOD of 50 cells/mL was conducted. Multiplex analysis for the different biomarkers allows us to discover the cancer cell type. Such a simple, sensitive, multiplex, and rapid analytical tool can be applied for a variety of diagnostic research fields. This research was supported by the Converging Research Center Program funded by the Ministry of Education, Science and Technology (2013K000273), BioNano Health-Guard as Global Frontier Project (H-GUARD_2014M3A6B), and the Korea CCS R&D Center (KCRC) grant (2013M1A8A1040878) funded by the Ministry of Science, ICT & Future Planning (MSIP) of Korea. The authors have declared no conflict of interest.

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