Circulating Tumor Cell Microseparator Based on Lateral

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Circulating Tumor Cell Microseparator Based on Lateral Magnetophoresis and Immunomagnetic Nanobeads Seonyoung Kim,† Song-I Han,† Min-Jae Park,‡ Chang-Wan Jeon,§ Young-Don Joo,‡ In-Hak Choi,∥ and Ki-Ho Han*,† †

Department of Nano Engineering, Center for Nano Manufacturing, Inje University, Gimhae 621-749, Republic of Korea Department of Hemato-Oncology, Haeundae Paik Hospital, Inje University, Busan 612-030, Republic of Korea § Department of Surgery, Cancer Center, Dongnam Institute of Radiological and Medical Sciences, Busan 619-900, Republic of Korea ∥ Department of Microbiology and Immunology, Busan Paik Hospital, College of Medicine, Inje University, Busan 614-735, Republic of Korea ‡

S Supporting Information *

ABSTRACT: This paper presents a circulating tumor cell (CTC) microseparator for isolation of CTCs from human peripheral blood using immunomagnetic nanobeads with bound antiepithelial cell adhesive molecule (EpCAM) antibodies that specifically bind to epithelial cancer cells. The isolation is performed through lateral magnetophoresis, which is induced by high-gradient magnetic separation technology, involving a ferromagnetic wire array inlaid in the bottom substrate of a microchannel. Experimental results showed that the CTC microseparator isolates about 90% of spiked CTCs in human peripheral blood at a flow rate of up to 5 mL/h and purifies to approximately 97%. The overall isolation procedure was completed within 15 min for 200 μL of peripheral blood. CTCs from peripheral blood of patients with breast and lung cancers were isolated with the CTC microseparator, and the results were compared with those of healthy donors. Using a fluorescence-based viability assay, the viability of CTCs isolated from peripheral blood of patients with cancer was observed. In addition, the usefulness of the CTC microseparator for subsequent genetic assay was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR) amplification of cancer-specific genes using CTCs isolated from patients with cancer. beads,14−16 nucleic acid quantification,17 fiber-optic array scanning,18 laser scanning cytometers,19,20 microposts,21,22 microfluidic mixing structures,23 and meandering microchannels.24 The most commonly used method for separating CTCs involves immunomagnetic beads,3,5,6,25 which are currently commercialized for clinical applications in the CTC-based diagnosis and prognosis of solid tumors. However, because of the extremely low frequency of CTCs in peripheral blood (1−2 CTCs per 10 billion blood cells), 26 the most previous methods isolate CTCs with a very low recovery rate5 and low purity,6,14 which disturbs their abilities to select primary therapy in metastatic disease and monitor effectiveness of postsurgical therapy and recurrence. The low purity also increases the discrimination time and risk of error in distinguishing CTCs from enriched cells. The low performance of CTC detection also hampers its clinical standardization for routine cancer prognosis.9 Furthermore, discordant results from different methods of CTC detection make it difficult for CTC

M

any recent studies have shown that the number of circulating tumor cells (CTCs) in peripheral blood is directly correlated with the progression of cancer.1−5 Because CTCs persist after therapy, the presence of CTCs is an independent predictor of progression-free survival and overall survival of patients with metastatic cancer. Further, the presence of CTCs was found to be proportional to disease progression when a carcinoma recurs.6 Other studies have shown that primary cancers begin shedding neoplastic cells into the circulation even at an early stage,3,7,8 implying that the number of CTCs may reflect the tumor burden at all stages of cancer progression. A particularly interesting characteristic of CTC detection is that it is minimally invasive and can therefore be performed frequently, while conventional cancer detection methods still require repeated invasive procedures that may be associated with limited patient compliance.5 For this reason, CTC detection may become a significant method with which to refine the prognosis in patients with cancer. Furthermore, it may serve as a real-time tumor assay for individualized targeted therapies.9 Methods for the detection and/or isolation of CTCs from peripheral blood have been previously published, including microsized filters,10−12 density gradient,13 immunomagnetic © XXXX American Chemical Society

Received: November 12, 2012 Accepted: February 5, 2013

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detection to serve as a predictor of treatment strategy responses in patients with cancer.9,27 In this report, we introduce a CTC microseparator with advantages such as simplicity of use, rapid separation, and high performance based on immunomagnetic nanobeads coated with anti-EpCAM antibodies and lateral magnetophoresis technology.28 Lateral magnetophoresis is generated with a ferromagnetic wire array inlaid in the bottom floor of a microchannel. Quantitative results for the recovery rate of the CTC microseparator and purity of CTCs isolated from blood samples prepared with spiked breast cancer cell lines (SKBR-3) are reported. CTCs were isolated with the CTC microseparator from peripheral blood of patients with breast (n = 3) and lung (n = 3) cancers and healthy donors (n = 2). Using a fluorescence-based viability assay, the viability of CTCs isolated from peripheral blood of patients with cancer was observed. In addition, reverse-transcriptase polymerase chain reaction (RTPCR) amplification to detect cancer-specific genes from the CTCs was performed to demonstrate that the isolated CTCs could be used directly for substantial CTC-based molecular analysis.

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THEORY AND DESIGN Working Principle. The CTC microseparator proposed in this report was fabricated using two borofloat glass slides (0.7 mm thick; Schott AG, Mainz, Germany) and SU8-to-glass adhesive bonding (Supporting Information Figure S1). It comprises two inlets, two outlets, and a ferromagnetic permalloy wire array inlaid in a bottom substrate (Figure 1a). The sample and buffer inlets are used to inject blood samples mixed with magnetic nanobeads and phosphate buffer saline (PBS) with 2% fetal bovine serum (FBS), respectively. Outlet no. 1 is designed to collect CTCs, and outlet no. 2 is designed to dispose normal hematologic cells. Due to fabrication process limitations, the channel width of outlet no. 1 is set at 200 μm. To enrich CTCs, the channel width of outlet no. 2 is designed to be 800 μm, which is 4 times wider than outlet no. 1. To put several ferromagnetic wires in the microchannel, which was limited to a length of 30 mm by the size (25 × 75 mm2) of the CTC microseparator, and to allow for a stronger magnetic force than the hydrodynamic drag force in the lateral (y′) direction, the angle of the wire is set to 5.7°, which means that the ratio of flow (x′) to the lateral (y′) direction lengths of the wires in Figure 1a is 10 to 1. The width and thickness (50 and 20 μm, respectively) of the ferromagnetic wire at intervals of 300 μm generate a high-gradient magnetic field in CTCs with bound magnetic nanobeads. To avoid aggregation and stacking of CTCs at corners comprising the ferromagnetic wires and the sidewall of the microchannel, the ferromagnetic wires were bent at almost a right angle 100 μm before reaching the sidewall. The wires were then laid in parallel with the direction of the magnetic field. Because magnetic nanobeads coated with anti-EpCAM antibodies bind to epithelial-derived CTCs, the CTCs behave as paramagnetic particles. Consider a ferromagnetic wire array placed at an angle of θ to the direction of flow and inlaid over the whole area of a microchannel. When a uniform external magnetic field is applied to the ferromagnetic wire, the external magnetic field is deformed near the wire. The inlaid ferromagnetic wire array thus generates a high-gradient magnetic field over the whole area of the microchannel (Figure 1b). The paramagnetic CTCs passing over the wire then experience magnetic force Fm and hydrodynamic drag force Fd. The lateral magnetic force, Fl, on CTCs (Figure 1a) generates a vector sum of the magnetic force and the drag force. Consequently, with an

Figure 1. (a) Perspective view of the CTC microseparator, including the inlaid ferromagnetic wire array placed at an angle of θ (5.7°) to the direction of fluidic flow under an applied external magnetic field H0. The magnetic force acting on the CTCs is induced by a high magnetic field gradient generated near the ferromagnetic wire array with an external magnetic field. Lateral displacement (del y′) is created by the magnetic force Fm and hydrodynamic drag force Fd. (b) Crosssectional view of the inlaid ferromagnetic wire array and the gradient magnetic field along the A−A′ cross section in panel a. (c) A photograph of the fabricated CTC microseparator. The enlarged view shows that CTCs spiked into peripheral blood are flowing into outlet no. 1 with an external magnetic field, while normal blood cells are flowing into outlet no. 2.

external magnetic field, CTCs with bound magnetic nanobeads are forced laterally and flow into outlet no. 1, while normal blood cells flow into outlet no. 2 (Figure 1c). B

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Theoretical Analysis. The x-directional magnetic force Fmx (Figure 1a), which acts on a CTC with bound N number of

magnetic nanobeads, generated by a rectangular ferromagnetic wire is expressed as follows (see the Supporting Information text):

8NVbMbxka 2B0

Fmx = −

2

( wh )a2(x 2 − z 2) + k2( wh ) a4

π (x 2 + z 2)2 (x 2 + z 2)2 + 2k

where Vb represents the volume of a magnetic nanobead, Mb is the saturation magnetization field of the magnetic nanobead, μB is the permeability of the buffer solution, μW is the permeability of the ferromagnetic wire, a is the effective radius of the ferromagnetic wire, and B0 is an applied external magnetic flux. If the levitation height z is constant, the x- and y-directional displacements are dx =

1 (Fmx + Fd sin θ )dt 6πηd

(2)

1 Fd cos θt 6πηd

(3)

where η represents the viscosity of the buffer solution, d is the radius of the CTC, and θ is the angle between the ferromagnetic wire and the direction of flow. The y′-directional lateral displacement del y′ (Figure 1a) of the CTC can be obtained using the numerical results of eqs 2 and 3 as follows: del y′ = x cos θ + y sin θ

k=

μ W − μB μ W + μB

(1)

on ice for 20 and 15 min, respectively. Before injection of the blood sample into the CTC microseparator, blood was fixed with 100 μL of 4% paraformaldehyde for 5 min and subsequently permeabilized with 100 μL of 0.2% Triton X-100 (AMRESCO) for 5 min. Blood cells were then stained with membrane-permeable nucleic acid fluorescent dye (Hoechst 34580) for nuclei, anti-CK19-PE antibodies for epithelial cells, and anti-CD45-FITC antibodies for normal blood cells for 30 min. The blood sample preparation was completed by mixing with 800 μL of a PBS solution with 2% FBS. Fluorescence-Based Viability Assay. Cells collected from outlet no. 1 of the CTC microseparator were transferred to a 1.5 mL tube and enriched by 300g centrifugation for 5 min. The cells were stained for 30 min with 50 μL of 0.1% ethidium homodimer-1 (Live/Dead viability/cytotoxicity kit for mammalian cells, Invitrogen) for live cells and 50 μL of 0.1% calcein AM (Invitrogen) for dead cells. After staining, all cells were spread on a glass slide and monitored with a fluorescent microscope.

and y=−

⎛ w ⎞⎡ 2 ⎛ w ⎞ 2⎤ 2 ⎜ ⎟ x − 3z + k ⎜ ⎟a , ⎢ ⎝ h ⎠⎣ ⎝ h ⎠ ⎥⎦

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RESULTS AND DISCUSSION Recovery Rate and Purity. To determine the recovery rate and purity of the CTC microseparator, breast cancer cell lines (SKBR-3) in the range of 10−104 cells and stained by a nucleic acid fluorescent dye (SYTO 13, Invitrogen) were spiked into 200 μL of human peripheral blood. The blood was mixed with anti-EpCAM antibodies and magnetic nanobeads in sequence and incubated on ice for 20 and 15 min, respectively, according to the manufacturer’s instructions (STEMCELL Technologies). The cancer cell-spiked blood sample was prepared by dilution with a 4-fold volume of PBS solution. Two syringe pumps were used to inject the blood sample and PBS buffer with 2% FBS at the same flow rate into the two inlets. The CTC microseparator was placed under a microscope (ME600, Nikon Instruments Inc.) with a fluorescence detector (Y-FL, Nikon Instruments Inc.) to count CTCs flowing into outlet no. 1 and to capture images of cells passing through the microchannel. A stack of two neodymium−iron−boron (NeFeB) permanent magnets was placed underneath the CTC microseparator, generating an external magnetic flux of 0.2 T, which was applied at a horizontal direction to the microchannel. Then, CTCs with bound magnetic nanobeads were drawn laterally along the edge of the wire; meanwhile, normal blood cells flowed into outlet no. 2 (Figure 2a). The CTCs moving along the wire (Figure 2b) had no sooner reached the bending point of the wire than they followed the flow direction and flowed into outlet no. 1 (Figure 2c). During injection of the blood sample, the cells that flowed into outlet no. 1 were recorded through the fluorescence microscope and collected in a tube. The recovery rate of the CTC microseparator was evaluated by monitoring the number of CTCs, and the purity was determined by fluorescence observation of the cells collected from outlet no. 1.

(4)

According to eqs 2 and 4, the lateral displacement del y′ increases as the magnitude of the x-directional magnetic force Fmx on the CTC increases and the angle θ decreases. As shown in Supporting Information Figure S4a, the negative x-directional magnetic force increases as CTCs approach the ferromagnetic wire based on the assumption that the levitation height of the CTC is constant. With a drag force that is generated by fluidic flow, the CTCs are positioned at a location to satisfy Fmx + Fd sin θ = 0 (eq 2). If the negative x-directional magnetic force is lower than the drag force, the CTCs move slightly laterally and eventually pass the wire.28 In contrast, for Fmx + Fd sin θ < 0, the CTCs cannot pass over the ferromagnetic wire and move laterally until they approach the bending point of the ferromagnetic wire (Figure 2).

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MATERIALS AND METHODS See the Supporting Information text for detailed descriptions of the following methods: (a) fabrication process, (b) instrument setup, and (c) RT-PCR protocol. Blood Collection and Sample Preparation. Healthy human peripheral blood samples were drawn from two healthy donors, and cancer blood samples were drawn from patients with lung and breast cancer using a protocol (IRB no. 2011104) approved by the Institutional Review Board (IRB) of Haeundae Paik Hospital in South Korea. The characteristics of the peripheral blood donors are summarized in Supporting Information Table S1. All specimens were collected in Vacutainer tubes containing the anticoagulant EDTA and processed within 12 h. According to the manufacturer’s instructions (STEMCELL Technologies), 200 μL of peripheral blood was mixed with anti-EpCAM antibodies and magnetic nanobeads in sequence and incubated C

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Figure 3. (a) Recovery rates of the CTC microseparator at various sample flow rates. Breast cancer cell lines (SKBR-3), which were spiked into 200 μL of human peripheral blood diluted with 800 μL of a PBS solution and tagged by magnetic nanobeads with bound antiEpCAM antibodies, are separated by the CTC microseparator at a sample and buffer flow rate of 2−5 mL/h with an external magnetic flux of 0.2 T. The error bars represent one standard deviation calculated from three data sets. (b) Regression analysis of the isolation recovery rate for varying SKBR-3 cell concentrations between 14 and 12 000 cells at a sample and buffer flow rate of 4 mL/h.

Figure 2. Photomicrographs showing a normal white blood cell and a cell from the breast cancer cell line SKBR-3, with bound magnetic nanobeads, passing through the microchannel of the CTC microseparator at a sample and buffer flow rate of 3 mL/h with an external magnetic field. (a) The breast cancer cell is drawn laterally along with the ferromagnetic wire, while the normal white blood cell moves with the fluidic flow. (b) When the cancer cell approaches the bending point, it leaves the ferromagnetic wire and flows with the fluid. (c) Eventually, the cancer cell with bound magnetic nanobeads flows into outlet no. 1, whereas the normal white blood cell is shunted into outlet no. 2. Tracking of the cells was performed at 33.3 ms intervals, and each trajectory of the normal white blood cell and cancer cell was observed at different moments and combined.

microscope. Red fluorescent cells were then counted as spiked CTCs and green fluorescent cells were counted as contaminated normal blood cells (Figure 4a). From the observed results, we calculated that the purity of separated CTCs was 97%. In addition, this high purity was maintained even at various sample flow rates of 2−5 mL/h (Figure 4b). Considering the high purity (97%) of the separated CTCs, the CTC microseparator provides a great opportunity for CTC-based molecular analysis. To verify the advantage of the CTC microseparator for molecular analysis, RT-PCR amplifications were performed to detect keratin 19 transcript using blood samples spiked with SKBR-3 cell lines and cell lines isolated from blood samples, as shown in Figure 5. The results showed that RT-PCR amplifications using the isolated cell lines are more sensitive than those using the blood samples. For the experiment, 200 μL of peripheral blood was spiked with 104−10 cancer cells; the blood also contained about 106 WBCs and 109 RBCs. Therefore, the CTCs constituted only 0.001−0.000001% of the cells in the blood sample and 1−0.001% of the nucleated cells. For this reason, significant RNA loss would likely occur during the nucleated cell lysis and RNA extraction steps. In addition, the RT-PCR performance is reduced by the presence of untargeted mRNA. Consequently, the proposed CTC microseparator can be used to separate and detect extremely rare cells from peripheral blood. Furthermore, it can be expected that the CTC microseparator allows for more sensitive genetic assays using separated rare cells. The high-gradient magnetic separation (HGMS) method used in this study produces an optimized high-gradient magnetic flux

Figure 3a shows the recovery rate of the CTC microseparator as measured by counting CTCs flowing into outlet no. 1 at various sample flow rates of 2−5 mL/h. The CTC microseparator isolated approximately 90% of CTCs spiked into blood samples with a flow rate of up to 5 mL/h, which is the maximum flow rate to count cells flowing into outlet no. 1 with the monitoring system used herein. The recovery rate was also measured for various numbers of CTCs spiked into blood samples from 14 to 12 000 per 200 μL of peripheral blood. The result (Figure 3b) shows that the recovery rate was consistent at 90% for various spiked numbers of CTCs. To determine the purity of CTCs separated by the CTC microseparator, 1000 CTCs probed with PE-conjugated anticytokeratin 19 (anti-CK19-PE) antibodies (red color; BD Biosciences) were spiked into 200 μL of human peripheral blood stained with FITC-conjugated anti-CD45 (anti-CD45FITC) antibodies (green; BD Biosciences). After separation of the spiked CTCs, all cells collected from output no. 1 were spread on a glass slide and monitored with a fluorescence D

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strong magnetic force. In the current study, the maximum x-directional magnetic force, Fmx, was approximately 2.1 nN (Supporting Information Figure S4a) based on the assumption that 100 000 magnetic nanobeads are bound on a single CTC32 levitated at a 10 μm height from the bottom. This is a larger magnitude than the Stokes drag (0.47−1.2 nN) on a CTC at a flow rate of 2−5 mL/h. Furthermore, because of the ferromagnetic wires inlaid in the bottom substrate of the microchannel, the lateral magnetic force is generated regularly over the whole area of the microchannel. Therefore, the CTC microseparator would potentially have a greater separation efficiency and throughput than previously reported magnetophoretic microseparators,29,33 which used a single ferromagnetic wire only. The majority of CTCs are separated laterally by the first and second ferromagnetic wires. However, a few CTCs would not be strongly influenced by the magnetic forces of the forward wires due to their initial levitation height from the bottom, as shown in Supporting Information Figure S4a. In addition to the x-directional magnetic force, Fmx (eq 1), there is a z-directional (vertical) magnetic force, Fmz (Supporting Information eq S11) that pulls the CTCs toward the bottom of the microchannel. According to the theoretical analysis (Supporting Information Figure S4b), the maximum vertical magnetic force on a CTC, bound to 100 000 magnetic nanobeads (50 nm in diameter) and levitated 10 μm from the bottom, is approximately 3 nN toward the bottom, as generated by a vertical magnetic flux gradient of approximately 15 kT/m. Therefore, as the CTCs approach the ferromagnetic wire, both the lateral and vertical magnetic forces increase simultaneously, followed by an increase in the lateral displacement. For this reason, although some CTCs pass over the forward ferromagnetic wires due to their levitation height, they are subsequently separated by the rearward ferromagnetic wires. As the flow rate increases, more CTCs pass over the forward wires and the separation performance decreases. Nevertheless, the results showed that the separation performance of the proposed CTC microseparator is maintained at a flow rate of 5 mL/h. Because of the strong induced magnetic force, some CTCs with bound magnetic nanobeads could be stacked at the corners between the ferromagnetic wires and the sidewall of the microchannel, thereby getting lost in the microchannel, which is a serious cause of reduced recovery rates. To avoid this problem and obtain a high recovery rate, the ferromagnetic wire was bent at almost a right angle 100 μm before reaching the sidewall of the microchannel. As a result, the portion of the ferromagnetic wire behind the bending point is placed in parallel with the external magnetic field. When the ferromagnetic wire and the external magnetic field are parallel to each other, a magnetic gradient does not develop on the wire; therefore, CTCs do not stack on the wire as shown in Figure 2, parts b and c. The Reynolds number was calculated to be approximately 5 for the highest flow rate (5 mL/h) of the present CTC microseparator. Thus, even with this high flow rate, the fluidic flow of the CTC microseparator is laminar.34 Laminar flow is crucial for the high purity of the CTC microseparator because it allows for a multistream fluid in which the CTCs are selectively transported across the sample and buffer stream boundary into outlet no. 1. In addition, the continuous separation by lateral magnetophoresis allows for the purity of the CTC microseparator to be 97%, which is several hundred times higher than that of other approaches.6,14 The high recovery rate (90%) and purity (>97%) of the CTC microseparator makes it an ideal

Figure 4. (a) Cells collected into outlet no. 1 were immunofluorescently labeled with anticytokeratin19-PE (red color) for SKBR-3 cells, anti-CD45-FITC (green) for normal blood cells, and Hoechst 34580 (blue) for DNA content. (b) Separation purity of the CTC microseparator at various sample flow rates. Approximately 103 of SKBR-3 cell lines were spiked into the blood sample and isolated through the CTC microseparator at varying flow rates of 2−5 mL/h. The error bars represent one standard deviation calculated from three data sets.

Figure 5. RT-PCR amplifications (211 bp) of keratin 19 transcript using blood samples spiked with 104, 103, 102, and 10 SKBR-3 cell lines (written “Blood”) and cell lines isolated from each blood sample (written “CTCs”).

(