High throughput capture of circulating tumor cells using an integrated microfluidic system

Biosensors and Bioelectronics 47 (2013) 113–119

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High throughput capture of circulating tumor cells using an integrated microfluidic system Zongbin Liu a, Wang Zhang a,b, Fei Huang a,c, Hongtao Feng a, Weiliang Shu a, Xiaoping Xu d,n, Yan Chen a,nn a

Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, PR China Medical Department of Nanchang University Graduate School, Nanchang 330006, PR China c College of Chemical Engineering, Sichuan University, Chengdu 610065, PR China d Hong Kong University Shenzhen Hospital, Shenzhen 518053, PR China b

art ic l e i nf o

a b s t r a c t

Article history: Received 7 December 2012 Received in revised form 4 March 2013 Accepted 8 March 2013 Available online 21 March 2013

In this work, we introduced an integrated microfluidic system for fast and efficient circulating tumor cell (CTC) isolation and capture. In this microfluidic platform, a combination of microfluidic deterministic lateral displacement array and affinity-based cell capture architecture, allows for the high efficiency cancer cell enrichment and continuous high throughput and purity cancer cell capture. Using this device to isolate breast cancer cells from spiked blood samples, we achieved an enrichment factor of 1500
, and a high processing throughput of 9.6 mL/min with 90% capture yield and more than 50% capture purity at cell concentration 102 cells/mL. This integrated platform offers a promising approach for CTC capture with high recovery rates, purity and stability, and exhibits potential capability in cancer cell culture and drug screening. & 2013 Elsevier B.V. All rights reserved.

Keywords: Microfluidics Cancer cells Deterministic lateral displacement Antibody

1. Introduction Cancer is a leading cause of death worldwide, and more than 90% cancer deaths are the results of metastasis. Cancer metastasis is a set of events that include cells escaping from primary cancer, spreading through circulation system, and seeding of tumors at secondary sites (den Toonder, 2011; Kaiser, 2010). The lack of early warning on metastasis limits the clinical treatment efficacy. Tumor cells that are identified in circulation system are referred to as circulating tumor cells (CTCs). Recent studies show that CTCs levels in the peripheral blood is a good measure for progress and overall survival (Cristofanilli et al., 2004; de Bono et al., 2008). The CTCs quantity was correlated in several tumor types with survival of patients with metastases. Besides the predictive power, CTCs can also be regarded as a liquid biopsy to non-invasively perform the molecular characterization of CTCs, and determine the personalized medicine (Camara et al., 2007; den Toonder, 2011; Maheswaran et al., 2008; Pantel et al., 2008). As a result, CTCs detection provides wealthy information, making them valuable in both clinical and basic research.

n

Corresponding author. Tel.: þ86 755 8691 3191; fax: þ 86 755 8691 3191. Corresponding author. Tel.: þ 86 755 8639 2288; fax: þ 86 755 8639 2299. E-mail addresses: [email protected] (X. Xu), [email protected] (Y. Chen).

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0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.03.017

The greatest challenge in the detection of CTCs is their rareness at the level of 1–100 CTCs in over 1
109 blood cells (den Toonder, 2011; Pantel et al., 2008). A variety of technologies has been developed to detect CTCs from blood, such as affinity-based (Choi et al., 2012; Gleghorn et al., 2010; Kang et al., 2012; Launiere et al., 2012; Ross and Stodkowska, 2009; Stott et al., 2010; Wang et al., 2011) and size-based methods (Beech et al., 2012; Bhagat et al., 2011; Doh et al., 2012; Hur et al., 2011; Kyung-A Hyun et al., 2012; Liu et al., 2013; Tan et al., 2010; Zheng et al., 2011). Affinitybased capture is by far the most commonly used technology to isolate CTCs from blood stream. Affinity-based technique employs antibodies that are expressed only on cancer cells, but not blood cells. Up to now, the only affinity-based technique approved by FDA (USA) is CellSearch (Cristofanilli et al., 2004; Riethdorf et al., 2007), which is also the gold standard in current CTCs detection. The CellSearch platform uses magnetic particles functionalized with anti-epithelial cell adhesion molecule (EpCAM) for positive capture. Recently, microfluidic chip-based affinity techniques have been developed (Autebert et al., 2012; Chen et al., 2012; Choi et al., 2012; Gleghorn et al., 2010; Kang et al., 2012; Lien et al., 2010; Nagrath et al., 2007; Stott et al., 2010; Wang et al., 2011; Yu et al., 2011). The advantages of these microfluidic chip system include high throughput, disposability, low cost and portability which offers new opportunity for CTCs detection. Nagrath et al. developed a microfluidic device with anti-EpCAM modified microposts (Nagrath et al., 2007). Captured CTCs on microposts were visualized by staining.

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The CTCs-chip achieved more than 60% capture yields and more than 40% capture purity for several cancer types. The same research group further developed a chip with herringbone structure to improve CTCs isolation efficiency (Stott et al., 2010). The herringbone structure can generate microvortex mixing to improve cell capture efficiency by enhancing cells contact with antibody-coated device. This herringbone structure was also adopted by Wang et al. for CTCs capture on nano-structured silicon substrate (Wang et al., 2011). The above research demonstrates that microfluidic-based affinity techniques have great capability to efficiently and selectively isolate CTCs from blood. However, these microfluidic devices must be operated with low flow velocity (∼mL/h) to ensure maximum cell-substrate attachment for better capture efficiency. Based on the flow velocity in the research of Nagrath et al., (2007), it would take 5 h to process 10 mL volume of blood. This low throughput cannot satisfy clinical requirements. Therefore, it is critical to develop a high throughput platform to rapidly isolate and capture CTCs from large volume of peripheral blood sample. In a previous study, we reported a microfluidic device with deterministic lateral displacement (DLD) arrays allowing continuous cancer cell enrichment from peripheral blood (Liu et al., 2013). Our system achieved 2 mL/min throughput with high isolation efficiency, and cancer cells had ∼40
enrichment over blood cells. However, the cancer cell purity was still very low after enrichment. In this paper, we combined the microfluidic DLD array and affinity-based technique to develop a microfluidic device for continuous high throughput cancer cell capture with high yield and purity. Cancer cells were rapidly enriched in the DLD array and then efficiently captured on the antibody coated substrate surface. Our research shows the first use of integrating size-based and affinity-based techniques for CTCs capture in a single chip. It is also first time to achieve CTCs capture in several minutes with 10– 100 mL blood sample using microfluidic affinity-based method. In addition, our device requires only simple dilution of the blood sample and cancer cell capture can be achieved in a continuous single step. All together, we constructed an attractive microfluidic platform for cancer detection.

2. Materials and methods 2.1. Device design and fabrication The microfluidic device was fabricated with one inlet, twelve outlets, nine cancer cell enrichment chambers and two cancer cell capture chambers (Fig. 1(a)) using standard photolithography and soft lithography. With a photomask, Su8-3025 (Microchem Corp., Naton, MA) pattern was fabricated on silicon substrate. The patterned silicon substrate was then poured with polydimethylsiloxane (PDMS) prepolymer solution (Sylgard 184 silicone elastomer kit, Dow Corning; A and B in 10 to 1 ratio) and curried at 80 1C for 1 h. The PDMS mold was next punched for inlet and outlet holes, and bonded to glass slide after oxygen plasma treatment (PDC-M, Chengdu Mingheng Science. & Technology Co., LTD, PR China). The surface of glass substrate was modified with 3-mercaptopropyl trimethoxysilane (4% v/v in ethanol at room temperature for 30 min), and then treated with N-maleimidobutyryloxy succinimide ester (GMBS, 0.1 mM in dimethyul sulfoxide (DMSO)) for 30 min. Next, the substrate surface was incorporated with streptavidin (10 μg/ml in phosphate buffer solution for 1 h). Finally, the modified substrate was grafted with anti-EpCAM (10 μg/mL in phosphate buffered solution (PBS) with 1.0% (w/v) bovine serum albumin (BSA) and 0.1% (w/v) sodium azide for 30 min) before use. The whole surface modification process is illustrated in Fig. 2. The microfluidic platform can be divided into two regions: (i) the cell enrichment region and (ii) the cell capture region. In the

cell enrichment region, there were eight parallel chambers connected to one single chamber (Fig. 1(b)). Each chamber was 42 mm long, 3.5 mm wide and 30 μm high with 85 μm outlet channel. The cell enrichment chamber was designed with deterministic lateral displacement (DLD) structure featuring mirrored triangular micropost array. The microarray consisted of microposts with 25 μm radius and 25 μm gaps, and had a tilted angle 3.2 deg to the fluid flow direction. Microfluidic DLD structure has been used for sizebased cell separation (Holm et al., 2011; Huang et al., 2004; Inglis, 2009; Inglis et al., 2006; Loutherback et al., 2010; Morton et al., 2008a, 2008b). In our previous research, DLD structure was used for breast cancer cell isolation with 99% capture yield, 40
enrichment factor and 2 mL/min throughput (Liu et al., 2013). In the present study, the cancer cells were first enriched in the eight parallel chambers and then further enriched in a single chamber. After the two enrichments, cancer cells were flown through the antibody-coated chambers (45 mm long and 3.9 mm wide) and were captured on the substrate surface. Fishbone structure was designed in the overload PDMS mold for promoting cell-substrate contact frequency (Fig. 1(b)). It has been reported that fishbone structure can greatly improve cell capture efficiency (Stott et al., 2010; Wang et al., 2011). 2.2. Cell culture and preparation Human breast cancer cell lines MCF7 (HTB-22™, human breast cancer cell line, ATCC, USA) were used to characterize the efficacy of the microfluidic device. Cancer cells culture was in low-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. Cell culture was maintained at 37 1C with 5% (v/v) CO2 and medium was changed every 2–3 days. Cells were harvested through incubation in 0.05% Trysin-0.53 mM EDTA at 37 1C for 5 min. The cell suspension was then diluted to the desired concentration. 2.3. Microfluidic cancer cells capture Cancer cells were spiked into phosphate buffered saline (PBS) at the concentration about 500 cells/mL. A syringe pump was connected to the inlet of device to control the fluid velocity. Outlets A, B1 and B2 were connected to another pump to withdraw fluid (Fig. 1(b)). As the width of the enrichment chamber (3.5 mm) was about 40 times that of outlet channel (85 μm), the velocity of inlet, both outlets B1 and B2, outlet A can be set in the ratio of 1600 to 40 to 1. If the inlet velocity is 9.6 mL/min, the velocity of both outlets B1 and B2 will be 0.24 mL/min and the velocity of outlet A will be 6 μL/min. After cancer cells were captured on the microchannel surface, the device was rinsed with PBS solution with the same velocity and the waste solution was collected. The captured cells were counted under a microscope. By measuring the cell concentration and volume of the waste solution from all the outlets, we can calculate the capture yield with the following equation: Capture yield ¼

NC NC þ C W V W

where Nc is the amount of cancer cells captured on the channel surface, Cw and Vw are the cancer cell concentration and volume of the waste solution. 2.4. Cancer cell isolation and capture from blood specimens Blood samples were provided by our collaborators in Hong Kong University Shenzhen Hospital, PR China. All blood specimens were collected into ethylene diamine tetraacetic (EDTA)-containing tubes and used in 12 h. The blood samples were diluted 10

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Fig. 1. (a) Overall process for cancer cell isolation and capture. 75 mL diluted blood sample was injected into the device at 9.6 mL/min. It would take about 8 min to complete the processing. The photo shows the device design, consisting of one inlet, twelve outlets, nine enrichment chambers and two capture chambers. (b) Schematic illustration of cancer cell enrichment and capture principle. The microfluidic deterministic lateral displacement (DLD) chamber was composed of mirrored triangular micropost array. In blood samples, large cells (cancer cells and part of leukocytes) were concentrated in the center of chamber, while small cells (erythrocytes and most of leukocytes) follow the streamline direction. The capture chamber consisted of anti-EpCAM modified substrate and an overlaid PDMS layer with fishbone structure capable of promoting cancer cell capture.

Fig. 2. Schematic illustration of surface modification and cancer cell capture.

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times with physiological saline to reduce viscosity before use. Cancer cells were pre-stained by Vybrant® DyeCycle™ Green (Life Technologies, Carlsbad, CA) and then spiked into the diluted blood specimens with the concentrations from 10 to 102 cells/mL. After cancer cell capture, the capture yield was calculated using the same methods described in the previous part. By counting cancer cells and blood cells captured on the channel surface, the capture purity was then calculated using the formula: Capture purity ¼

NC NC þN b

where Nc is the amount of cancer cells captured on the channel surface, Nb is the amount of blood cells adsorbed on the surface. 3. Results and discussion 3.1. Cancer cell enrichment One of greatest challenges in CTCs detection is their extreme rarity in the blood. Very few CTCs (1 in 109 blood cells) are present in patients with metastatic disease. Owing to CTCs' rarity, enrichment is essential before detection. A lot of techniques, such as microfiltration and inertial microfluidics (Bhagat et al., 2011; Kyung-A Hyun et al., 2012; Zheng et al., 2011), have been employed. In our study, deterministic lateral displacement (DLD) structure (Fig. 1(b)) was used for CTCs enrichment. DLD structure was designed with mirrored impost array, which formed a tilt angle 3.2 deg to the fluid direction. The mechanism of cell separation in DLD array is based on the interaction of cells with microposts under low Reynolds number conditions. As shown in Fig. 3(b), microposts can divide the fluid flow into several narrow streams. When cells pass through the gaps of microposts, small cells with radius less than the width of first stream L are able to continue following this stream. On the contrary, large cells with radius larger than the width of first stream L are forced to follow the second stream with a deterministic path. According to this analysis, 2 L can be regarded as the critical cell size. Based on the theory proposed by Loutherback et al. (2010), the critical separation size of our DLD structure was about 5–6 μm. In blood samples,

large cells (cancer cells and part of white blood cells) moved through the tilt direction and finally concentrated in the center of chamber, while small cells (red blood cells and most white blood cells) moved through the fluid direction and flowed away from the chamber (Fig. 1(b)). In order to characterize the enrichment efficiency, microfluidic device without surface modification was used. Cancer cells MCF-7 were spiked into PBS solution and injected into the device. As there was no antibody modified on the channel surface, cancer cells were not captured on substrate and flowed out through outlet A. By measuring the cell concentration in the collected solution from outlet A, the cancer cell enrichment factor can be calculated with the formula: Enrichment factor¼CO/CI, where CO is cancer cell concentration of collected solution from outlet A, and CI is the initial cancer cell concentration in the inlet. The cell enrichment results are shown in Fig. 3. Fig. 3(a) is a video frame showing that all MCF-7 cancer cells were concentrated to the center of chamber and collected into a narrow outlet channel. Fig. 3(b) shows the calculated enrichment factor at different flow rates of 1.6, 4.8, 9.6 and 16 mL/min. The enrichment factor was about 1500 and had little change with the increase of flow rate. The results indicated that cancer cells were significantly enriched in DLD chambers. Cancer cell enrichment resulted in the increase of cancer cell concentration and the decrease of flow velocity in the antibody-modified chamber. The increase of cell concentration can improve the detection limit, while the decrease of flow velocity can lead to enhanced throughput of device. 3.2. Cancer cell capture efficiency On the surface of cancer cells, there exist a lot of antigens which have specific interactions with anti-EpCAM antibodies. After enrichment in the DLD chambers, concentrated cancer cells were flown into antibody modified chambers. When cancer cells contacted the antibodies, they bonded to the substrate surface through strong antigen–antibody interaction. However, the flow in the chamber was in a laminar pattern, it is difficult for cells to contact the chamber surface. In order to increase capture efficiency, fish-bone

Fig. 3. (a) Image of MCF-7 cancer cells concentrated in the center of enrichment chamber with DLD structure at flow rate of 9.6 mL/min, and enrichment factor of MCF-7 at flow rates of 1.6, 4.8, 9.6 and 16 mL/min. (b) Schematic illustration of cell separation in DLD micropost array.

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structure was designed and fabricated in the cover layer by twostep photolithography process. Previous research by Nagrath et al. (2007) and Wang et al. (2011) has shown that fish-bone structure can give rise to chaotic mixing of solution, and increase cellsubstrate contact frequency to improve cell capture efficiency. In our study, the cell capture performance of this device was characterized by introducing MCF-7 cancer cell spiked PBS solution. The cell capture yields were then analyzed and the results were summarized in Fig. 4. Fig. 4(a, b) shows the effect of EpCAM antibody on cancer cell capture efficiency. Without antibody modification, there was no cell capture in the chamber (Fig. 4 (a)). On the contrary, substrate modified with antibody can easily capture cancer cells (Fig. 4(b)). The results indicated that antiEpCAM is an effective antibody for cancer cell capture. Fig. 4 (c) shows the relationship between capture yields and different flow rates. At flow rates of 1.6, 4.8 and 9.6 mL/min, high capture yield 90% was achieved. If the flow rate increased to 16 mL/min, the capture yield decreased to about 80%. Moreover, the spatial distribution of captured cells for 4.8, 9.6 and 16 mL/min was further analyzed. At flow rate of 4.8 mL/min, most cells were captured on the first 0–3 cm substrate surface. With the increase of flow rate to 9.6 and 16 mL/min, most cells were captured on the middle substrate surface. High flow rate induced enhanced shear force, which reduced cell capture efficiency. From the results, an optimal flow rate of 9.6 mL/min was determined. At flow rate of

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9.6 mL/min, high capture yield with relative high throughput can be achieved. Affinity-based microfluidic techniques have shown great potential to effectively isolate cancer cells with high yields. However, these devices must be operated with low throughput (∼mL/h) for better capture efficiency. As for our microfluidic platform, it not only realized the high capture efficiency, but also achieved high throughput processing. Our device showed the possibility of satisfying both high capture efficiency and throughput, which presented significant advance in clinical applications compared to other affinity-based devices. 3.3. Cancer cell capture from Blood specimens The high-throughput microfluidic platform was applied for cancer cell capture from blood specimens. To mimic the presence of cancer cells in peripheral blood, MCF-7 cells were pre-stained by Vybrant® DyeCycle™ Green and spiked into 10
diluted blood samples at the concentration of 102 cells/mL. The capture yield was then calculated (Fig. 5). Fig. 5(a) shows the brightfield, fluorescent and merged images of cancer cells and blood cells. As cancer cells were pre-stained, they were able to be discriminated from other blood cells by fluorescence microscope. Fig. 5 (b) shows cancer cell capture yields at different flow velocities. The capture yield was about 90% at flow rates from 1.6 to 9.6 mL/ min, and decreased to about 70% at flow rate of 16 mL/min. The

Fig. 4. MCF-7 cancer cell capture from spiked PBS solution. (a, b) Image of cancer cell capture on substrate surface without or with antibody modification at flow rate of 9.6 mL/min. (c) Cancer cell capture yields at flow rates of 1.6, 4.8, 9.6 and 16 mL/min. (d, f) Spatial distribution of captured cancer cells along the substrate at different flow rates.

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Fig. 5. (a) Fluorescent and brightfield images of cancer cells and bloods. Cancer cells were pre-stained by Vybrant® DyeCycle™ Green. (b) Cancer cell capture yields at flow rates of 1.6, 4.8, 9.6 and 16 mL/min in spiked blood samples. (c, d) Capture efficiency and purity of cancer cells at different spiked cell numbers ranging from 10 to 100 cells/mL.

results of capture yields in blood samples were consistent with that in PBS solutions. MCF-7 cells were also spiked into blood samples with cell densities from 10 to 100 cells/mL. The results of capture recovery were shown in Fig. 5(c). More than 90% recovery was achieved and maintained with the increase of spiked cell numbers. Capture purity is a critical parameter in CTCs detection. The presence of blood cells can contaminate DNA sequencing, therefore, CTC separation technologies must offer higher performance in capture purity. In our previous research, microfluidic device with DLD structure achieved efficient isolation of cancer cells with high throughput and capture efficiency. However, the cell purity (less than 1%) was very low with a fraction of blood cells (Liu et al., 2013). In this study, the purpose of antibody modification was to capture cancer cells with high purity. To determine the capture purity, the optimized flow velocity 9.6 mL/min was used. The results of capture purity were summarized in Fig. 5(d). The capture purity increased with the increase of spiked cell concentration. The average capture purity ranging from 17% to 52% is much higher than the results in our previous research. The main advantage of the developed device is capable to process large volume of blood with high capture yields and purity in several minutes. In clinical application, it is important to high-throughput process milliliters of blood samples within a short period of time. While most published affinity-based techniques work with low throughput (∼mL/h), our platform is capable of achieving comparable high throughput. For the experiments in this work, there were many outlets connected to several pumps with different flowing velocities, which made the manipulation of device challenging. Further optimization of the design would be necessary if such a device were to become useful for practical point-of-care applications in the future.

with high yield and purity. Cancer cells were first enriched by the DLD structure and then captured on the anti-EpCAM modified substrate surface. The DLD structure can efficiently concentrate MCF-7 cancer cells with 1500
enrichment factor. The device was used for MCF-7 isolation from spiked blood samples, achieving 9.6 mL/min throughput with 90% capture yield and more than 50% capture purity at cell concentration 102 cells/mL. The microfluidic platform was capable of performing cancer cells capture in a continuous single step with the combined advantages of high throughput, capture yield and capture purity. The ultimate goal of such a microfluidic device is to isolate circulating tumor cells from large sample volumes of peripheral blood, and collect tumor cells for further cell culture, drug screening and molecular analysis. Therefore, the microfluidic device provides an attractive platform for clinical cancer diagnosis, its high performance to recover cancer cells with high throughput and specificity would greatly facilitate disease intervention and prognosis monitoring of known metastasis.

Acknowledgment This work is supported by the National Natural Science Foundation of China (Grant 61106128), the Knowledge Innovation Project of the Chinese Academy of Science (Grant KGCX2-YW904), the Guangdong Innovation Research Team Fund for Low-cost Healthcare Technologies (GIRTF-LCHT), the ShenZhen Development and Reform Commission Project (Grant 2012-385) and the ShenZhen Science and Technology Plan Project (Grant 201101004).

References 4. Conclusions In summary, we described a microfluidic chip with the integration of deterministic lateral displacement (DLD) and affinity-based techniques for continuous high throughput cancer cell capture

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