Integrative Cancer Biology & ResearchResearch Article Open Access

Integrated Microfluidic Chip for Rapid Capture and Drug Screening of Cancer Cells from Peripheral Blood

Zongbin Liu1,2†, Weiliang Shu1†, Rui Chen3, Hongtao Feng1, Zongqian Hu4, Yuanxiang Wang5*, Geng Tian6*, Aixue Liu6* and Yan Chen1*1Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, 518000, China

2Zigzag Biotechnology Co. LTD., Shenzhen, 518000, China

3The Third Affiliated Hospital, Guangzhou Medical University, Guangzhou, 510150, China

4Beijing Institute of Radiation Medicine, Beijing, 100850, China

5Department of Cardiothoracic Surgery, Shenzhen Children’s Hospital, Shenzhen, 518000, China

6Department of Oncology, Shenzhen Second People’s Hospital, Shenzhen, 518000, China

†These authors contributed equally to this work

*Corresponding authors: Yan Chen, Email: yan.chen@siat.ac.cn; Aixue Liu, Email: liuaixue_76@sina.com; Geng Tian, Email: tiangeng666@aliyun.com; Yuanxiang Wang, Email: yuanxiangw@126.com

Received: 03 January 2018; Accepted: 23 January 2018; Published: 05 February 2018

Abstract Objective: Circulating tumor cells (CTCs) play a key role in

metastasis. The detection and characterization of CTCs have important implications for diagnosis and prognosis. However, it is challenging to capture the rare CTCs and do the downstream analysis. The objective of this paper is to develop an integrated microfluidic chip to isolate, capture and culture cancer cells from human whole blood for anti-cancer drug screening.

Methods: A microfluidic system with the combination of deterministic lateral displacement array and affinity based capture is used for the isolation and capture of cancer cells. Deterministic lateral displacement is used to isolate cancer cells from whole blood samples utilizing the size differences between cancer cells and blood cells. The isolated cancer cells are further captured by EpCAM modified surface.

Results: The whole process, including cancer cell capture, culture and drug screening, is performed within an integrated microfluidic chip with simple operation. A capture yield of 90% was achieved at an optimized flow rate of 12 mL/h. The captured cancer cells were capable of proliferation. After cultivation, cancer cells were analyzed for anti-cancer drug screening with the anti-cancer drugs, MMC and TAM. Results indicated that MMC had a greater cytotoxic effect on cancer cells than TAM.

Conclusions: We have demonstrated the feasibility of CTC capture, cultivation and anti-cancer drug screening complied sequentially into a single device. The system has the combined advantages of fast CTC isolation and efficient cancer cell capture, and maintained cell viability for downstream analysis. The microfluidic device with DLD structure and antibody modified architecture may provide an attractive platform for the use of personalized medicine for cancer patients.

Keywords: Circulating tumor cells; Microfluidic chip; Deterministic lateral displacement; Drug screening

Copyright © 2018 The Authors. Published by Scientific Open Access Journals LLC.

IntroductionCirculating tumor cells (CTCs), which disseminate from primary

tumors and circulate in the bloodstream or lymphatic vessels, play a key role in cancer metastasis. CTCs are essentially a liquid biopsy which can be used to non-invasively monitor cancer metastasis [1,2]. The detection and characterization of CTCs have important implications for diagnosis and prognosis of disease, assessment of personalized medicine, and evaluation of treatment efficacy [1,3]. However, there is tremendous challenge of isolating CTCs from blood due to the extremely rareness of CTCs in the bloodstream (1 per 109 blood cells). The processing of a relatively large total volume of blood (typically 7.5 mL or more) is usually necessary. Another big challenge is to harvest CTCs in a suitable form for subsequent molecular analysis and drug screening for therapy guidance.

Recently, microfluidic technologies have been used to isolate and detect CTCs based on magnetic susceptibility [4-6], cell adhesion [7], cell size and deformability [8-12], dielectrophoresis [13,14] and impedance [15,16]. However, the present research wok is primarily focused on the capture and enumeration of CTCs. It is challenging to characterize the behavior and function of captured cancer cells. The characterization of CTCs, rather than only enumeration, plays a crucial role in providing the personalized medicine analysis and evaluating treatment effects. For example, if the captured CTCs could be cultured in vitro, anti-cancer drugs could be screened for each individual patient. Kang et al. reported a combined microfluidic micromagnetic device which allowed efficient isolation, detection and culture of CTCs from blood and showed that captured CTCs could grow as a single colony in vitro culture [17]. Biohsel et al. developed a microfluidic chip to culture captured CTCs as clonogenic three-dimensional spheroids for functional assays.18 The challenge is to build a system that makes it possible to perform both the rapid isolation and the drug effect characterization of CTCs. Moreover, CTCs are very vulnerable cells. It’s difficult to maintain viability after CTCs isolation. In order to reduce the damage of cells caused by complex fluid handling, the design of the system should enable cell culture directly on a single device.

In a previous study, we introduced a microfluidic system that utilized a deterministic lateral displacement (DLD) array for fast and efficient isolation of CTCs based on differences of cell size [10]. The microfluidic system achieved high capture yield. More importantly, the device had the capability of processing a large volume of blood in short time, which indicated that it could potentially meet the clinical requirement. In the current study, we extend the investigation of this microfluidic system to integrate affinity-based cell capture architecture for cancer cell capture. The integrated microfluidic platform consists of two functional units: a cell-enrichment chamber and a cell-capture chamber. The cell-enrichment chamber is designed with DLD structure, allowing rapid CTC separation from blood samples at high throughput. The following cell-capture chamber consists of a fishbone structure to maximize cancer cell capture efficiency on the surface. The captured cancer cells are further cultured in vitro for anti-cancer drug screening. The capability to capture and culture cancer cells, coupled with the capability to perform drug sensitivity screening, offers a potentially promising approach for personalized medicine for cancer patients.

Materials and MethodsDevice design and fabricationThe microfluidic device was fabricated using standard

photolithography and soft lithography. Su8-3025 (Microchem Corp., Naton, MA) pattern was fabricated on silicon substrate using

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Citation: Liu Z, Shu W, Chen R, et al. Integrated Microfluidic Chip for Rapid Capture and Drug Screening of Cancer Cells from Peripheral Blood. Integr Canc Biol Res 2018; 1:009.

collected. Cancer cells captured on the surface were observed and counted using microscope. The capture yield was calculated using the following formula, 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.

Capture yield =

Nc

Nc+CwVwCapture yield =

Nc

Nc+CwVw

Cancer cells culture and drug treatmentAfter cancer cells were captured on the substrate surface, pipette

tips with culture medium were inserted into the inlet and outlet holes for long-term cell culture. Cell culture was maintained at 37℃ with 5% (v/v) CO2. When the cells reached 40%-50% density, cell culture medium was replaced with medium supplemented with mitomycin C (MMC) or tamoxifen (TAM) at concentrations of 5, 50, 100 and 150 μM. After drug exposure for 24 hr, immunostaining was performed to analyse the extent of cell death and apoptosis.

Analysis of cell viabilityAfter cancer cells were treated with the drugs, as described in

previous section, they were washed with PBS solution, and then stained with 1 μg/mL Propidium Iodide (PI, Sigma-Aldrich Co., USA) and 5 μg/mL Hoechst 33258 (Sigma-Aldrich Co., USA). PI is membrane impermeant and commonly used to stain DNA of apoptotic cells. Hoechst 33258 can be used to stain DNA of both live and apoptotic cells. A Nikon Eclipse 80i fluorescence microscope (Nikon, Japan) was used to record fluorescence images of stained cells.

Results and DiscussionCharacterization of cancer cell capture efficiencyThe microfluidic chip in this study consisted of two functional

units: (i) the cell-enrichment chamber and (ii) the cell-capture chamber (Figure 1a). The cell-enrichment chamber (45 mm long, 4 mm wide and 30 µm high) was designed with deterministic lateral displacement (DLD) microarray, which consisted of microposts with 25 µm radius,

a photomask. PDMS prepolymer solution (Sylgard 184 silicone elastomer kit, Dow Corning; A and B in 10:1 ratio) was poured onto the silicon substrate and curried at 80℃ for 1 hr. The PDMS mold was peeled off from the silicon substrate and punched for holes of inlets and outlets. The PDMS mold was then bonded with glass slide after oxygen plasma treatment.

Surface functionalizationThe substrate of microfluidic chamber 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 DMSO) for 30 min. Next, the substrate surface was incorporated with streptavidin (10 μg/ml in phosphate buffer solution for 1 hour). Finally, the modified substrate was grafted with anti-EpCAM (10 μg/mL in PBS with 1.0 % (w/v) BSA and 0.1 % (w/v) sodium azide for 30 min). In the above steps, appropriate solvents or buffer solutions were used to wash away the excess chemicals.

Cell cultureHuman breast cancer cell line MCF7 (HTB-22TM) was purchased

from ATCC. MCF7 cancer cells were cultured 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℃ with 5% (v/v) CO2 and medium was changed every 1-2 days. Cancer cells were harvested by incubation in 0.05% Trysin-0.53 mM EDTA at 37℃ for 5 min.

Quantifying cancer cell capture efficiencyBlood specimens were provided by Shenzhen Second People’s

Hospital and Shenzhen Children’s Hospital, P. R. China. EDTA-containing tubes were used to collect blood specimens. MCF7 cells pre-stained by Vybrant® DyeCycle™ Green (Life Technologies, Carlsbad, CA) were spiked into blood samples at concentrations from 102 to 103/mL. A syringe pump was used to perfuse blood samples into the device. After capture of cancer cells on the substrate surface, the device was rinsed with PBS solution and the waste solution was

Figure 1: Schematic illustration of the microfluidic chip and the work principle. (a) Overview of the chip. The microfluidic chip consists of two modules: the module for cancer cell enrichment, and the module for capture and culture of cancer cells. (b) Enrichment of cancer cells by the DLD microarray in the first module. Large cancer cells are concentrated to the center, while red blood cells and most white blood cells follow the direction of fluid flow. (c) Capture of cancer cells by anti-EpCAM antibody in the second module. Fish-bone structure is designed in the cover PDMS layer to improve capture efficiency.

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Citation: Liu Z, Shu W, Chen R, et al. Integrated Microfluidic Chip for Rapid Capture and Drug Screening of Cancer Cells from Peripheral Blood. Integr Canc Biol Res 2018; 1:009.

Figure 4: Culture of cancer cells in the microfluidic chip. (a) Photo of cell culture in the microfluidic chip. Medium is perfused into chip by gravity flow. (b) Images of cancer cells cultured for 0, 24 and 48 hr. Scale bar, 50 µm. (c) Cancer cell proliferation as a function of cell culture time.

25 µm gaps and a tilted angle 3.2°C to the fluid flow direction (Figure 1b). Cells above the critical size followed a bump migration path and concentrated to the center of chamber. Cells that were smaller than the critical size followed the fluid direction and flowed out of chip. In this study, the critical size of the DLD design was 5-6 µm. In blood samples, cancer cells and some blood cells larger than 6 µm, can be concentrated and enriched by the DLD structure. Thus, the enrichment of cancer cell can not only result in an increase in cancer cell concentration but also actually improve the throughput of the device. After the enrichment in DLD chamber, cancer cells flowed into the antibody-modified chamber. The cell-capture chamber (37 mm long, 4.5 mm wide and 45µm high) was designed with fish bone structure (15 µm high) in the cover PDMS layer to improve cell capture efficiency (Figure 1c).

Breast cancer cells MCF-7, pre-stained by Vybrant® DyeCycle™ Green to allow their discrimination from blood cells, were spiked into the blood sample. Fig. 2a is a video frame of capturing cancer cells from a blood sample. The capture yields were analyzed and were summarized in Figure 2b. At a flow rate between 6 and 12 mL/hr, the chip achieved a high capture yield (>90%). If the flow rate was increased to 18 and 24 mL/hr, the capture yield decreased dramatically. A high flow rate led to an increase in shear stress and may reduce the capture yields. Cell viability was also assessed. At the flow rate of above 12 mL/hr, the ratio of dead cells increased significantly (Figure 3). A high flow rate may induce more damage of cell membrane. Considering both capture yield and cell viability, 12 mL/hr was regarded as the optimal flow rate. At this flow rate, the capture efficiency was around 95%. S. Nagrath et al. [19] and S. Wang et al. [20] have developed chips with anti-EpCAM modified microchannels and achived 1~2 mL/hr throughput. Compared with these platforms, our microfluidic system realized high throughput processing.

Culture of cancer cells in the microfluidic chipThe maximal amount of cancer cells cultured on chip was

determined by the surface area. In this study, it is estimated that the maximal amount of cancer cells was ~80,000. MCF-7 cancer cells were captured in the antibody-modified chamber and maintained at 37℃ with 5% (v/v) CO2. Figure 4a shows the operation of the microfluidic device. Figure 4b demonstrates the cancer cell growth at 0, 24 and 48

Figure 2: Effect of flow velocity on capture yield. (a) Video frame of capturing MCF-7 cells from blood sample at the flow velocity of 12 mL/hr. Scale bar, 50 µm. (b) Capture yields as a function of flow velocity.

Figure 3: Effect of flow velocity on cell viability. (a) Images of live/dead assay of cancer cells at different flow velocities. Red dots indicate dead cells. Scale bar, 100 µm. (b) Percentage of cell death as a function of flow velocity.

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Citation: Liu Z, Shu W, Chen R, et al. Integrated Microfluidic Chip for Rapid Capture and Drug Screening of Cancer Cells from Peripheral Blood. Integr Canc Biol Res 2018; 1:009.

hr. There was an obvious increase in cell numbers from 0 to 48 hr. Moreover, cancer cells can adhere and spread well on the substrate surface. The results of cell proliferation after cell culture for 0, 12, 24, 48, and 96 hr are further summarized in Figure 4c. After cell culture for 96 hr, there were about 2.5 fold more cells than baseline, which indicated the increase of cell proliferation. Due to the extreme rareness of CTCs in bloodstream, it’s better to expand cells in vitro before further analytical studies can be attempted. The proliferation results in Figure 4 demonstrated that the microfluidic chip may provide a suitable environment for the culture and expansion of cancer cells.

Drug induced apoptosis of cancer cellsWhen the labeled, co-cultured MCF-7 cells reached 40%-50%

density, the MMC and TAM drugs were introduced into the microfluidic chip. MMC and TAM are widely used anticancer drugs, and can induce the apoptosis of a variety of cancer cells, such as breast cancer cells and hepatocellular cells [21-24]. During the process of apoptosis, the plasma membrane becomes increasingly permeable and allows PI enter and bind to DNA. Another nucleic acid dye Hochest 33258 was used for

the identification of total live cells. Figure 5 shows the cytotoxic effect of the MMC and TAM drugs on MCF-7 cancer cells. In the fluorescent images in Figure 5a, PI staining positive (red) cells can be regarded as the apoptotic cells. Cells cultured in chip and in dish were compared. Without drugs, there were few apoptotic cells. Then with the increase of drug concentration from 0 to 150 µM, there was a significant increase in the quantity of apoptotic cells. Compared to cell culture in chip, there were more apoptotic cells in dish. The percentage of surviving cells was calculated and the results were shown in Figure 5b-e. The results show that both drugs led to increased cancer cell mortality. However, MMC had a greater cytotoxic effect than TAM. The half maximal inhibitory concentration (IC50) of MMC was smaller than that of TAM. The effects of MMC and TAM on cancer cells were consistent with that found by HX Song et al. [25] The microfluidic chip consisted with small-dimension michochannels and was filled with little volume of drug solution. In contrast, the dish was filled with large volume of drug solution. The more drug amount in dish may induce more apoptosis in cancer cells. Overall, the apoptosis results demonstrate the feasibility of anti-cancer drug screening using our microfluidic chip.

Figure 5: The cytotoxic effect of MMC and TAM drugs on MCF-7 cancer cells. (a) Apoptosis induced by MMC and TAM drugs. Cancer cells are cultured on chip and on petri-dish respectively. Red dots indicate dead cells. Scale bar, 100 µm. (b-e) Percentage of cell survival as a function of drug dose of MMC and TAM respectively. IC50 values are indicated in (d-e).

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Citation: Liu Z, Shu W, Chen R, et al. Integrated Microfluidic Chip for Rapid Capture and Drug Screening of Cancer Cells from Peripheral Blood. Integr Canc Biol Res 2018; 1:009.

Affinity-based microfluidic technique is a promising method for CTCs capture [20,26-28]. After cell capture, the release of viable cancer cells is important for subsequent cell cultivation and molecular analysis. However, due to the strong adhesion force between cells and antibodies, it is challenging to detach cells from microfluidic substrate surface. Many techniques, such as enzyme degradation and thermodynamic methods, have been employed for the release of captured cancer cells [29,30]. However, these techniques may have potential harmful effect on cell structure and viability. Using our microfluidic system, there was no need for the viable release of cancer cells from the antibodies used for their capture. The subsequent cell cultivation and anti-cancer drug screening can be completed sequentially in this chip. Though our previous design can realize high efficiency cell capture, the operation was complicated. The current design has only two modules for enrichment and capture, and enables more simple operation [11]. Therefore, the microfluidic system utilized in this study serves as a simple and promising platform for CTCs capture, cultivation, drug susceptibility, and molecular analysis.

ConclusionsIn this study, we developed a microfluidic chip that was composed of

a deterministic lateral displacement (DLD) array and an affinity-based cell capture architecture for efficient cancer cell capture, cultivation, and anti-cancer drug screening. Cancer cells were first enriched by DLD array, and then captured onto an antibody modified substrate surface. A capture yield of 90% was achieved at an optimized flow rate of 12 mL/h. The captured cancer cells were capable of proliferation in the microfluidic environment. After cultivation, cancer cells were further analyzed for anti-cancer drug screening with the anti-cancer drugs, MMC and TAM. The application of the DLD structure provides an opportunity for rapid CTC isolation with high recovery yield and high cell viability for cancer cell drug screening. Results indicated that MMC had a greater cytotoxic effect on MCF-7 cancer cells than TAM. In this proof-of-concept study, survival rates of cultured cancer cells in the platform can be calculated by immunostaining, providing a convenient method for selecting anti-cancer drugs. Taken together, we have demonstrated the feasibility of CTC capture, cultivation and anti-cancer drug screening complied sequentially into a single device. The system has the combined advantages of fast CTC isolation and efficient cancer cell capture, and maintained cell viability for downstream analysis. The microfluidic device with DLD structure and antibody modified architecture may provide an attractive platform for the use of personalized medicine for cancer patients.

Author ContributionsZ. L. designed the research; Z. L., W. S. performed the experiments;

Z. L., W. S., R. C., H. F., Z. H., Y. W., G. T., A. L., Y. C. analysed the data; Z. L., Y. C. wrote the manuscript. The authors declare no conflicts of interest.

AcknowledgementsThis work is supported by the Ministry of Science and Technology

of China (2015AA020409), the National Natural Science Foundation of China (31570861), the Major Program of Guangdong Science and Technology Project (2015B020227002), the Fundamental Research Program of Shenzhen (JCYJ20170413153034718), the Youth Innovation Promotion Association CAS, and SIAT Innovation Program (No. 201710).

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