High-Efficiency Capture of Individual and Cluster of Circulating Tumor

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High-Efficient Capture of Individual and Cluster of Circulating Tumor Cells by a Microchip Embedded with Three-Dimensional PDMS Scaffold Shi-Bo Cheng, Min Xie, Jiaquan Xu, Jing Wang, Song-Wei Lv, Shan Guo, Ying Shu, Ming Wang, Wei-Guo Dong, and Wei-Hua Huang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01130 • Publication Date (Web): 11 Jun 2016 Downloaded from http://pubs.acs.org on June 12, 2016

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Analytical Chemistry

High-Efficient Capture of Individual and Cluster of Circulating Tumor Cells by a Microchip Embedded with Three-Dimensional PDMS Scaffold Shi-Bo Cheng, †,‡ Min Xie, †,‡ Jia-Quan Xu, † Jing Wang, # Song-Wei Lv, † Shan Guo, † Ying Shu,# Ming Wang,# Wei-Guo Dong, # *and Wei-Hua Huang†* † Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China # Renmin Hospital of Wuhan University, Wuhan 430060, China ‡ These two authors contributed equally to this work. * Address correspondence to [email protected] or [email protected]

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ABSTRACT: Effective isolation of circulating tumor cells (CTCs) has great significance for cancer research, but is highly challenged. Here, we developed a microchip embedded with three-dimensional (3D) PDMS scaffold by a quadratic-sacrificing template method for high-efficient capture of CTCs. The microchip was gifted with 3D interconnected macroporous structure, strong toughness and excellent flexibility and transparency, enabling fast isolation and convenient observation of CTCs. Especially, 3D scaffold chip perfectly integrates the two main strategies currently used for enhancement of cell capture efficiency. Spatially distributed 3D scaffold compels cells undergoing chaotic or vortex migration in the channel, and spatially distributed nanorough skeleton offers ample binding sites, which synergistically and significantly improve CTCs capture efficiency. Our results showed that 1–118 CTCs/mL were identified from 14 cancer patients’ blood and 5 out of these cancer patients showed 1–14 CTC clusters/mL. This work demonstrates for the first time the development of microchip with transparent interconnected 3D scaffold for isolation of CTCs and CTC clusters, which may promote in-depth analysis of CTCs. KEYWORDS: three dimensional scaffold, microchip, circulating tumor cells, CTC clusters, CTC isolation

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INTRODUCTION Circulating tumor cells (CTCs) are the tumor cells escaped from the tumor mass and able to enter into the blood stream, which may respond for metastasis.1, 2 CTCs have received a lot of interest for early diagnosis of cancer and cancer therapy.3, 4 However, the low occurrence of CTCs in the bloodstream challenges the enrichment and characterization of CTCs.5, 6 So far, a variety of techniques have been developed to isolate CTCs, such as cell size-based filter devices,7-9 cell density-based method,10-11 and protein expression-based specific isolation methodologies.12-16 Among which, affinity interaction between CTCs and biofunctionalized magnetic beads,16-19 micro/nanosubstrates,6, 20-27 or microchips28-31 are the strategies that have been well developed and frequently used to specifically isolate CTCs. Notably, microchips showed great clinical application potential because it could integrate microfabrication, microfluidic, nanotechnology, and magnetic manipulation techniques in one chip,32-41 achieving fast, high-throughput and highly efficient capture of CTCs. Currently, two main strategies have been developed in fabricating effective microchips to improve capture efficiency of CTCs. One strategy relies on increasing the contact frequency of cells/substrates by changing migration of cells from laminar flow to chaotic or vortex migration pattern, representing by “herringbone-chip”42 proposed by Toner et al.. Second one is focus on enhancing the interaction force of cells/substrates by incorporation of nanostructures into microchips, like “NanoVelcro” cell-affinity substrates43-48 put forward by Tseng et al.. With the development of microchip fabrication techniques, it is conceivable that by combination of these two 3



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strategies together, capture efficiency would be far more improved. However, to achieve these goals, complicated microfabricating procedures and elaborate operations are usually needed, and development of simple, low-cost and easily-manipulated microfluidic platform for fast and highly efficient capture of CTCs is still a very attractive target. Herein, we prepared a tough, flexible and transparent three dimensional PDMS scaffold, and coalesced this high-performance material into a microchip to develop a novel platform (called “3D scaffold chip” below) for capture of individual and cluster of CTCs. The 3D scaffold chip could simply realize the combination of above mentioned two strategies without complicated microfabrication and elaborate operation procedures. The change of cell migration pattern and enhanced interaction between cells and substrate synergistically and significantly improve the capture efficiency of CTCs (Scheme 1). Well-adjustable interconnected macropores allow CTC clusters to transit freely, while flexible PDMS skeleton preserve integrity of CTC clusters. Furthermore, the strong toughness of 3D scaffold could sustain high velocity of blood, and transparency of the chip allows convenient observation of captured cells. These unique features facilitate fast, efficient and high-throughput isolation of individual and cluster of CTCs from real clinical samples.

Scheme 1. Scheme of capturing CTCs by 3D scaffold chip. 4


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EXPERIMENTAL SECTION Materials and Instruments. Ni foam was purchased from Kunshan Jiayisheng Electronics Co., Ltd.. Gelatin was purchased from Sinopharm Chemical Reagent Co., Ltd.. Bovine serum albumin (BSA), anti-EpCAM mouse monoclonal antibody, FITC-labeled goat anti-mouse secondary antibody, and 4, 6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich. 1, 1-dioctadecy l-3, 3, 3, 3-tetramethylindocarbocyanine perchlorate (DiI) was purchased from ThermoFisher. FITC-labeled mouse anti-human cytokeratin (FITC-CK), and PE-labeled mouse anti-human CD45 (PE-CD45) were obtained from Abcam Company. MCF-7 cells and Hela cells were purchased from China Type Culture Collection. All blood samples were obtained from Renmin Hospital of Wuhan University. All the media used for cell culture was obtained from Gibco Corp. All other chemicals were supplied by Shanghai Chemical Reagent Company. SEM images were obtained using a Zeiss Sigma field-emission scanning electron microscope (SEM). Fluorescent microscopic images were obtained using a Zeiss microscope (AxioObserver Z1, Zeiss, Germany). Preparation of 3D Porous PDMS Scaffold. The Ni foam slice (20 mm × 4 mm × 0.5 mm) was sonicated in acetone, ethanol and water for 30 min, respectively, then dried in the drying oven. Subsequently, newly washed Ni foam was immersed in a 2 mL centrifuge tube containing freshly prepared poly (dimethylsiloxane) (PDMS) and centrifuged at 8000 rpm for 5 min. By this 5



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centrifugation, PDMS was filled into the Ni foam scaffold and completely covered onto the surface of the Ni foam skeleton. Then PDMS-stuffed Ni foam was transferred into another empty Eppendorf tube for centrifugation for 3 min which is intended to remove the body stuffed PDMS while maintaining surface covered PDMS layer. To optimize centrifugal speed, 2000, 3000, 4000 and 6000 rpm were tried, respectively. After that, the Ni foam coated with PDMS was heated to 80 °C for 3 h in order to make PDMS solidification, which was then immersed in the 7 mol/L HNO3 for 2 h to etch the Ni foam completely.49 Thus far, porous PDMS scaffold was obtained and washed with water and dried. Integration of 3D Porous PDMS Scaffold into Microchip Before integration of porous PDMS scaffold into microchip, it was functionalized with carboxyl groups, facilitating chemical conjugation with biological molecules, like anti-EpCAM antibody. Here, porous PDMS scaffold was plasma treated for 3 min, and then dipped into the solution of 5% carboxyethylsilanetriol Na salt for 4 h to couple carboxyl. After that, porous PDMS scaffold coupled with carboxyl groups was transferred into 15% fluidic gelatin (37 °C) and totally immerged. Subsequently, the gelatin in porous PDMS scaffold was solidified at room temperature for blocking the porous structure of PDMS. Then gelatin-blocked PDMS scaffold was transferred onto the silicon wafer and covered by none crosslinked PDMS which was solidified at room temperature for 12 h. By these procedures, gelatin-blocked PDMS was coalesced with PDMS, which was cut and bonded with the clean glass to form microchip. Finally, the chip was heated to 50 °C and washed with warm water at the 6


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flow rate of 100 µL/min for 40 min to completely wash out gelatin filled in the porous PDMS. Thus far, 3D scaffold chip was successfully prepared. For comparison, three kinds of Ni foam were used as templates to fabricate 3D scaffold chips with size distributions of 125 ± 50 µm, 134 ± 54 µm, 162 ± 42 µm, respectively. Functionalization of 3D Scaffold Chip with Anti-EpCAM Antibody The carboxyl-functionalized 3D scaffold chip was washed by PBS and activated by 10 mmol/L EDC and 20 mmol/L NHS. After that, 10 µg/mL mouse anti-human anti-EpCAM antibody were introduced into the chip and incubated for 4 h, then washed by PBS at the flow rate of 100 µL/min for 3 min. Finally, FITC-labeled goat anti-mouse IgG was employed to identify the successful conjugation of anti-EpCAM antibody with the 3D scaffold chip, where microchips without anti-EpCAM antibody were used as control. Evaluating the Performance of 3D Scaffold Chips. MCF-7 cells were selected as CTC model cells to evaluate the capture performance of the 3D scaffold chip. Before capture, the cells were washed with PBS and re-suspended at 106 cells/mL, followed by staining with 1, 1-dioctadecy l-3, 3, 3, 3-tetramethylindocarbocyanine perchlorate (DiI). The labeled cells were stored at the 4 °C and further diluted to the desired concentration. Before experiment, PBS was used to wash the extra anti-EpCAM antibody in the chips. After that, 5% BSA and 0.2% Tween-20 were introduced into the chip to block none-specific binding sites for 1 h, followed by PBS washing at the flow rate of 100 7



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µL/min for 3 min. Finally, 1 mL of PBS containing 102 of MCF-7 cells prestained by DiI was continuously pumped into the chip (pore size of 125 ± 50 µm) at different flow rate of 20, 60, 100, 140 µL/min, respectively. The same amount of cell suspension was distributed in 96-well plate to calculate the mean of cells used. Then the chip was washed by PBS at the flow rate of 100 µL/min for 3 min to remove the nonspecifically captured cells. Finally, MCF-7 cells captured in the chips were counted for calculating capture efficiency and its location in the chips was recorded and mapped to know the distribution of MCF-7 cells in the chips. Meanwhile, 1mL PBS containing 102 DiI-prestained MCF-7 cells was used to flow through the 3D scaffold chip for estimation of the nonspecific effect of porous structure. (ZEISS, German). To know the size effect in capture efficiency, three kinds of 3D scaffold chips with pore size distributions of 125 ± 50 µm, 134 ± 54 µm, 162 ± 42 µm were used under the same conditions where MCF-7 cells were in a concentration of 102 cells/mL and flow rate was 100 µL/min. Hela cells and white blood cells (WBCs) were used as controls to test the specificity of anti-EpCAM antibody-functionalized 3D scaffold chip. Hela cells were cultured, collected and suspended in PBS. WBCs were obtained from lysed human blood. They were stained by DiI and kept in a concentration of 106 cells/mL in PBS, respectively. After that, 1 mL of PBS containing 106 Hela cells or WBCs was pumped into microchips at flow rate of 100 µL/min. Hela cells or WBCs in PBS flowed out of the chips was collected and counted by hemacytometer. 8


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Capture of Spiked Cells from Mimic Cancer Patient Blood Samples 1 mL of whole blood spiked with 120 of MCF-7 cells was used as mimic cancer patient blood samples which was injected into microchips (pore size of 125 ± 50 µm) at flow rate of 100 µL/min. After that, PBS was used to wash out nonspecific binding cells at flow rate of 100 µL/min for 3 min. Then captured cells were fixed with 4% paraformaldehyde (30min, 4°C), permeabilized with 0.2% Triton-X 100 (30 min, 4°C), and stained with 10 µg/mL DAPI, FITC-labeled anti-CK19 monoclonal antibody, and PE-labeled anti-CD45 monoclonal antibody (according to kit instruction) for 2 h at 4 °C. Captured cancer cells were classified as DAPI+/CK+/CD45-, and white blood cells were defined as DAPI+/CK-/CD45+. To know the capture efficiency of 3D scaffold chip, 1 mL of whole blood spiked with 10-170 of DiI-stained MCF-7 cells was tested by capture and counting procedures and quantitative data were obtained. To know the state of MCF-7 cells, 1×105 MCF-7 cells were spiked into 1 mL of blood and proceeded for isolation which were then fixed, dehydrated and lyophilized for SEM imaging. Isolation of CTCs in the Whole Blood. The EDTA-anticoagulated whole blood samples (15 cancer patients, including lung cancer, nasopharyngeal cancer, cervical cancer, breast cancer, esophageal cancer, colorectal cancer, endometrial cancer and 6 routine health checkup persons) were obtained from Renmin Hospital of Wuhan University and used without any pretreatment. Before sample proceeding, anti-EpCAM antibody-functionalized 3D

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scaffold chip was blocked by 5% BSA and 0.2% Tween-20, which could avoid the none-specific binding of the blood cells onto the skeleton.

Then 1 mL of whole

blood samples were injected into the chip, and PBS was used to remove the hematologic cells. Then 4% paraformaldehyde was introduced into the chip and incubated for 30 min at 4°C, followed by PBS washing at the flow rate of 100 µL/min for 3 min. Next, 0.2% Triton X-100 was pumped into the chip to incubate for 30 min at 4 °C and washed by PBS at flow rate of 100 µL/min for 3 min. Then 5% BSA was introduced into the chip and incubated for 1 h at room temperature, followed by PBS washing at the flow rate of 100 µL/min for 3 min. Finally, CK-FITC, CD45-PE and DAPI were pumped into the chip at the flow rate of 50 µL/min and incubated for 2 h at 4 °C, which were then washed by PBS at the flow rate of 100 µL/min for 3 min to remove the extra fluorescent dyes. Finally, 3D scaffold chip were observed under fluorescent microscope. The CTCs were classified as DAPI+/CK+/CD45-, and WBCs were defined as DAPI+/CK-/CD45+. CTC clusters were classified as cellular groupings that more than three cells band together. RESULTS AND DISCUSSION Preparation and Characterization of 3D Scaffold Chips We put forward a quadratic-sacrificing template method to prepare 3D PDMS scaffold chip. Firstly, Ni foam as sacrifice template was filled with non-crosslinked PDMS and then centrifuged to remove body stuffed PDMS, while maintaining surface covered PDMS layer. Here, the centrifugal speed must be optimized, since slow

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centrifugation induces dead pores in replicated 3D PDMS structure, while fast centrifugation does not benefit for coating uniform PDMS layer (Figure S1). Uniformly covered PDMS on Ni skeleton crosslinked at 80 °C, which was followed by nitric acid corrosion to remove Ni foam for preparing transparent 3D PDMS scaffold. Subsequently, macropores of 3D PDMS was blocked by solidified gelatin and then immerged by none crosslinked PDMS for integration with microchip. Finally, solidified gelatin in PDMS scaffold was washed out by warm water to obtain 3D scaffold chip (Figure 1a). The blood was injected and flowed through the microchip easily (Figure 1b), confirming the good permeability and connectivity of inter pores. The dented impressions (Figure S2) on both the upper cover and lower base of the chip indicate

Figure 1. a) Procedure for preparation of 3D scaffold chip; b) blood stuffed 3D scaffold chip; c) SEM image of 3D scaffold (inset: magnified SEM image of PDMS scaffold); d) characterization of anti-EpCAM antibody-functionalized 3D scaffold chip by FITC-labeled goat anti-mouse IgG. 11



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that 3D PDMS scaffold was tightly embedded into the chip, and spatially distributed through the whole channel rather than only used as channel substrate. The interconnected porous structure of 3D PDMS scaffold was clearly demonstrated in SEM images of Figure 1c, indicating that 3D scaffold chip was successfully prepared with excellent permeability, connectivity and integrity. An interesting result is that the PDMS scaffold was produced with nanorough surface (inset of Figure 1c, and Figure S3c). Considering that the surfaces of Ni form and PDMS-coated Ni foam were flat without subtle structure (Figure S3a,b), the nanostructure on 3D PDMS scaffold could be generated by nitric acid corrosion during Ni dissolution process. Figure S4 and S5 showed that 3D PDMS scaffold and Ni foam had similar porous size distributions, suggesting that porous PDMS replicated Ni foam structure in high fidelity but generating elaborate nanostructure, which will be beneficial to efficiently enhance the cell-substrate interactions during cell recognition. Besides, as-prepared 3D PDMS scaffold was tough and characterized with good flexibility. When the 3D PDMS scaffold was recovered from being stretched by 25%, the 3D structure was well preserved (Figure S6). This is an important feature for the 3D scaffold to withstand high velocity during cell capture. To specifically recognize CTCs, 3D scaffold chip was functionalized with anti-EpCAM antibody, which was characterized by FITC-labeled goat anti-mouse IgG. As shown in Figure 1d, strong fluorescence was observed on the PDMS scaffold coated with anti-EpCAM antibody, while the fluorescence on the PDMS scaffold

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without anti-EpCAM antibody was negligible (Figure S7), confirming the successful functionalization of 3D scaffold chip with anti-EpCAM antibody. Evaluating the Performance of 3D Scaffold Chip in Cancer Cells Capture MCF-7 cells, over-expressed of EpCAM, were selected as model of CTCs to evaluate the performance of anti-EpCAM antibody-functionalized 3D scaffold chip, while Hela cells and WBCs were used as negative control. Compared with control cells, DiI-prestained MCF-7 cells could be found in same horizontal position but at different focal planes of the 3D scaffold chip (Figure 2a and b). To test how the flow rates influence the capture efficiency, 1 mL PBS containing 100 MCF-7 cells were introduced into the device, and the effect of different flow rates

Figure 2. a, b) Fluorescent images of MCF-7 cells in same horizontal position but at different focal depth of 3D scaffold chip;(c) Effect of flow rate on MCF-7 cells capture efficiency in 3D scaffold chip; d) capture efficiencies of 3D scaffold chips with different pore size distributions or chips without 3D scaffold; (e) performance of anti-EpCAM antibody-functionalized 3D scaffold chip in capturing WBCs, Hela cells, MCF-7 cells, respectively; blank column represents MCF-7 cells trapped in 3D scaffold without anti-EpCAM antibody immobilization; f) an example of distribution mapping of captured MCF-7 cells. 13



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(20, 60, 100, 140 µL/min) on the CTC-capture efficiency was investigated. As shown in Figure 2c, for a 3D scaffold chip with the size of 20 mm × 4 mm × 0.5 mm, more than 90% CTCs capture was achieved when the flow rate was less than 100 µL/min (Figure 2c). The high cell capture efficiency at such a high flow rate is attributed to the unique 3D macroporous structure of the PDMS scaffold as well as their excellent mechanical stability. The capture efficiency decreased to 60% when the flow rate increased to 140 µL/min. This could be explained by the decreased duration of contact between cells and the substrates and increased shear force under large flow rate. Benefitting from the excellent mechanical strength of PDMS, larger flow rate could be used in the 3D scaffold chip. However, to get an equilibrium between capture efficiency and detection throughput, 100 µL/min was selected as the optimum condition in the following experiments. In order to know the influence of pore size in capture efficiency, three kinds of 3D scaffold chips with size distributions of 125 ± 50 µm, 134 ± 54 µm, 162 ± 42 µm were prepared to capture MCF-7 cells under same conditions (note that the minimum size is limited by the commercially available Ni foam). As shown in Figure 2d, the capture efficiency was tested to be 92%, 89%, 75%, respectively, which is gradually decreased with the increase of pore size. Actually, 3D macroporous PDMS scaffold generates chaotic cell migration pattern by interconnected irregular super macropores which significantly increases the cells and substrates contact frequency. Meanwhile, 3D scaffold offers spatial distributed nanorough surface which increases the capacity of EpCAM antibody immobilization and also enhances the local topographic 14


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interaction between cells and substrates. Combined these two advantages, 3D scaffold could significantly increase cell capture efficiency. Along with the increase of the pore size, the contact frequency between cells and substrates is decreased, resulting in the lower capture efficiency. It is deserved to know that the chip without 3D scaffold only obtained 4% of capture efficiency under the same experimental condition, suggesting the effectiveness of 3D scaffold in enhancing capture efficiency. To keep the high capture efficiency of the device, 3D scaffold chips with 125 ± 50 µm pore size were finally selected for the subsequent experiments.

The specificity of anti-EpCAM antibody-functionalized 3D scaffold chip was quantitatively evaluated by Hela cells and WBCs under flow rate of 100 µL/min. When 1 mL of PBS containing 106 Hela cells or WBCs cells (comparable to 1 mL of lysed human whole blood ) were injected into the chip, almost all of Hela cells or WBCs flowed out of the channel as shown in Figure 2e. Meanwhile, few MCF-7 cells were trapped by 3D scaffold chip without anti-EpCAM antibody (Figure 2e, blank column). These results suggested that 3D scaffold chip scarcely physically obstructed non-target cells, which was echoed by more than 90% of MCF-7 cells capture efficiency in anti-EpCAM antibody-functionalized chip. All these data support that anti-EpCAM antibody-functionalized 3D scaffold chip could be used for specifically capturing EpCAM-positive cells. We mapped the location of captured MCF-7 cells along the 20 mm long scaffold to know the distribution of captured cells. As shown in Figure 2f, captured MCF-7 cells were sparsely distributed in the scaffold although 67% of the cells were captured in the first half of the channel. These data further suggested 15



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Figure 3. Capture of MCF-7 cells from mimic clinical blood samples. (a) MCF-7 cells are DAPI+/CK-FITC+/CD45-PE-, while WBCs are DAPI+/CK-FITC-/CD45-PE+; (b) Recovery rate of MCF-7 cells spiked in blood samples; (c, d) The SEM images of representative MCF-7 cells captured in the 3D PDMS scaffold chip from mimic blood sample.

that MCF-7 cells transited through the interconnected pores smoothly and easily, while specifically and randomly captured by PDMS skeleton.

Capture of Cancer Cells from the Mimic Cancer Patient Blood Samples

Mimic cancer patient blood samples were used to evaluate the potential application of this chip in clinical blood. As shown in Figure 3a, this chip could successfully isolate rare MCF-7 cells from mimic cancer patient blood samples which could be enumerated by three-color immunocytochemistry (ICC) identification using CK-FITC (a marker for epithelial cells), CD45-PE (a marker for WBCs) and DAPI nuclear staining. As few as 10 MCF-7 cells in 1 mL of blood can be successfully recovered, and quantitative data show that the average capture efficiency of MCF-7 cells spiked in the whole blood is 88.4% (Figure 3b). SEM was used to further characterize the 16


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isolated MCF-7 cells from mimic blood. As shown in Figure 3c and d, MCF-7 cells hang upon or stick onto the skeleton of 3D scaffold. The substructures of MCF-7 cells are clearly observed which is helpful to confirm the specific and local topographic interaction between cells and substrates.

Isolation of CTCs from the Cancer Patients’ Blood. 3D scaffold chip was finally applied for real clinical blood samples. Here, 15 cancer patients and 6 routine health checkup persons were investigated. The captured cells in the channel were identified with the three-color ICC staining (Figure 4a-g). Furthermore, morphology and sizes of cells (>10 µm) offer auxiliary help for CTC identification, and the combined information was used to delineate CTCs. As shown in Figure 4h and summarized results in Table S1, rare CTCs and CTC clusters were found in 14 cancer patients, although there was no CTC found in 1 patient with cervical cancer. As to the results for 6 routine health checkup persons, 5 of them were CTC-negative, but 1 of them showed CTC-positive (1 CTC in 1 mL of blood). Actually, the person showed CTC-positive is not a “really” healthy control, since she was suffering gynecological disease when she had body checkup. Later she was further diagnosed with an early tumor of hysteromyoma by medical image and biopsy. This suggests that our platform has potential capability in early warning and diagnosis of tumors, despite that the relationship between CTC and hysteromyoma is currently unclear.

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Figure 4. Capture of CTCs in the whole blood samples from cancer patients. Pictures a-g) represent the CTC, WBC, and these cells were fluorescently stained with DAPI (blue), CK-FITC (green), CD45-PE (red); h) quantification of CTCs from cancer patients’ blood samples. Scale bar = 10 µm.

Besides single CTCs, several CTC clusters were detected in the blood of metastatic cancer patients, as shown in the Figure 5. CTC clusters are multicellular groupings of CTC which greatly contribute to the metastatic spread of cancer.50 Actually, as many as 14 CTCs clusters were captured in 1 mL blood of cancer patient with bone metastasis (patient with nasopharyngeal cancer). We think there are several reasons to explain that we could observe intact CTC clusters. Firstly, 3D PDMS replicates Ni foam structure with high fidelity and has pore size distribution of 125 ± 50 µm, allowing CTC clusters to pass through freely and smoothly. Secondly, the interconnected macropores of PDMS scaffold provide tough but flexible skeleton to 18


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avoid mechanical damage or break of CTC clusters at high flow rate which assists to preserve integrity of CTC clusters. Finally, anti-EpCAM antibody-functionalized 3D scaffold chip could capture intact CTC clusters specifically but physical obstructing effect of 3D scaffold would also be favorable for CTC cluster isolation based on a recent report that “cluster-chip”51 could capture CTC cluster by physical effect. It was reported that the aggregation of tumor cells can improve the ability of CTCs to survive in the blood-borne dissemination of cancer. The presence of circulating tumor cell clusters was found to be a more important prognostic factor in the metastatic process as compared with single CTCs.50 We prospect that our 3D scaffold chip could become a useful tool for effective isolation of CTCs and sustaining of CTC clusters in microfluidic channel, which may facilitate cancer diagnosis and metastatic judgement. Beyond capture of CTCs, next-generation devices and materials benefiting acquisition of more information of CTCs are required.52 Actually, as-prepared 3D scaffold chip is a versatile platform that can be integrated with chemical, enzymatic, and thermal release system for capture and release of CTCs. Our ongoing work focus on high-efficient capture, release, reculture and molecular analysis of CTCs and CTC cluster from clinical samples, and preliminary results showed that thermal-sensitive gelatin coated 3D scaffold chip could successfully capture and release of CTCs with high efficiency, facilitating subsequent analysis of CTCs.

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Figure 5. Representative of the CTC clusters captured from cancer patients’ blood. a, e), b, f), c, g) Microscopic images of nucleus (DAPI), CK (FITC), CD45 (PE) respectively; d, h, i-l) Merged images of nucleus (DAPI), CK (FITC), CD45 (PE). Scale bar = 10 µm.

CONCLUSIONS In conclusion, we prepared high-performance 3D PDMS scaffold and coalesced it into microchip by a quadratic sacrifice template method for highly efficient isolation of individual and cluster of CTCs from the whole blood. The 3D scaffold chip was characterized with unique 3D structure with interconnected macropores and great mechanic stability. 3D structure significantly increases the contact frequency between cells and substrates and meanwhile provides a number of binding sites for target cells, thus immensely improving the capture efficiency. The 3D scaffold chip achieves high capture efficiency of CTCs at high flow rate, which offers a simple, high efficient and high-throughput approach for fast isolation of CTCs from the whole blood. In addition, the 3D porous features of our microfluidic platform benefits 20


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capturing of CTC clusters which may be utilizable for judgement of metastatic menace. We predict that 3D scaffold chip would provide a new strategy for CTCs isolation and its integration with various release system could possibly promote CTC-related research. ASSOCIATED CONTENT Supporting Information. Additional Figures S1 to S7 and Table S1. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] or [email protected] Author Contributions ‡ These two authors contributed equally to this work. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENTS This work was supported by the National Basic Research Program of China (973 Program, No. 2012CB720603), National Natural Science Foundation of China (Nos.

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21575110, 21375099) and Specialized Research Fund for the Doctoral Program of Higher Education (20120141110031).

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Graphic Table of Contents:

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High-Efficiency Capture of Individual and Cluster of Circulating Tumor

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