Modular Chamber Assembled with Cell-Replicated Surface for

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Modular Chamber Assembled with CellReplicated Surface for Capture of Cancer Cells He Sun, Lulu Han, Liwei Yang, Yan Yang, Wenning Jiang, Ting Xu, and Lingyun Jia ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01605 • Publication Date (Web): 29 Mar 2019 Downloaded from http://pubs.acs.org on March 29, 2019

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ACS Biomaterials Science & Engineering

Modular Chamber Assembled with Cell-Replicated Surface for Capture of Cancer Cells He Sun, Lulu Han, Liwei Yang, Yan Yang, Wenning Jiang, Ting Xu, Lingyun Jia*

Liaoning Key Laboratory of Molecular Recognition and Imaging, School of Life Science and Biotechnology, Dalian University of Technology, NO. 2 Linggong Road, Dalian, Liaoning 116023, P. R. China

*Corresponding authors E-mail address: [email protected] Phone: (86) 411 84706125 Fax: (86) 411 84706125

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ABSTRACT The capture of circulating tumor cells (CTCs) is mainly carried out with a small volume of blood using magnetic nanoparticles and complex microfluidics. In this study, we propose a CTC-capture apparatus based on a modular-design and called this apparatus as the CTC-chamber. Distinct from other CTC-capture apparatuses, the capacity of the CTC-chamber could be altered by varying the number of CTC-capture modules to accommodate the different volumes of blood sample. The core component of the CTCcapture module was a polydimethylsiloxane (PDMS) film with cell-replicated topological structure and anti-EpCAM antibody coating. Both synergistic roles can enhance the capture yield of cancer cells. Furthermore, the CTC-chamber was assembled with one or three CTC-capture modules for the capture of cancer cells from spiked blood samples representing late-stage (3 mL blood, 10 cancer cells mL-1) cancer or middle-early stage (9 mL blood, 1 cancer cell mL-1) cancer. The results showed that high capture yield (EpCAM-positive: ~80%, EpCAM-negative: ~65%) and purity (EpCAM-positive: ~90%, EpCAM-negative: ~80%) could be obtained within 1 h. This economic and facile CTC-chamber could therefore open up opportunities for designing the next-generation CTC detection devices suitable for the diagnosis of different stages of cancer. KEYWORDS: CTC-chamber, Cell-Replicated Surface, PDMS, Capture, Cancer Cells

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Introduction Circulating tumor cells (CTCs) are rare tumor cells that are shed from solid tumor lesions and flowed into peripheral blood whit a process of epithelial-mesenchymal transformation (EMT), 1-3 where they would then spread to other parts of the body. 4-7 Since CTCs are closely related to cancer metastasis,

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and direct capturing of CTCs

from blood is a minimally invasive detection method,9-10 their isolation and characterization can deeply understand the effect of chemotherapies and realize the early diagnosis of cancers.

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However, the number of CTCs in peripheral blood is

extremely low, especially for early-stage cancer patients.15-16 For instance, one previous study reported for 75% of the stage I and II breast cancer patients, merely 1-4 CTCs were detected in 30 mL of blood.17 Although the number of CTCs in the blood of latestage cancer patients may range from a few to a few hundred per mL of blood, which still pales in comparison with the presence of millions of white blood cells and billions of red blood cells.18-19 At present, the volume of blood commonly used for the isolation of CTCs is about 2-7.5 mL. Since the numbers of CTCs in the blood are extremely low, using a larger volume of blood for the isolation of CTCs should provide more accurate information for liquid biopsy, especially during the early diagnosis of cancer.20-21 So far, magnetic beads and microfluidic chips are the two major approaches for CTC capture.22-23 The only FDA-approved CTC-capture method, Johnson & Johnson's CellSearch system, is based on anti-EpCAM-modified magnetic beads to recognize the surface antigen of cancer cells and to allow CTC isolation from the blood.24-25 By increasing the amount of magnetic beads, CellSearch can process a large volume of 3



blood sample.26-27 Nonetheless, the magnetic separation may cause cell loss, while the nano-scale (100 nm) magnetic beads may mediate leukocytes endocytosis, consequently leading to high false negative rate and low selectivity.28 The microfluidic chip method, which is based on the complex chaotic mixing channels and cancer cellrecognizing biomolecules, has higher CTC-capture yield and selectivity,29-31 when capturing CTCs from 1 mL of blood.32-33 However, if the CTC-capture yield reaches more than 90%, the blood flow is generally set to slower than 2 mL h-1.34 As a result, CTC capture from a large volume of blood is time consuming.35 Besides, this method requires complicated operation and expensive experimental setup.36-37 To overcome the drawbacks of the two aforementioned methods, it is essential to develop an economic and facile method that enables the fast and efficient capture of CTCs not only from a small, but also from a large volume of blood such that it can be used as liquid biopsy for patients at different cancer stages. Recent years, cancer-cell capture depends largely on the micro-nano topological structure of the capturing surface,38-44 while CTC-recognizing molecules, such as antiEpCAM can further increase its efficiency and specificity.45-47 Artificial nanomaterials and nanoparticles often feature a uniform topological structure,48 whereas cells usually consist of nano-scale filopodias and micro-scale asperities, 49-50 forming a multi-scale, complex and natural micro-nano structure, that can be replicated to improve the efficiency of CTC capture.51 Existing cell-replicated technology is mainly based on the soft lithography, imprinting and silicification on the cells.52-54 Even though both have proven to be promising methods in the preparation of cell-replicated surfaces,55 several 4


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drawbacks still remain, which include the complicated manipulation of the silicification process54 and the inability to preserve the surface nanostructures of mammalian cells during soft lithography and imprinting.56-59 In this study, inspired by the flexibility of modular design, in which modules can be added to or removed from a system to change the processing capacity of the system, we proposed a new design for constructing a CTC-chamber for the capture of CTCs from the blood. The CTC-chamber composed of varying numbers of modules capable of capturing extremely low numbers of cancer cells present in the blood of cancer patients. In addition, a well-defined cell-replicated topological structure of alcohol-dehydrated cells was created on the surface of polydimethylsiloxane (PDMS) (CellRePDMS) via soft lithography. This well-defined feature was generated because dehydration with anhydrous alcohol not only maintained the cell topological structure but also quickly reduced the water content in the cells, shrinking the cells and further increasing the detailed micro-nano topological structure of the cells. After coating with anti-EpCAM antibody on the CellRePDMS (CellRePDMS/Anti-EpCAM), the CellRePDMS/AntiEpCAM film and silicone washer used to build the CTC-capture module, and several such modules were further stacked together to form the CTC-chamber. Furthermore, the CTC-chamber was evaluated to rare cancer cell-capture performance from blood samples spiked with cancer cells for simulating the diagnosis of cancer patients in the middle-early stage and the late stage of cancer.

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Experimental section Materials and Reagents Polydimethylsiloxane (PDMS) prepolymer (Sylgard 184) was purchased from Dow Corning (USA). N-Octadecyltriethoxysilane (C18) was purchased from Alfa Aesar (USA). Biotinylated bovine serum albumin (biotin-BSA) was obtained from Carexinc Co., Ltd. (Beijing, China). TRITC-phalloidin, triton X-100, biotinylated monoclonal anti-EpCAM antibody, monoclonal anti-vinculin antibody and fluorescein-labeled donkey anti-mouse IgG were purchased from Sigma-Aldrich (USA). 4',6-diamidino-2phenylindole (DAPI), fluorescein diacetate (FDA) and 1,1'-dioctadecyl-3,3,3',3'tetramethylindocarbocyanine (DIL) were obtained from Beyotime Biotechnology (Shanghai, China). Trypsin-EDTA (0.25%) and penicillin-streptomycin were obtained from Hyclone (USA). Paraformaldehyde (POM) was purchased from Aladdin Biotechnology (Shanghai, China). Phosphate buffer saline (PBS, pH 7.4) was obtained from GE Healthcare Life Sciences (Logan, UT). Streptavidin (SA), Ficoll-Paque and whole blood erythrocyte lysing reagent were purchased from Solarbio Technology Co., Ltd. (Beijing, China). Fetal bovine serum (FBS) was purchased from Gibco (USA). Cy5-labeled anti-CD45 monoclonal antibody and fluorescein isothiocyanate (FITC)conjugated monoclonal anti-cytokeratin 8 antibody were purchased from Abcam (Cambridge, UK). Ultrapure water with a resistivity of 18.2 MΩ.cm (Millipore, U.S.A.) was used. All other used reagents were analytical grade. Cell Culture Human breast cancer cell line (MCF-7), lung cancer cell line (A549), liver carcinoma 6


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cell line (HepG2) and cervical cancer cell line (HeLa) were obtained from Shanghai Institute for Biological sciences, Chinese Academy of Science. MCF-7 and HeLa cells were cultured in DMEM (HyClone) whereas HepG2 and A431 cells were cultured in RPMI 1640 (Gibco). All cultures were supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin (HyClone. Final concentrations of penicillin and streptomycin are 100 U mL-1 and 100 μg mL-1, respectively) and maintained at 37°C in a humidified incubator with 5% CO2. The cells were passaged at approximately 90% cell confluency. Preparation of CellRePDMS MCF-7 cells were placed in tissue culture dishes at different cell densities (8×103, 1.4×104, 2×104 and 2.5×104 cells cm-2). After two days of incubation, the cells were gently rinsed three times with PBS and fixed in 4% paraformaldehyde PBS solution for 20 min. The fixed cells were dehydrated with anhydrous alcohol for 30 min and used as template cells for the subsequent preparation of CellRePDMS. CellRePDMS was fabricated through casting a mixture of PDMS prepolymer onto the template cells prepared above. First, a PDMS prepolymer mixture (monomer/crosslinker = 10:1, m/m) was defoamed in a vacuum-drying oven, and then poured over the template cells. The polymer was cross-linked and solidified in an 80 °C oven for 6 h. After that, the obtained PDMS with a cell-replicated pattern was peeled off and designated as CellRePDMS. Anti-EpCAM Surface Modification CellRePDMS and flat PDMS films (0.5 cm × 0.5 cm) were pretreated with ozone and UV ray for 10 min using an ultraviolet ozone machine (Guoda BZZ250G-T, 7



Shanghai). Subsequently, the pretreated films were each placed in separate wells of a 96-well culture plate and treated at 20 °C for 45 min with N-octadecyltriethoxysilane (C18, 0.4 mg/mL in ethanol). After washing with ethanol and water, 100 μL of biotinBSA solution (0.5 mg mL-1 in PBS) was added to each well and the plate was incubated at 37 °C for 2 h followed by washing with PBS (100 μL per well), subsequent addition of 100 μL of streptavidin solution (10 μg mL-1 in PBS) and further incubation at 37°C for 30 min. Finally, the films were incubated in 50 μL biotin-anti-EpCAM solution (10 μg mL-1 in PBS) at 37°C for 30 min, and the resulting anti-EpCAM-modified CellRePDMS and flat PDMS were designated as CellRePDMS/Anti-EpCAM and PDMS/Anti-EpCAM, respectively. In addition, the anti-EpCAM-modified PDMS was further dipped in 100 μL of fluorescein-labeled donkey anti-mouse IgG (secondary antibody) solution (10 μg mL-1 in PBS) at 37 °C for 2 h followed by washing in PBS and dried with nitrogen gas for subsequent fluorescence analysis. The extent of antiEpCAM modification was monitored by the intensity and homogeneity of green fluorescence under a fluorescence microscope (Olympus 71, Japan). The fluorescence images were quantitatively analyzed using the software of ImageJ. Surface Characterization The template cells, CellRePDMS, flat PDMS and CellRePDMS/Anti-EpCAM films were completely vacuum-dried and characterized using a 3D measuring laser confocal microscope (Olympus LEXT OLS4000). SEM images were taken on a Quanta 450 scanning electron microscope (FEI, U.S.A.) at an accelerating voltage of 20 kV. The samples were sputter coated with gold before measurement. XPS experiments were 8


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performed with an ESCALAB 250Xi X-ray photon-electron spectrometer (Thermo Scientiﬁc, U.S.A.) using Mg Kα radiation under a vacuum of 2 × 10−8 Pa. The binding energy (BE) scale was calibrated by comparing with the neutral adventitious C 1s peak at 284.6 eV. The wettability of each sample was studied by measuring the water contact angle using an optical contact angle measuring instrument (KRüSS-DSA100). Thickness of air-dried films was performed using a spectroscopic ellipsometer (J. A. Woollam M-2000, U.S.A.). Blood Collection and Processing Blood from healthy donors was collected and stored in 5-mL blood collection tubes containing sodium citrate as an anticoagulant (All agreements related to the use of blood were approved by the Ethical Committee of Dalian University of Technology). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient separation with the addition of lymphocyte solution to fresh anticoagulant. After the separation process, PBMC suspension was obtained and the concentration of PBMCs was about 0.8 × 106 cells mL−1. Cell Capture in Culture Medium The culture mediums were implemented for cell-capture experiments. For a typical capture assay performed in culture medium, the CellRePDMS, PDMS/Anti-EpCAM or CellRePDMS/Anti-EpCAM films were placed in a 96-well cell culture plate with the addition of 100 μL per well of cancer-cell (MCF-7, HepG2, HeLa or A549) suspension (1.6 × 105 cells mL-1). After incubation for 60 min at 37 °C, the cells captured on the surface of the films were washed with PBS for three times, and then stained with 9



fluorescein diacetate (FDA) solution (1 µg mL-1 in PBS) for 5 min followed by washing three times with PBS. The cells were then photographed and counted under an inverted fluorescence microscope (Olympus 71, Japan). Cell capture yield represents the total number of cells of a given type captured divided by the total number of cells seeded of that type. PBMCs and HUVECs Adhesion The CellRePDMS, PDMS/Anti-EpCAM or CellRePDMS/Anti-EpCAM films were placed in a 96-well cell culture plate with the addition of 100 μL of PBMC suspension or human umbilical vein endothelial cells (HUVECs) suspension (1.6 × 105 cells mL-1, isolated from the vein of normal human umbilical). The PBMCs adhered on the surface of the films were washed three times with PBS after incubation at 37 °C for 60 min, and then fixed in a solution of 4% paraformaldehyde (diluted with PBS) for 30 min. 0.2% Triton-X 100 (diluted with PBS) was used for 5 min to increase the permeability of the cell membrane, and then treated whit 1% BSA (diluted with PBS) at 20 °C for 30 min to seal the non-specific antigen-determining clusters. After that, the PBMCs were incubated with Cy5-labeled anti-CD45 monoclonal antibody (10 µg mL1

in PBS) for 1 h at 37 °C. The PBMCs were then stained with DAPI (10 µg mL-1 in

PBS) for 30 min and observed under a confocal laser scanning microscope (Leica TCS SP8). The HUVECs captured on the CellRePDMS/Anti-EpCAM were fixed for 30 min in PBS containing 4% paraformaldehyde followed by staining with DAPI (10 µg mL1 in PBS) for 30 min and washing three times with PBS. Image-pro Plus software was used to count the numbers of adherent PBMCs or HUVECs of 10 randomly collected 10


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images. Cancer Cells Capture from PBMC-spiked samples First, a known number (10, 20, 50, 100, 200, 400, 600, 800 and 1000) of MCF-7 cells were mixed with a suspension of PBMCs. Then, the CellRePDMS/Anti-EpCAM films were placed in a 96-well culture plate with the addition of 100 μL MCF-7 cells-spiked PBMC suspension to each well. The cells (MCF-7 cells and PBMCs) captured on the surface of the films were washed three times with PBS after incubation at 37 °C for 60 min, and then fixed in a solution of 4% paraformaldehyde (diluted with PBS) for 30 min. 0.2% Triton-X 100 (diluted with PBS) was used for 5 min to increase the permeability of the cell membrane, and then treated whit 1% BSA (diluted with PBS) at 20 °C for 30 min to seal the non-specific antigen-determining clusters. After that, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-cytokeratin 8 antibody (10 µg mL-1 in PBS) for 1 h at 37 °C followed by incubation with Cy5-labeled anti-CD45 monoclonal antibody (10 µg mL-1 in PBS) for 1 h at 37 °C. The cells were then stained in a DAPI solution (10 µg mL-1 in PBS) for 30 min. Finally, the cells were observed under a confocal laser scanning microscope. The capture yield was defined as the total number of MCF-7 cells captured divided by the total number of MCF-7 cells seeded, and the capture purity was defined as the number of cancer cell captured divided by the total number of cells (cancer cells and PBMCs) captured within the same image. SEM Observation of Cell Morphology To observe the morphology of the adherent cells under SEM, the captured cancer 11



cells and adherent PBMCs on the CellRePDMS, PDMS/Anti-EpCAM or CellRePDMS/Anti-EpCAM films were fixed with paraformaldehyde (4% in PBS) for 20 min at room temperature. After washing with PBS for three times, the cells were dehydrated through a gradient series of alcohol (30%, 50%, 70%, 85%, 95% and 100%), each for 15 min. Finally, liquid CO2 was used with a supercritical point dryer to maintain the morphology of the samples for gold sputtering in the subsequent SEM (FEI-QUANTA 450, USA) observation. Immunofluorescent Staining of Captured MCF-7 Cells and PBMCs The actin cytoskeleton and focal adhesion of MCF-7 cells and PBMCs were detected by immunofluorescence staining. The captured cells were first fixed in paraformaldehyde (4% in PBS) for 20 min at room temperature and subsequently permeabilized with 0.2% Triton-X100 (in PBS) for 5 min. After washing three times with PBS, the cells were treated with blocking solution (1% BSA in PBS) for 30 min, followed by incubation with primary antibody (anti-vinculin mouse monoclonal antibody, diluted 1:400 in PBS) for 1 h and secondary antibody (FITC-conjugated goat anti-mouse antibody, diluted 1:30 in PBS) for 30 min. After that, the cells were incubated with 100 μL of TRITC-conjugated phalloidin (15 μg in 250 μL methanol and diluted 1:40 in 1% BSA in PBS) and 100 μL of DAPI (2 μg mL-1 diluted by PBS). The nuclei, actin cytoskeletons and focal adhesions of the cancer cells and PBMCs were visualized and photographed using a laser scanning confocal microscope. Preparation of CTC-chamber CellRePDMS/Anti-EpCAM and a silicone washer were used to build the CTC12


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capture module (Figure S1). Modules and the top and bottom plastic boards were stacked to form the CTC-chamber. First, plastic boards were cut to the size of 60 mm (L) × 40 mm (W) × 5 mm (H) as the top and bottom seal plates, embedded with syringe needles as flow inlet and outlet as indicated in Figure S1. CellRePDMS/Anti-EpCAM with a size of 50 mm (L) × 30 mm (W) × 3 mm (H) and a silicone washer with a size of 40 mm (inner L)/43 mm (outer L) × 25 mm (inner W)/28 mm (outer W) × 3 mm (H) were used to build the CTC-capture module. Each CellRePDMS/Anti-EpCAM had one open pore for connecting to the modules above and below (Figure S1a, S1b). When the number of modules was set to 2 and above, the pores of the two adjacent CTC-capture modules were set in a diagonal position in order to realize the maximum liquid fills in each module without leaving bubbles. Certain number of modules and the top and bottom seal plates were assembled with four screws to form the final CTC-chamber. Cell Capture in CTC-chamber A known number of DIL-pre-stained live cancer cells (EpCAM-positive: MCF-7 and HepG2, EpCAM-negative: HeLa and A549) were mixed with the fresh blood containing anticoagulant. Subsequently, the erythrocytes were removed by erythrocytelysing reagent. A mixture of cancer cells and PBMCs was obtained after centrifugation. For simulating the middle-early stage of cancer, 9 mL of blood was spiked with 9 cancer cells (1 cancer cell mL-1) and a CTC-chamber with three modules was used in the cell capture experiment. In order to accurately control the cell number at 1 cell per mL blood, DIL per-staining cancer cells were first diluted with culture medium to a very low number, and then 1 μL of cancer cell suspension was added into one well of the 24-well 13



cell culture plate and counted under the microscope. When the added cancer cells were 9, 1 mL of blood was injected into the well and completely mixed with the cancer cells. Subsequently, the obtained mixture was added to 8 mL of blood with a final concentration of cancer cells at 1 cancer cell per mL blood. To simulate the late stage cancer, 3 mL of blood was spiked with 30 cancer cells (10 cancer cells mL-1) and a CTC-chamber with one module was used in the cell capture experiment. First, the bloods spiked with DIL-pre-stained cancer cells were slowly injected into the CTC-chamber via a syringe connected to the bottom inlet at a rate of 3 mL min-1. When the CTC-chamber was fully filled with the blood, the syringe was removed, and the inlet was sealed with a sealing membrane and the chamber was incubated in a 37°C incubator with 5% CO2 for 60 min. After washing with PBS at a rate of 3 mL min-1, trypsin-EDTA (0.25%) was injected gently into the CTC-chamber and incubated under the same condition for 5 min. After incubation, the liquid fraction containing the released cells was drawn at a rate of 3 mL min-1 and collected in a centrifuge tube with the addition of fresh DMEM cell culture medium. All the cells (DIL-pre-stained cancer cells and PBMCs) were stained with fluorescein diacetate solution (FDA) for 2 min. Finally, the cells were imaged and counted using the inverted fluorescence microscope (Olympus 71, Japan). The yield and purity of the captured cells were determined as described in section 2.9. Statistical Analysis All experiments were performed at least three times (n ≥ 3) for each sample, and the mean values (± standard deviations, SDs) were reported. All data were compared with 14


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one-way ANOVA tests to evaluate the statistical significances, which were considered at the P< 0.05, P< 0.01 and P< 0.001 levels. Results and Discussion Anti-EpCAM Coating on the CellRePDMS The preparation of CellRePDMS is schematically illustrated in Figure 1a. Human breast cancer (MCF-7) cells were used as template cells to fabricate a cell-replicated PDMS film (CellRePDMS) because MCF-7 cells have long dendritic pseudopodia and luxuriant asperities (Figure 1b). After dehydration with anhydrous EtOH, the cells displayed a clearer micro-nano surface structure (Figure 1b) compared to the gradient EtOH-dehydrated cells, and this might be attributable to the fact that anhydrous EtOH adequately removes water from the cells (Figure S2). When CellRePDMS was peeled off from the template cells, its surface retained the imprint of the template cell surface (Figure 1c), while no cell was detached from the template even after six rounds of castings (Figure S3), suggesting good reusability of the template. Besides, CellRePDMS casted on anhydrous EtOH-dehydrated template cells displayed a higher cell capture capability than the one casted on gradient EtOH-dehydrated template cells (Figure S2e), implying the defined and detailed micro-nano topological structure of the former better facilitated the cell capturing process. Moreover, because the better dehydrate pretreatment strategy using anhydrous alcohol, the capture yields of cancer cells are more efficient than the previous reported natural cell-replicated surfaces (Table S1).43-44, 60

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Figure 1. Schematic illustration and characterization of CellRePDMS. (a) Schematic illustration of the preparation of CellRePDMS. Briefly, MCF-7 cells were cultured in a cell culture plate and dehydrated with ethanol. PDMS prepolymer was then poured onto the dehydrated cells and allowed to polymerize. The polymer film was peeled off from the cells and designated as CellRePDMS. (b) SEM images showing the surfaces of template cells. (c) SEM images showing the surface of CellRePDMS. (d) Cell-capture yields of CellRePDMS with increasing cell replica ratios (the area of a PDMS film replicated by the template cells divided by the total area of the PDMS film). The blue and polychromatic insets are the 3D structures of CellRePDMS in a 0 (without cells) and 90% cell replica ratio, respectively. Error bars denote the standard deviation.

When the cell replica ratio increased from 0 (flat), 20%, 50% and 65% to 90%, the capture yield of MCF-7 by the corresponding CellRePDMS gradually increased, up to a maximum of 71 ± 8% (Figure 1d), possibly because more cells resulted in more 16


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filiform pseudopodia micro-nano features casted on the CellRePDMS (Figure 1d inset), which further promoted the cell capture (Figure 1d). Although with an increase in cell replica ratio, the contour sizes of the cellular structures in the CellRePDMS would overlap at the cell edge (Figure 1c), but there was little influence on the average projected area of individual cell replica, because of cell contact inhibition (Figure S4). Therefore, CellRePDMS with a 90% cell replica ratio was chosen for the subsequent cell capture experiments. The surface of CellRePDMS was further modified by coating with anti-EpCAM via biotin-avidin interaction (Figure 2a) to enhance the cell capture yield. After treatment with ozone and UV rays, the ratio of oxygen on CellRePDMS increased (Figure 2b), while the water contact angle (WCA) of CellRePDMS decreased from the original value of 113.5o to 67.2o (Figure 2c), implying the formation of silanol (CellRePDMS/OH). Subsequent coupling of C18 to CellRePDMS/OH through silanization was confirmed by the increased ratio of elemental C on CellRePDMS/C18 and the significantly increased surface hydrophobicity (WCA was 97.6 ± 5.4°). This process avoided the replacement of surface-bound BSA of the biomaterials by other proteins that are naturally present in the blood (Vroman effect),61 such as human fibrinogen (HFg), because albumin exhibits a high affinity for circulating free fatty acids, primarily those consist of 16-18 carbon chains.62 The sequential binding of biotin-BSA (b-BSA), avidin and biotin-anti-EpCAM to the substrates via biotin-avidin interaction led to the decrease in WCA, reaching a value of 38.6 ± 3.2° (Figure 2c). Finally, the CellRePDMS/Anti-EpCAM surface was obtained. 17



Figure 2. Schematic illustration and characterization of anti-EpCAM-modified CellRePDMS. (a) Steps illustrating the modification of CellRePDMS surface with antiEpCAM. (b) Superficial elemental ratios of Si, C, O on CellRePDMS (Blank), CellRePDMS/OH (OH) and CellRePDMS/C18 (C18) measured under XPS. (c) Water contact angle of the CellRePDMS (Blank) and CellRePDMS modified with OH, C18, biotinlated-BSA (B-BSA) and anti-EpCAM. (d) Fluorescence intensity of blank CellRePDMS and CellRePDMS/Anti-EpCAM stained with FITC-labeled secondary antibody from the background subtraction. Error bars denote the standard deviation.

Anti-EpCAM was homogeneously distributed on the surface of CellRePDMS/AntiEpCAM with a thickness of 6 nm (Figure 2d, S5 and S6), and maintained the appropriate sites for recognition of cancer cells. Moreover, anti-EpCAM could stably exist in the cell culture condition (Figure S6), which is suitable for further cell capture. Broad-Spectrum Capture for Cancer Cells with High Efficiencies MCF-7 cells were used as a cell model to study the cancer cells capture performance of PDMS/Anti-EpCAM, CellRePDMS and CellRePDMS/Anti-EpCAM. The capture 18


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yields

of

MCF-7

cells

on

the

PDMS/Anti-EpCAM,

CellRePDMS

and

CellRePDMS/Anti-EpCAM films increased with longer capture time and reached a maximum at 60 min while the maximum yields differed among the three polymers (p

Modular Chamber Assembled with Cell-Replicated Surface for

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