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A Chemical Method for Specific Capture of Circulating Tumor Cells Using Label-Free Polyphenol-Functionalized Films Liwei Yang, He Sun, Wenning Jiang, Ting Xu, Bing Song, Ruilian Peng, Lulu Han, and Lingyun Jia Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.8b01646 • Publication Date (Web): 06 Jun 2018 Downloaded from http://pubs.acs.org on June 6, 2018

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Chemistry of Materials

A Chemical Method for Specific Capture of Circulating Tumor Cells Using Label-Free Polyphenol-Functionalized Films Liwei Yang, He Sun, Wenning Jiang, Ting Xu, Bing Song, Ruilian Peng, Lulu Han*, Lingyun Jia Liaoning Key Laboratory of Molecular Recognition and Imaging, School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116023, P. R. China ABSTRACT: The capture of circulating tumor cells (CTCs) mainly depends on the biological affinity and differences in physical properties between tumor cells and peripheral blood mononuclear cells (PBMCs), but the effective capture of CTCs using only chemical method is still rare. Inspired by the excellent anti-inflammatory effects of polyphenols, we used polyphenol tannic acid (TA)-functionalized film to develop a simple and yet effective chemical strategy for label-free capture of CTCs. The film could be easily prepared within minutes by metal-polyphenol coordination interaction while tactfully integrating the effects of repelling PBMCs adhesion and enhancing cancer cells adhesion. The galloyl groups of TA were found to play a crucial role in the enhanced adhesion of cancer cells to the film. As a result of the preference of TA film for cancer cells over PBMCs, the film could effectively capture a broad range of cancer cells without the aid of capture antibodies (yield > 76%). Importantly, the viability of the captured cells was as high as 90%, which is conducive to subsequent biological assay. This easy and label-free platform would provide new clues for developing chemical-based CTC capture methods and open up opportunities in designing next-generation devices for the detection and analysis of CTCs.

INTRODUCTION The leading cause of carcinoma-related mortality is tumor metastasis.1 Circulating tumor cells (CTCs) are rare cancer cells that have detached from primary or metastatic tumors and migrated into the peripheral bloodstream, carrying significant information of cancer progression and metastasis.2, 3 Therefore, the isolation and characterization of CTCs has been regarded as a liquid biopsy technology for cancer diagnosis, genotyping, and prognosis.4-6 However, the isolation of CTCs is particularly challenging because of the extremely low concentration of CTCs in peripheral blood (usually in the range of 1-100 cells mL-1).7 Over the past decade, there has been great interest in developing strategies for the selective isolation of CTCs. Label-dependent (biological affinity) isolation is the most commonly adopted method for the capture of CTCs, which uses bioactive molecules (antibodies, polypeptides, or DNA aptamers) to specifically bind to the surface biomarkers of the CTCs.8-10 However, for efficient or high throughout capture, these biomolecules need to be conjugated with magnetic beads,11, 12 nanostructured substrates,13-16 or microfluidic chips,17-19 thus requiring multistep fabrication processes related to surface etching, biomolecules immobilization and devices assembly. In addition, the active proteins and aptamers are expensive and easy to deactivate or degrade. Furthermore, these isolation approaches mostly rely on a single biomarker, such as the epithelial cell adhesion molecule (EpCAM).20 Hence, the efficiency of CTC capture can be compromised because the expression of EpCAM is downregulated in many CTC subpopulations that undergo epithelial-mesenchymal transition (EMT).1, 21 Employing a cocktail of antibodies may increase the capture efficiency of CTC subpopula-

tions,22-24 but it is experimentally time-consuming and cost-ineffective. The capture bias of biological affinity methods encountered by researchers has encouraged the development of label-free CTC isolation techniques that are mainly based on physical characteristics such as size,25, 26 density,27 deformability,28 acoustic,29 dielectric,30, 31 and adhesion strength.32 Even though these methods have proven promising for detecting CTCs in patients with metastatic cancers, several drawbacks still exist, such as the loss of CTCs or low sample purity due to the leukocyte contamination, and limited viability of the captured CTCs because of elevated temperatures, and the use of external force fields as well as complicated experimental setup. Thus, further efforts should be devoted to label-free CTC isolation techniques more efficient and convenient.7-9, 19, 33 Surface modification by chemical groups, which acts as a facile and label-free method, may open a new avenue to achieve these requirements. So far, phenylboronic acid (PBA) is the only chemical group that has been used for CTC capture, as it relies on the fast and stable formation of boronate esters between PBA and the glycans (i.e. sialic acid) on the surface of the cancer cells.34, 35 However, effective CTC capture merely occurs when PBA is constructed on substrates with nanostructure features,34, 35 and the capture purity is also limited. Few studies have reported the efficient capture of cancer cells by a single chemical group. Polyphenols are naturally occurring compounds found in fruits and vegetables.36 They are bulky molecules composed of abundant phenolic hydroxyl groups in the forms of catechol and galloyl groups, which can strongly

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Figure 1. Physiochemical characterization of TA film. (a) Tannic acid (TA), a water-soluble natural polyphenol extracted from plants (e.g. fruits and tea leaves). (b) Photograph of glasses, polydimethylsiloxane (PDMS) and silicone tubes before and after TA coating. Scale = 1 cm. (c) AFM topographical image of TA film modified on glass substrate, and the measured average roughness (Ra) of the film is indicated. (d) SEM image of TA film and the corresponding elemental mapping images of C, O, and Fe elements. Scale = 10 μm. (e) XPS spectra of glass substrate before and after TA coating. Insets are highresolution Fe 2p spectra of TA film. interact with polysaccharides and proteins via hydrogen bonds and hydrophobic interaction.36-38 As the extracellular matrix of cancer cells consists of a variety of glycoproteins (e.g., glycans and fibrous proteins) known as the glycocalyx, a 500-nm gel-like layer of biologically inert macromolecules formed during cancer metastasis,39, 40 we speculated that polyphenols may exhibit good affinity for cancer cells through interaction between the phenolic hydroxyl groups of polyphenol and the glycocalyx of cancer cells. Most importantly, polyphenols have demonstrated excellent anti-inflammatory effects in in vitro and in vivo experiments,41-43 by down-regulating the level of proinflammatory cytokines and inhibiting the expression of leukocyte adhesion molecules, further reducing the ability of leukocytes to attach to a surface.44, 45 Thus, these properties of polyphenols may offer a possibility for developing a novel and label-free chemical method for capturing CTCs. To verify our hypothesis, we chose tannic acid (TA, Figure 1a), a natural polyphenol with abundant catechol and galloyl functional groups for surface coating via metal ion coordination, as a representative polyphenol molecule.46-48 Besides, TA is readily available and inexpensive, and its usage is already approved by the U.S. Food and Drug Administration (FDA). We first fabricated a TAfunctionalized film through coordination-driven assem-

bly.49 The peripheral blood mononuclear cells (PBMCs) adhesion and anti-inflammatory effects on the prepared film were then investigated. For the proof-of-concept study, MCF-7 breast cancer cell line was employed to exam the capture performance of the film, and the influences of chemical groups of the film on the cell capture was further elucidated. Finally, by taking advantage of the differential adhesion preference between PBMCs and cancer cells, a broad-spectrum capture for artificial CTCs from PBMCs on the TA film was achieved. This work provided a new chemical strategy for the effective and label-free capture of CTCs through a facile and low-cost construction of polyphenol-functionalized film. RESULTS AND DISCUSSION Preparation and Characterization of TA Film. TA film was prepared on the basis of the coordination between polyphenol (TA) and FeIII ions (Figure S1, Supporting Information).46 Briefly, TA acted as an organic ligand, while FeIII ions served as an inorganic cross-linker. The adjacent hydroxyl groups of TA provided the chelating sites for the FeIII ions, and the large number of galloyl groups of TA facilitated the efficient coordination-driven assembly. After a simple incubation of substrate in the mixture of the TA and FeIII ions aqueous solutions for several minutes, a three dimensionally cross-linked supramolecular film


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could form. Moreover, due to the general surface binding affinity of TA,50 this coating method could be applied to a wide variety of substrates (i.e., glass, polydimethylsiloxane (PDMS) and silicone tube) regardless of composition and shape (Figure 1b). Furthermore, the resulting film was stable in physiological environment because of the coordination interaction between TA and FeIII ions (Figure S2). Using flat glass slide as a substrate (Figure S3), AFM and SEM analyses of the TA surface morphology revealed a microscopically corrugated surface with short peaks, but appeared flat at a macroscopic level, with a 41.4-nm roughness (Figure 1c and 1d). The chemical composition of the TA film was investigated by XPS (Figure 1e and Table 1). Following the modification of the film on the glass substrate, the Si 2p signal (100.1 eV) decreased while C 1s signal (284.6 eV) increased. Besides, a distinct signal of Fe 2p at ~711 eV with a peak separation of ~12-14 eV was detected, and the Fe EDS signal homogeneously dispersed over the substrate surface (Figure 1d), indicating a uniform film deposition. Considering the structural formula of TA as C76H52O46, the molar ratio of FeIII to TA was calculated to be 1.17 for the TA film (Table 1). Previous study reported the galloyl-FeIII complexes ranged from mono-type to tris-type complexes (one, two and three polyphenol ligands coordinated to a single iron ion, respectively), depending on a given pH.46 The low molar ratio of FeIII to TA on the TA film suggested a large amount of the free galloyl groups on the surface, which was also confirmed by an antioxidant test (Figure S4).51 Table 1. XPS data for surface elemental composition.

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%Si 2p

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55.92 0.72 0.96

35.39 33.49 39.53

8.69 64.79 50.08

0 1.00 9.43

N/A 1.17 14.31

Anti-PBMCs Adhesion and Anti-inflammatory Effects of TA Film. The undesirable leukocyte adhesion not only causes a low capture purity of CTCs but also hinders the use of CTCs for further biochemical assays.3, 7 Therefore, the suppression of leukocyte adhesion on material surfaces is a crucial issue for CTC capture. Existing methods for reducing leukocyte adhesion generally rely on the construction of passive surfaces by employing hydrophilic polymer like poly(ethylene glycol) (PEG)52, 53 and zwitterionic poly(carboxybetaine methacrylate) (pCBMA)54 and inert biological molecules (e.g., bovine serum albumin (BSA)).55 However, these methods not only suffer from multistep treatments and the limited applicability to a given substrate type, but also prevent the adhesion of cancer cells. Inspired by the anti-inflammatory effects of TA,44, 45 which can down-regulate the expression of leukocyte adhesion molecules, as well as the ease and versatility of the TA coating process,46 the anti-PBMCs adhesion property of the TA film was investigated. PBMCs readily adhered to the pristine glass substrate, and the average PBMCs density was ~74 cells mm-2 (Figure 2a). In contrast, the number of adherent PBMCs on the TA film gradually decreased with increasing number of TA coating cycle, and reached a balance of ~11 cells mm-2 after the

fifth coating cycle, similar to the number of leukocyte adhered to the inert BSA surface reported in a previous study.55 We further investigated the levels of proinflammatory cytokines (TNF-α and IL-6) released by PBMCs to evaluate the anti-inflammatory effects of the TA film. In the presence of TA film, the concentration of TNF-α in the medium was 22.5 pg mL-1, a two-fold decrease compared to the concentration measured in the medium containing the glass substrate (Figure 2b). Besides, adding TA (40 μM) to the glass substrate also led to a decrease in TNF-α concentration from 44.9 to 10.3 pg mL-1. Similarly, the concentration of IL-6 in the medium decreased from 2.7 to 2.3 pg mL-1, and to 1.7 pg mL-1 when the PBMCs were treated with the TA film, and 40 μM TA, respectively (Figure 2c). Thus, the TA film exhibited good antiinflammatory property as a result of the incorporation of bioactive TA molecule, thus may open up a new and facile route for designing leukocyte-repellent surfaces. Effect of Galloyl Groups of TA Film on Capture Yields of Cancer Cells. MCF-7 cancer cells (EpCAM-positive) were used to investigate the cell-capture performance of the TA film. Compared to the glass substrate, the TAfunctionalized surface significantly enhanced the cellcapture yield (Figure 3a). Notably, the capture yield gradually increased with increasing number of coating cycle, reaching a maximum after the fifth coating cycle (76.2% ± 5.2%), similar to the capture yield of EpCAMfunctionalized silicon-nanostructured substrate reported in previous studies.13, 54 In particular, the captured MCF-7 cells on the TA film (inset in Figure 3a) not only held larger spreading area but also stretched out more filopodia than those on the glass substrate (Figure S5), revealing an enhanced cell interaction with the TA film. Moreover, no significant difference was found between the capture yields obtained in medium containing 10% serum and in serum-free medium (Figure S6), implying that the chemical composition of the TA film promoted cancer cell adhesion rather than the adsorption of serum proteins. The TA film is composed of two kinds of molecules, TA and FeIII ions, and the TA molecules in the film may comprise of three types of phenolic hydroxyl groups in the forms of phenol, catechol and galloyl groups.46 To exclude the effect of FeIII ions on the efficient capture of cancer cells on the TA film, the three types of phenolic groups (phenol, catechol and galloyl) were modified on the glass substrates directly, and the capture yields obtained by the three surfaces were then determined. The capture yields reached ~34.1% and ~48.1% for the phenol and catecholmodified surfaces, respectively (Figure 3b), while on the galloyl-modified surface, the capture yield increased to ~74.3%, close to that achieved on the TA film. To further verify the contribution of the galloyl groups on the TA film to cell capture, the galloyl groups of the film were blocked by FeIII ion coordination,56 and the resulting film was denoted as TA/Fe film (Figure 3c). XPS analysis of the TA/Fe film showed that after the immersion of FeIII ions, the content of Fe element on the surface of the TA/Fe film increased from ~1.0% to ~9.4%, and the molar ratio of


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Figure 2. Anti-PBMCs adhesion and anti-inflammatory effects of TA Film. (a) Quantitative analysis of PBMCs adhesion on TA film as a function of coating cycle. (b) TNF-α release and (c) IL-6 release of PBMCs after incubation with glass substrate, TA film and glass substrate supplemented with 40 μM TA. Data are presented as mean ± standard deviation, n = 3, error bars are within symbol size if not shown. Statistical analysis was performed by employing one-way ANOVA test, *: p < 0.05, **: p < 0.01, ***: p < 0.001, (N. S.) denotes not significant at p > 0.05.

Figure 3. Effect of phenolic hydroxyl groups on capture yield and focal adhesion of MCF-7 cells. (a) Capture yield of MCF-7 cells on TA film as a function of coating cycle in culture medium. Capture time = 90 min. Inset is pseudocolored SEM image of the captured MCF-7 cells on the TA film the fifth coating cycle. Scale = 10 μm. (b) Capture yield of MCF-7 cells on different phenolic hydroxyl-functionalized surfaces. Capture time = 90 min. (c) Schematic illustration showing the different chemical compositions of TA and TA/Fe films. (d) Representative immunofluorescence images of MCF-7 cells captured on TA and TA/Fe films. The captured cells were costained for the nucleus (DAPI, blue), focal adhesions (Alexa Fluor 488, green) and actin cytoskeleton (rhodamine, red) in the fluorescent images. Scale = 20 μm. (e-g) Nucleus area per cell (e), vinculin area per cell (f), and cytoskeleton area per cell (g) of MCF-7 cells captured on TA and TA/Fe films. Data are presented as mean ± standard deviation, n = 5. Statistical analysis was performed by employing one-way ANOVA test, **: p < 0.01.


toward cancer cells, especially in the test with rare target cells.

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FeIII to TA was calculated to be 14.31 for the TA/Fe film (Table 1), meaning a substantial decrease of the amount of free galloyl groups compared with that of the TA film (Figure S4). This phenomenon could be explained by the fact that the oxygen atoms of the galloyl groups in TA can chelate the heavy metal ions by forming coordination structures.56, 57 The capture yield of the TA/Fe film was dramatically reduced to 12.1% ± 0.9% compared to that of the TA film (~76%) (Figure 3b). Notably, the TA/Fe and TA films were found to have similar physical properties, such as topography, roughness (Figure S7), and surface wettability,56 which could exclude the effects of physical properties on the cell capture (Figure S8). The results above suggested that the chemical groups of the TA film, especially the galloyl groups, played an important role in improving the cell-capture yield. The cytoskeletons and focal adhesions of MCF-7 cells captured on the two films were evaluated by immunofluorescent staining to understand and explore the enhancement mechanism of the TA film in capture yield (Figure 3d and S9). The cells captured on the TA/Fe film remained spherical with few filopodia, indicating a weak interaction between cells and TA/Fe film. However, the cells on the TA film protruded more filopodia that were well distributed across the film, meaning that the cells interacted intimately with the film. Morphometric analysis also revealed a larger nucleus and higher abundance of vinculin (involving in the adhesion strengthening response)58 and cytoskeleton (essential for adhesion, migration and metastasis)59 per cell than those on the TA/Fe film (Figure 3e-g). Taken together, the introduction of galloyl groups effectively enhanced the cell-material interactions, resulting in a high CTC capture yield of the TA film. This enhancement may be attributed to the interaction between glycocalyx on the surface of the cancer cells and the galloyl groups, which could potentially act as an adhesion ligand for the glycocalyx, thereby facilitating the binding between the TA film and cancer cells. Broad-Spectrum Capture of Cancer Cells with High Efficiency. To further optimize the time for optimal cell capture, we evaluated the cell-capture performance of the TA film at different incubation time (Figure 4a). The results showed that the maximal capture yield was achieved after 90 min of incubation with the TA film (~76.2%), and no significant differences in capture yields were found after 90, 120, and 240 min of incubations (p > 0.05). Considering that there was no significant difference between in adherent PBMCs densities on the TA film after 30, 60, 90, 120, and 240 min of incubations (p > 0.05, Figure S10), 90 min was chosen as the incubation time in the subsequent experiments of cancer cell capture. We then performed a cell capture experiment to investigate the capture sensitivity of the TA film using a series of artificial mixture samples spiked with a low number of MCF-7 cells. The number of captured cells was linearly correlated with the number of cells loaded, and this linear relationship was maintained up to 1000 spiked cancer cells (R2 = 0.996) (Figure 4b). Besides, ~73.9% ± 7.7% of the spiked cells could be captured from these samples over a wide range of cell density (Figure 4c). Therefore, the TA film displayed considerable capture yield and sensitivity

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Figure 4. Quantitative evaluation of the capture performance of TA film for MCF-7 cells in culture medium. (a) Capture yield of MCF-7 cells on TA film as a function of capture time. A glass substrate was used as a control. (b) Number of MCF-7 cells captured on TA film as a function of loaded cell number. (c) Capture yield of MCF-7 cell on TA film as a function of loaded cell number. Data are presented as mean ± standard deviation, n = 5, error bars are within symbol size if not shown. In the case of label-dependent CTC capture that relies on EpCAM expression, the main limitation is that the most commonly used capture agent, anti-EpCAM antibody, is only suitable for EpCAM-positive cancer cells, causing a substantial loss of informative CTCs.7 In addition, for efficient cell capture, the anti-EpCAM antibody needs to be modified on a micro or nanostructured substrate (usually hundreds of nanometers to several micrometers) to enhance the local topographic interaction between the substrate and extracellular matrix.13, 14 Therefore, it is necessary to develop a new film material for the efficient capture of broad-spectrum cancer cells on a flat substrate.



Figure 5. Captured yields of various cancer cells (EpCAMpositive cells: MCF-7, A431; EpCAM-negative cells: HeLa, A549) on TA-modified and anti-EpCAM-modified glass substrates. Inset is the schematic representation of antiEpCAM-modified glass. Data are presented as mean ± standard deviation, n = 5, error bars are within symbol size if not shown. The capture performance of TA film modified on a flat glass substrate was measured for EpCAM-positive (MCF-7 and human epidermoid cancer cell (A431)) and EpCAMnegative (human cervical cancer cell (HeLa) and human lung cancer cell (A549)) cancer cell lines. An anti-EpCAMmodified flat glass surface was also evaluated in parallel, and it was found to exhibit low capture yields for all types of cancer cells (0.3% to 5.7%) (Figure 5 and S11). However, the TA film modified on a flat substrate showed vastly improved capture yields for both EpCAM-positive and EpCAM-negative cancer cells without using any capture antibodies (>76%). The surface of TA film is enriched in galloyl groups (three phenolic hydroxy groups tethered to an aromatic ring), and the glycocalyx of cancer cell consists of a variety of glycans and fibrous proteins.39 The phenolic hydroxy groups of the TA film can interact with glycans and proteins through hydrogen bonds,60, 61 and the aromatic rings of the TA film can form hydrophobic interaction with proteins.37, 38 Therefore, the nonselective capture of cancer cells may be attributed to the multiple hydrogen bonds and hydrophobic interactions between the galloyl groups of the TA film and the glycocalyx commonly found on the extracellular matrix of various cancer cell types.

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Figure 6. Schematic illustration of engineering TAfunctionaized film for label-free capture of cancer cells, which integrates the effects of repelling PBMCs adhesion while enhancing cancer cells adhesion. Label-free Capture of Cancer Cells with High Efficiency and Purity. Difference in cell adhesion is a key parameter to enable the separation and enrichment of cancer cells.32, 62 According to the results in Figure 2 and Figure 3, the TAfunctional surface appeared to show a significant preference for the adhesion of cancer cells than for PBMCs. Thus, we proposed that the TA film could potentially act as a label-free platform for the capture of CTCs (Figure 6). Quantitative evaluation of the capture performance of TA film for MCF-7 cells from PBMCs was carried out to explore the potential clinical application of the film (inset in Figure 7a). The correlation between the number of captured cancer cells and loaded cancer cells was almost linear (Figure 7a, R2 = 0.995), similar to the results of the capture experiments performed in culture medium, meaning that adding PBMCs to the cancer cells had little effect on the capture capacity of the TA film. In addition, a considerably high capture yield (~76%) was obtained from the PBMC samples containing MCF-7 cells (Figure 7b), comparable to the capture yield obtained with previous label-free approaches,8, 33 but higher than the commercial Cellsearch technique.63 The captured MCF-7 cells and non-specifically adhered PBMCs were further identified using a typical immunocytochemistry method. DAPI-positive, nucleated cells that also stained positive for cytokeratin were deemed CTCs (Figure 7c). PBMCs were identified as DAPIpositive but cytokeratin-negative nucleated cells. By counting the total number of adherent cancer cells and PBMCs from all acquired images, the purities of the captured cancer cells were determined to be 53.2% ± 11.0%, 68.5% ± 15.8%, and 81.7% ± 2.3%, respectively, for samples spiked with 100, 200, and 400 CTCs, indicating the TA film was capable of efficiently isolating cancer cells from PBMCs with a higher percentage of CTCs compared to the existing label-free approaches for CTC isolation.33 This suggested that TA film might have great potential in clinical application.


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Figure 7. Quantitative evaluation of capture performance of TA film for MCF-7 cells from PBMCs. (a) Number of MCF-7 cells captured on TA film as a function of loaded cell number. (b) Capture yield of MCF-7 cells from PBMCs on TA film as a function of loaded MCF-7 cell number. (c) Representative immunofluorescence images of MCF-7 cells captured from PBMCs (number of loaded MCF-7 cells = 200). The captured cells were costained for nucleus (DAPI, blue) and cytokeratin (FITC, green) in the fluorescent images. Scale = 100 μm. (d) Capture purity of MCF-7 cells from PBMCs on TA film as a function of loaded MCF-7 cell number. Data are presented as mean ± standard deviation, n = 5, error bars are within symbol size if not shown.

Figure 8. Cytocompatibility test of TA film. (a) MTT assay of various cells (EpCAM-positive cells: MCF-7, A431; EpCAMnegative cells: HeLa, A549) captured on TA film. Cells were incubated with TA film for 48 h. (b) Representative fluorescence images of MCF-7 cells captured on TA film after incubation with the film for 0, 12, 24, and 48 h. The viable cells were stained in green by fluorescein diacetate (FDA). Scale = 100 μm. (c) Cell coverage (the area of TA film covered by MCF-7 cells) as a function of incubation time according to the fluorescent images. Data are presented as mean ± standard deviation, n = 5, error bars are within symbol size if not shown. Cytocompatibility and Enabling Culture of Cancer Cells. Downstream molecular biological analysis of CTCs for metastatic potential requires the captured cells be keptalive and cultured.9 Establishing cultures from primary CTCs has also enabled drug or chemo sensitivity to be monitored.64 To assess the cytocompatibility of the TA film, the viabilities of the four types of cancer cells captured on the TA film were first determined by MTT assay. All four cell types exhibited up to ~90% viability (Figure 8a), indicating that the TA film-based CTC isolation method did not harm the cancer cells. This observation was further confirmed by the live/dead cell staining with

fluorescein diacetate/propidium iodide (FDA/PI) dyes (Figure S12), which showed viabilities higher than 95% for the four types of cancer cells captured on the TA film. Then, a continuous culture of the MCF-7 cells captured on the TA film was conducted. The captured cancer cells exhibited good proliferation on the film under prolonged incubation (Figure 8b), with the area of the film covered by the cells increased from 4.3% ± 0.7% to 12.7% ± 2.0%, 28.9% ± 7.1%, and 54.1% ± 11.9% after 12, 24, and 48 h of incubations, respectively (Figure 8c), indicating the excellent cytocompatibility of the TA film.



CONCLUSION In summary, our work provided a new chemical strategy for the capture of CTCs using TA-functionalized film, without the introduction of traditional nanostructured surfaces or any recognition molecules. The film was effective for capturing CTCs (yield > 76%), regardless of the EpCAM expression status on the cell surfaces. This likely originated from the enhanced interaction between the galloyl groups of TA and the glycocalyx on the cancer cell surface, in addition to the anti-inflammatory effects of TA, which could reduce the ability of leukocytes to attach to the surface of the film. The TA-functionalized film also displayed excellent cytocompatibility, thus enabling the cell enrichment without compromising cell viability (> 90%) and the following cell culture. This may be very useful for isolating a large number of cancer cells for anticancer drug assays designed for personalized therapy and to improve the care of cancer patients. Besides, due to its low cost and wide applicability on solid material surfaces with different shapes and compositions, we believe that the TA-functionalized film could provide new clues and opportunities for designing the next-generation devices for CTC capture and detection. EXPERIMENTAL SECTION Materials and Reagents: Tannic acid (TA, Mw = 1701.23 Da), iron (III) chloride hexahydrate (FeCl3·6H2O), rhodamine-conjugated phalloidin and mouse monoclonal anti-vinculin antibody were purchased from Sigma-Aldrich (U.S.A.). 4-Hydroxybenzaldehyde, 3,4dihydroxybenzaldehyde, 3,4,5-trihydroxybenzaldehyde, 3aminopropyl triethoxysilane, sodium borohydride, paraformaldehyde (PFA), glutaraldehyde, 3mercaptopropyl trimethoxysilane, N-maleimidobutyryloxy succinimide ester, fluorescein diacetate (FDA), 4’,6diamidino-2-phenylindole (DAPI) and 2,2-diphenyl-1picrylhydrazyl (DPPH) were bought from J&K Chemical (Beijing, China). Propidium iodide (PI) and methylthiazolyldiphenyl-tetrazolium bromide (MTT) were bought from Beyotime Institute of Biotechnology (Shanghai, China). Alexa Fluor 488-conjugated goat antimouse IgG antibody, fluorescein isothiocyanate (FITC)conjugated monoclonal anti-cytokeratin 8 antibody and biotinylated monoclonal anti-EpCAM antibody were purchased from Abcam (Cambridge, UK). Fetal bovine serum (FBS), penicillin-streptomycin, streptavidin, trypsin-ethylenediaminetetraacetic acid (0.25%), highglucose Dulbecco's Modified Eagle's Medium (DMEM) with L-glutamine and Roswell Park Memorial Institute (RPMI) 1640 medium with L-glutamine were purchased from Thermo Scientific (Waltham, MA). Phosphate buffer saline (PBS, pH 7.4) was bought from GE Healthcare Life Sciences (Logan, UT). All other reagents were of analytical grade. The water used in all experiments was obtained from an ultrapure water purification system (Millipore, U.S.A.) with a resistivity of 18.2 MΩ.cm. All aqueous solutions were filtered with 220 nm diameter membranes before use. Sample Preparation: Flat glass slides were cut into 1 cm × 1 cm pieces and used as substrates for film fabrication. Prior to surface modification, the substrates were immersed in boiling piranha solution (H2SO4 (98%):H2O2 (30%) = 3:1, v/v) for 30 min to oxidize the organic

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residues (Caution: piranha solution reacts violently with most organic materials and must be handled with extreme care!). After that, the substrates were washed with ultrapure water and dried with nitrogen gas. TA surface: The TA film was fabricated on the basis of the coordination between polyphenol and metal ions.49 Briefly, the substrate was first placed in a tube, and 1 mL of FeCl3·6H2O aqueous solution (0.8 mg mL-1) and 1 mL of TA aqueous solution (3.2 mg mL-1) were then successively added to the tube, followed by gentle shaking for 3 min. Subsequently, the substrate was removed and thoroughly rinsed with ultrapure water. This coating process was repeated five times, leading to the formation of the TA film. TA/Fe surface: To block the phenolic hydroxyl groups on the TA film, the as-prepared TA film was immersed in FeCl3·6H2O aqueous solution (0.8 mg mL-1) at room temperature for 4 h.56 Afterward, the film was washed with ultrapure water and dried with nitrogen gas, and the resulting film was denoted as the TA/Fe film. Phenolic hydroxyl-functionalized surfaces: Phenol, catechol and galloyl groups were introduced onto the substrates through nucleophilic addition. Typically, the piranha solution-treated glass substrates were first immersed in 3-aminopropyl triethoxysilane solution (0.01% in ethanol) at room temperature for 2 h. After washing with ethanol under ultrasonication, the silanized substrates were reacted with 4-hydroxybenzaldehyde (1% in ethanol), 3, 4-dihydroxybenzaldehyde (1% in ethanol) and 3, 4, 5-trihydroxybenzaldehyde (1% in ethanol) for 2 h, and then washed with ethanol followed by ultrapure water. Subsequently, they were treated with a solution of sodium borohydride at a concentration of 1.0 mg mL-1 for 30 min. The substrates were then taken out, thoroughly washed with ultrapure water and dried with nitrogen gas. Surface Characterization: All the samples were completely vacuum-dried prior to characterization. AFM experiments were carried out with a Dimension Icon AFM (Bruker, U.S.A.). The roughness and morphology of the samples were analyzed using NanoScope Analysis software. XPS experiments were 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 to the neutral adventitious C 1s peak at 284.6 eV. SEM images were recorded 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. Energy dispersive X-ray spectroscopy (EDS) profiles were acquired by using an Oxford X-Max EDS detector. Cell Culture: Breast cancer cell line MCF-7, epidermoid cancer cell line A431, cervical cancer cell line HeLa, and lung cancer cell line A549 were purchased from Shanghai Institute for Biological sciences, Chinese Academy of Science. The cells were cultured in medium (DMEM for MCF-7, A431 and HeLa; RPMI 1640 for A549) supplemented with 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin. These cells were maintained in a humidified incubator at 37 °C and in the presence of 5% CO2.


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Blood Collection and Processing: Blood was drawn from healthy donors and collected in 5-mL blood collection tubes containing sodium citrate as an anticoagulant (All protocols pertaining 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 Ficoll-Paque (Beijing Solarbio Co. Ltd., China). Every 1 mL of whole blood yielded ~100 μL of isolated PBMCs. PBMCs Adhesion and Anti-inflammatory Effects: The TA film was first placed inside a 24-well cell culture plate, and 0.5 mL of PBMCs suspension was then added to the film surface. After incubation at 37 °C for 90 min, the film was washed three times with PBS. For the PBMCs adhesion assay, the adherent PBMCs were fixed with 4% paraformaldehyde in PBS for 30 min, and then stained with DAPI (10 µg mL-1 in PBS) for 30 min in the dark. After washing with PBS, the adherent PBMCs were observed under an Olympus IX71 fluorescence microscope. The numbers of adherent PBMCs from five randomly taken images were counted using the software of Image-Pro Plus. For the anti-inflammatory test, the PBMC suspensions were centrifuged and the supernatants were harvested for further cytokines tests according to the instruction of TNFα/IL-6 ELISA kit (Beyotime Institute of Biotechnology, China). For the positive control, 10 μL of TA solution (final concentration: 40 μM) was added into the PBMCs suspensions to induce anti-inflammatory effects. Anti-EpCAM Antibody Coating: Glass substrates were functionalized with anti-EpCAM antibodies using avidinbiotin chemistry.17 Briefly, the piranha solution-treated glass substrates were modified with 4% (v/v) 3mercaptopropyl trimethoxysilane in ethanol at room temperature for 45 min. After being rinsed with ethanol, the substrates were treated with N-maleimidobutyryloxy succinimide ester (GMBS, 0.25 mM) in DMSO for 30 min. The substrates were then incubated with 10 µg mL-1 of streptavidin solution in PBS at room temperature for 30 min, leading to immobilization of streptavidin onto GMBS. The substrates were washed with PBS, air-dried, and stored at 4 °C till later use. Before the cell capture experiments, biotinylated anti-EpCAM antibody (10 µg mL1 in PBS with 1% (w/v) bovine serum albumin and 0.09% (w/v) sodium azide) was added to the substrates for 30 min for the specific recognition targeted cancer cells. Cell Capture Experiments: Cell capture experiments were performed in culture medium and isolated PBMCs suspensions. For the capture assays in culture medium, the samples were first placed into a 24-well cell culture plate, and 0.5 mL of cancer cell suspensions (1 × 105 cells mL-1) were then loaded. After 90 min incubation at 37 °C and 5% CO2, the samples were washed three times with PBS. The captured cells on the surfaces of the samples were fixed with 4% paraformaldehyde in PBS for 30 min. After that, the samples were incubated with a DAPI solution (10 µg mL-1 in PBS) for 30 min, followed by three times of PBS washes. Finally, the capture cells were observed and counted with a fluorescence microscope. To avoid surface blocking caused by plasma proteins and the impediment of dense blood cells, PBMCs isolation was performed by blood fractionation. For capture assays in

isolated PBMCs suspensions, a known number of MCF-7 cells were first mixed with the suspensions, and then incubated with the samples at 37 °C and 5% CO2 for 90 min. Afterward, the captured cells were washed three times with PBS, fixed with 4% paraformaldehyde in PBS for 30 min, permeabilized with 0.2% Triton-X 100 in PBS for 5 min, and incubated with 1% BSA in PBS for 30 min at room temperature. After that, the captured cells were incubated with fluorescein isothiocyanate (FITC)conjugated monoclonal anti-cytokeratin 8 antibody (10 µg mL-1 in PBS) at 37 °C for 1 h. Then, the cells were maintained with DAPI solution (10 µg mL-1 in PBS) for 30 min. Finally, immunofluorescence images of the captured cells were recorded under a confocal laser scanning microscope (Olympus FV1000). MCF-7 cells captured on the substrate were defined as the cells of dual stains (blue: DAPI+ and green: cytokeratin+), and PBMCs were defined as the cells of single stain (blue: DAPI+ and green: cytokeratin-). SEM Specimen Preparation: After the cell capture experiments, the MCF-7 cells captured on the samples were washed with PBS, and fixed with glutaraldehyde (2.5% in PBS) for 2 h at room temperature. The cells were then dehydrated through a series of ethanol concentrations (30%, 50%, 70%, 80%, 90%, and 100%, 15 min each). Afterward, the cells were dried with liquid CO2 using a supercritical point dryer to maintain the morphologies of the captured cells. After complete drying, the samples were sputter coated with gold, and observed and photographed with a Quanta 450 SEM (FEI, U.S.A.). Immunofluorescence Staining for Actin Cytoskeleton and Focal Adhesions of Captured Cells: MCF-7 cells were first incubated with the samples for 90 min, followed by washing with PBS for three times. Then, the captured cells were sequentially treated with 4% paraformaldehyde in PBS for 30 min, 0.2% Triton X-100 in PBS for 5 min, and 1% BSA in PBS for 30 min at room temperature. After that, the cells were incubated with a primary antibody (antivinculin produced in mouse, diluted 1:400 in PBS) for 1 h at 37 °C, and then stained with a secondary antibody (Alexa Fluor 488 conjugated goat anti-mouse IgG, diluted 1:100 in PBS) for 1 h at 37 °C. Then, rhodamine-conjugated phalloidin (200 nM in PBS) and DAPI (10 µg mL-1 in PBS) were used to visualize the cytoskeleton and nucleus, respectively. Finally, immunofluorescence images of the captured cells were recorded under a confocal laser scanning microscope. The spread area (nucleus, cytoskeleton and vinculin) of each cell from five randomly taken images were quantitatively analyzed using the software of Image-Pro Plus. Cell Viability Assay: The viability of cancer cells captured on the TA film was investigated by MTT assay and Live/Dead staining method. For the MTT assay, the cells were seeded in a TA-modified 96-well plate at a density of 1 × 104 cells well-1 and cultured in growth medium for 48 h at 37 °C in 5% CO2. After washing with PBS, 180 μL of the medium and 20 μl of MTT solution (5 mg mL-1 in PBS) were added to the wells. The plates were further incubated at 37 °C in 5% CO2 for 4 h. After the addition of 130 μL of DMSO solution, the absorbance at 570 nm was measured with a plate reader (Thermo Scientific, U.S.A). The



absorbance of control wells without MTT was subtracted, and the relative cell viability was normalized relative to the untreated control cells. For the Live/Dead staining experiment, FDA/PI working solution was first prepared by mixing 1 μg mL-1 FDA solution and 0.5 μg mL-1 PI solution at a volume ratio of 1:1. Then, the cancer cells captured on the TA film were stained by incubating with 0.5 mL of the FDA/PI working solution for 5 min. Cell viability was then determined by fluorescence microscopy analysis. Cell Proliferation Assay: To test the cytocompatibility of the TA film, MCF-7 cells captured on the TA film were subjected to a long-term culture. Briefly, after the cell capture experiments, the film with the captured cells was immersed in fresh DMEM growth medium for culture (0 h). After a certain time of incubation (12, 24, and 48 h), the cells were stained by a FDA solution (1 μg mL-1) for 5 min, and then observed under a fluorescent microscope. The cell coverage, the ratio of the area of TA film covered by cancer cells to the total area of TA film, was calculated using the software of ImageJ. Statistical Analysis: All experiments were repeated at least three times (n ≥ 3), with each experiment done in triplicates, and the mean value (± standard deviation, SD) was reported. All data were compared using 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.

ASSOCIATED CONTENT Supporting Information. Schematic illustration of TA coating procedure; Stability test of TA film; Physiochemical characterization of glass substrate; Antioxidant abilities of TA and TA/Fe films. Immunofluorescence and SEM images of MCF-7 cells captured on glass substrate; Capture yields of MCF-7 and HeLa cells on TA films obtained in DMEM supplemented with 10% FBS and with 0% FBS; Physiochemical characterization of TA/Fe film; Capture performance of a smoother polyphenol-functionalized film for MCF-7 cells; Immunofluorescence images of MCF-7 cells captured on TA and TA/Fe films; PBMCs adhesion on TA film as a function of incubation time; Fluorescence images of various cancer cells captured on TA-modified and anti-EpCAMmodified glass substrates; Viability test of captured cancer cells. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Email: [email protected]

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors acknowledge the financial support of the National Natural Science Foundation of China (Project No. 51773026), the Fundamental Research Fund for the National Key Technologies R&D Program (2016YFC1103002) and the Fundamental Research Fund for the Central Universities (DUT17LAB06). The authors thank Dr. Alan K. Chang for editing the language of the manuscript.

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