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Degradable Zinc Phosphate-Based Hierarchical Nanosubstrates for Capture and Release of Circulating Tumor Cells Shan Guo, Jiaquan Xu, Min Xie, Wei Huang, Erfeng Yuan, Ya Liu, Liping Fan, Shi-Bo Cheng, Songmei Liu, Fubing Wang, Bifeng Yuan, Wei-Guo Dong, Xiao-Lian Zhang, Wei-Hua Huang, and Xiang Zhou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04002 • Publication Date (Web): 06 Jun 2016 Downloaded from http://pubs.acs.org on June 7, 2016

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ACS Applied Materials & Interfaces

Degradable Zinc Phosphate-Based Hierarchical Nanosubstrates for Capture and Release of Circulating Tumor Cells Shan Guo,†,◊ Jiaquan Xu,‡ ,◊ Min Xie,‡ Wei Huang, ‡ Erfeng Yuan,§ Ya Liu,# Liping Fan,‡ Shibo Cheng,‡ Songmei Liu,§ Fubing Wang,§ Bifeng Yuan,† Weiguo Dong,# Xiaolian Zhang,ǁ Weihua Huang,*, ‡ and Xiang Zhou*, † † College of Chemistry and Molecular Sciences, the Institute for Advanced Studies of Wuhan University, Wuhan University, Wuhan 430072, China ‡ Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China § Zhongnan Hospital, Wuhan University, Wuhan430072, China # Renmin Hospital of Wuhan University, Wuhan 430060, China ǁ State key laboratory of Virology, Department of Immunology, School of Medicine, Wuhan University, Wuhan 430072, China KEYWORDS hierarchical nanosubstrates, zinc phosphate, degradable, circulating tumor cells, capture, release, molecular analysis

ABSTRACT Circulating tumor cells (CTCs) play a significant role in cancer diagnosis and personalized therapy, and it is still a signifaicant challenge to efficiently capture and gently

ACS Paragon Plus Environment

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release CTCs from clinic samples for downstream manipulation and molecular analysis. Many CTC devices incorporating various nanostructures have been developed for CTC isolation with sufficient capture efficiency, while fabricating such nanostructured substrates often require elaborate design and complicated procedures. Here we fabricate a degradable zinc phosphatebased hierarchical nanosubstrate (HZnPNS), and demonstrate its excellent CTC-capture performance along with effective cell-release capability for downstream molecular analysis. This transparent hierarchical architecture prepared by a low-temperature hydrothermal method, enables substantially enhanced capture efficiency and convenient imaging. Biocompatible sodium citrate could rapidly dissolve the architecture at room temperature, allowing that 88 ± 4% of captured cells are gently released with a high viability of 92 ± 1%. Furthermore, anti-epithelial cell adhesion molecule antibody functionalized HZnPNS (anti-EpCAM/HZnPNS) was successfully applied to isolate CTCs from whole blood samples of cancer patients, as well as release CTCs for global DNA methylation analysis, indicating it will serve as a simple and reliable alternative platform for CTC detection.

INTRODUCTION

Circulating tumor cells (CTCs), which are shed from either primary tumor masses or metastatic sites, circulate in blood in the early stage and establish distant metastatic lesions at new organ sites.1 Several studies have shown that the number of CTCs in the blood is correlated with the prognosis of cancer patients and that monitoring the change of CTC numbers is useful to assess metastatic risk and response to treatment.2 Furthermore, molecular analysis of CTCs can provide more valuable and significant diagnostic-relevant information, which is desired to guide therapy


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and ultimately realize personalized treatment.3,4 However, as a result of the extremely low abundance and heterogeneity of CTCs, their isolation and detection are technically challenging. To date, a variety of approaches mainly including immunomagnetic beads,5-13 microfluidic devices,14-28 and nanostructured substrates29-38 have been developed for enumerating and isolating CTCs. Immunomagnetic bead-based approaches commonly rely on magnetic beads with biological specificity and fast magnetic response to recognize and separate CTCs from blood. The procedure is simple but suffers from relatively low capture efficiency and purity. Microfluidic-based devices affording the advantage of precise flow control and parameter manipulation allow for increased purity, yield and sensitivity. However, complicated fabrication processes are usually needed to prepare microfluidic devices. Nanostructured substrates integrated with the scale of extracellular structures, high surface-area-to-volume ratio, and increased capture agent presentations, have been developed and emerged as an ultrasensitive platform in CTC isolation. Currently, approaches such as lithography,30 etching,29,31 electrospinning,32,33 and electrochemical deposition34 have been used for fabricating various nanostructured substrates. However, these methods often require complex techniques and sophisticated and delicate manipulation. Recent results showed that hierarchical nanostructured substrates exhibited significantly enhanced cancer cell-capture efficiency,34,35 compared to single vertical nanostructures (like nanopillars29 and nanodots36) and single horizontal nanostructures (like nanofibers23,32). These substrates topographically matched better with the filopodia of cancer cells and provided more binding sites for cell capture.35 However, it is still a challenge to engineer a hierarchical nanostructured substrate with optimized performance for CTC detection. In addition to pursuing satisfied performance of CTC capture, gentle release of CTCs from substrates for further culture and downstream molecular analysis is another critical issue. Cell


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detachment has been achieved by a variety of approaches such as enzyme degradation,15,22 photosensitive-induced

cleavage,11,39,40

electrochemical

desorption,34,41

thermodynamic

release,42,43 chemical reagent-triggered substrate sacrifice,44 and competition-based ligand replacement,9,10,45 and so on. However, majority of these methods have certain limitations such as low release efficiency, poor cell viability and limited processing conditions. Furthermore, many nanostructured substrates for CTC isolation are relatively opaque, which is not convenient for direct observation, imaging and manipulation. Therefore, the development of transparent and versatile hierarchical nanosubstrates with effective cell-capture and -release properties is significantly essential and desired for CTC analysis. Here, we fabricated a transparent and degradable HZnPNS for capture and release of CTCs from whole blood samples (Scheme 1). Via a simple low-temperature hydrothermal method, zinc oxide nanowires (ZnO NW) grown on transparent glass substrates were transformed into flowerlike HZnPNS. Then, streptavidin (SA) was covalently introduced onto the surface of HZnPNS to facilitate the bioconjugation with biotinylated anti-EpCAM. Thus, antiEpCAM/HZnPNS simutaneously possessing affinitive and topographical interaction with the surface of cancer cells enabled effective isolation of rare cancer cells with a high purity. Such hierarchical nanosubstrates could be rapidly dissolved by biocompatible sodium citrate at room temperature, allowing gentle and efficient release of captured cancer cells with a high viability. Finally, anti-EpCAM/HZnPNS was used to sensitively detect CTCs from blood samples of cancer patients, as well as release captured CTCs for downstream global DNA methylation analysis. These results indicate that transparent and degradable HZnPNS will serve as a simple and reliable bio-platform for CTC analysis.


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Scheme 1. Schematic diagram illustrating the fabrication process of HZnPNS and antiEpCAM/HZnPNS for capture, release and molecular analysis of CTCs.

MATERIALS AND METHODS

Materials

and

instruments.

Zinc

acetate

dehydrate,

zinc

nitrate

hexahydrate,

hexamethylenetetramine and sodium citrate were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Carboxyethylsilanetriol Na salt (25% in water) was purchased from J&K Scientific Co. Ltd.. 10 mM PBS (pH 7.4, containing 82 mM NaCl, 2.7 mM KCl) was prepared in our facilities. 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), biotin (5-fluorescein) conjugate, 4’, 6-diamidino-2-phenylindole (DAPI), FITC-labelled goat anti-mouse secondary antibody, and streptavidin (SA) were purchased from Sigma-Aldrich. Biotin-labelled anti-human CD326 (EpCAM) monoclonal antibody was obtained from eBioscience. FITC-labelled anti-cytokeratin 19 antibody and PE-labelled anti-CD45 antibody were purchased from Abcam Company. Apoptosis detection kits were obtained from the Beyotime Institute of Biotechnology. Nucleic acid stain SYTO@13 and DiI282 were obtained from Invitrogen Corp.. Red blood cell lysing buffer was purchased from Boster company


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(Wuhan, China). Blood samples were obtained from Zhongnan Hospital of Wuhan University, except metastatic cancer patients' blood samples for CellSearch assay collected by Hubei Cancer Hospital. All the media of cell culture were purchased from Gibco Corp.. 4-well NUNC LabTekTMⅡChamber Slides were purchased from Thermo Fisher Scientific. UV−Vis absorption spectra were measured with an UV−Vis spectrophotometer (UV2550, Shimadzu). SEM images were obtained by a field-emission scanning electron microscope (Zeiss SIGMA). Inductively coupled plasma-atomic emission spectrometry (ICP-AES) was used to measure the composition of the dissolution products of HZnPNS. Electron diffraction spectroscopy (EDS, X-Max, Oxford) was taken to examine the elemental mapping of the substrates. X-ray diffraction (XRD) analysis was performed on a Rigaku SmartLab 9 kW X-ray diffractometer at room temperature, in Bragg-Brentano geometry employing Cu Kα lines focused radiation (1.54059 Å, 1.54439 Å) at 9 kW (45 kV, 200 mA) power. Fluorescence microscopy (AxioObserver Z1, Zeiss, Germany) was used to image and identify cells. CellSearch assay was tested by Celltracts analyzer II®. Analysis of DNA methylation was performed on liquid chromatography−electrospray ionization−tandem mass spectrometry (LC-ESI-MS/MS) system consisting of an AB 3200 QTRAP mass spectrometer (Applied Biosystems, Foster City, CA, USA) with an electrospray ionization source (Turbo Ionspray) and a Shimadzu LC-20AD HPLC (Tokyo, Japan) with two LC-20AD pumps, a SIL-20A autosampler, a CTO-20AC thermostated column compartment, and a DGU-20A3 degasser. Preparation of flowerlike HZnPNS. Glass slides were first washed by ethanol and dried by nitrogen stream. Then, via a modified previous method,46 zinc oxide nanowires (ZnO NW) were grown on the clean glass surface. Specifically, zinc oxide nanocrystals as a seed layer grew in aqueous solution containing 25 mM zinc nitrate hydrate and 25 mM hexamethylenetetramine at


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90 °C for 3 h. Subsequently, ZnO NW substrates (ZnO NWS) with a dimension of 1 cm × 2 cm were put into PBS containing 10 mM Mg2+ for 48 h at 37 °C. Finally, glass slides were entirely coated with flowerlike hierarchical architecture. In addition, another two solutions, PBS and solution containing Mg2+ without phosphate ions were prepared as control for the treatment of ZnO NWS. Modification of HZnPNS surfaces with anti-EpCAM antibody. HZnPNS (1 cm × 2 cm) was first treated with plasma for 1–2 min to activate the surface, and then was incubated with 2 mL of PBS containing 40 µL of carboxyethylsilanetriol for 3 h. After washing, carboxylic groups modified HZnPNS was obtained. Subsequently, the carboxylated substrates were immersed in a boric acid buffer (10 mM, pH 7.4) containing 25 mM EDC solution and incubated with SA (0.5 mg/mL, 200 µL) on ice for 4 h. To verify whether SA was successfully conjugated with carboxylated substrates, FITC-labelled biotin (50 µg/mL, 20µL) was incubated with SA-coated HZnPNS (SA/HZnPNS) or pure HZnPNS for 30 min, respectively. Finally, SA/HZnPNS was incubated with 0.5 mg/mL biotin-labelled anti-EpCAM for 30 min. Similarly, FITC-labelled goat anti-mouse secondary antibody was incubated with anti-EpCAM/HZnPNS or SA/HZnPNS for 30 min to evaluate whether HZnPNS was successfully functionalized with anti-EpCAM. Experiments of cancer cell capture and release. Prior to cell capture, SA/HZnPNS were incubated with 200 µL of biotinylated anti-EpCAM (10 µg/mL in PBS) for 30 min followed by rinsing with PBS. Then, a solution of 5% (50 mg/mL) bovine serum albumin and 0.2 % Tween20 in PBS was used to passivate the surface and reduce the non-specific cell adhesion. Cellcapture experiment was performed using the simple and convenient procedures described in a previous report.29 Anti-EpCAM/HZnPNS and control substrates (SA/HZnPNS and antiEpCAM/ZnO NWS) were placed into 4-well chamber slides, and then 0.5 mL of MCF-7 cell


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suspension (105 cell/mL) pre-stained by SYTO@13 nucleic acid dye (with a final concentration of 50 nmol/L) was loaded. After incubation for 45 min in a cell incubator, the unbound cells were gently removed from the substrates by shaking at 90 rpm for 2 min twice. Finally, fluorescence microscopy was employed to image and count the captured cells. Here, Hela cells pre-stained by SYTO@13 nucleic acid dye and incubated with Anti-EpCAM/HZnPNS were used as a negative control. For the cell-release experiment, we first investigated the degradation of HZnPNS treated by 1% sodium citrate in PBS (pH 6.5) at room temperature for 0 min, 5 min, 10 min, 15 min and 20 min. To clearly visualize citrate-assisted substrate dissolution, HZnPNS was labelled by FITC. FITC-labelled HZnPNS was produced by incubation SA/HZnPNS with FITC-labelled biotin for 0.5 h, and then the substrate was washed by PBS (pH7.4). Due to FITC is a pH-sensitive fluorophore and its quantum yield is close to zero at pH’s lower than 4,47 prior to imaging each time, substrates after incubation with sodium citrate in PBS (pH 6.5) were washed by PBS (pH 7.4) to reduce the effect of pH on the fluorescent intensity. To further confirm that 1% sodium citrate (pH 6.5) could dissolved as-prepared HZnPNS, ICP-AES was used to measure whether Zn, P and Mg elements existed in the dissolution product. To avoid the P element of PBS, HZnPNS (2.6 cm × 7.6 cm) was put into 10 mL of water containing 1% sodium citrate (pH 6.5) for 20 min. Then the solution was filtered and analyzed by ICP-AES. Finally, antiEpCAM/HZnPNS after capturing cancer cells was treated with optimized substrate-dissolution conditions, allowing the release of captured cancer cells from hierarchical nanosubstrates. Cells remaining on substrates were imaged and counted for calculating the release efficiency. The viability assay of retrieved cancer cells. First, methylthiazoletetrazolium (MTT) assay was employed to investigate the toxicity of sodium citrate. Different concentrations of sodium


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citrate in PBS ranging from 0.0625% to 2% (g/100 mL) were incubated with MCF-7 cell for 30 min at room temperature. Additionally, optimized cell-release condition (1% sodium citrate in PBS, pH 6.5) was also tested. To evaluate retrieved cell viability in detail, an annexin V-FITC kit was utilized for apoptotic analysis of released cells as illustrated in the manufacturer’s protocol. After staining, cells were immediately imaged by fluorescence microscopy to calculate the viability percentage. Meanwhile, retrieved cells were collected and further cultured. After cultivation for 48 h, cells were stained with calcein-AM/PI. Live cells were stained by calceinAM and dead cells were labelled by PI. Rare cancer cell capture from lysed blood samples. To prepare artificial CTC samples, healthy human whole blood was first treated with red blood cell lysing buffer according to the manufacturer's instructions. Then, a series of artificial blood samples were prepared by spiking DiI282-stained MCF-7 cells into lysed blood at densities of 12, 60, 477 and 3700 cells per 0.5 mL. The same amount of cell suspension was distributed on three wells in 96-well plate to calculate the mean of cells spiked into blood samples. Cell-capture experiments were operated as the conditions of pure cancer cell capture, the isolated MCF-7 cells were imaged and counted to quantify the recovery rate. To quantify the purity, captured cells on anti-EpCAM/HZnPNS were fixed with 4% paraformaldehyde (20 min), permeabilized with 0.1% Triton-X 100 (20 min), blocked with 5% BSA (30 min), and stained with 20 µg/mL DAPI, FITC-labelled anti-CK19 monoclonal antibody and PE-labelled anti-CD45 monoclonal antibody (30 min). MCF-7 Cells were identified as DAPI+/ CK+/ CD45- and WBCs were DAPI+/ CK-/ CD45+. Isolation of CTCs from cancer patient blood samples. To test the potential application of antiEpCAM/HZnPNS for CTC isolation from whole blood samples, we collected 13 patient blood samples (the volume of each blood sample was 1.0 mL). Then, 1.0 mL whole blood was loaded


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on as-prepared anti-EpCAM/HZnPNS, and incubated on ice for 45 min. After washing by PBS (the majority of red blood cells was washed away and the color of substrates was changed from red to colorless), captured cells were immunostained as above. CTCs were DAPI+/ CK+/ CD45and WBCs were DAPI+/ CK-/ CD45+. In addition, we compared the performance of antiEpCAM/HZnPNS with CellSearch assay for CTC isolation. 5 patient blood samples with metastatic breast, prostate, or colorectal cancer were collected. For each patient blood sample, 7.5 mL of whole blood was examined by CellSearch, and 1.0 mL of whole blood was tested by anti-EpCAM/HZnPNS. Isolated CTCs were imaged and enumerated by immunostaining. DNA methylation analysis of retrieved CTCs by LC-ESI-MS/MS. To analyze DNA methylation of CTCs, captured CTCs from each breast cancer patient’s whole blood (the volume of each blood sample was 1.0 mL, n=11) were released from anti-EpCAM/HZnPNS by optimized cell-release conditions and collected in a tube. The collected cell suspension was first centrifugated at 1000 rpm for 8 min, and then concentrated cells were washed by PBS once. Finally, retrieved cells were dispersed in 20 µL of sterilized water. Meanwhile, to obtain control blood cells, we collected 11 healthy human blood samples with matched genders and ages corresponding to breast cancer patients. To reduce the influence of experimental operation, healthy human blood samples were treated similar to cancer patient blood samples. 1.0 mL of each blood sample was loaded on carboxylic group modified HZnPNS and incubated on ice for 45 min. Thus, blood cells non-specifically adhered on the substrates. After washing, blood cells from healthy human blood were released and retrieved in 20 µL of sterilized water. Then, the retrieved CTCs from breast cancer patients and blood cells from healthy controls were treated by a reported strategy to quantitatively analyze DNA methylation by LC-ESI-MS/MS.48 Briefly, small amounts of cells in 15 µL of sterilized water were first incubated at 90 °C for 10


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min to break the cells, then proteinase K (2 µL, 20 mg/mL) was added followed with incubating at 58 °C for 1 h. The resulting sample was then incubated at 95 °C for 15 min to denature DNA as well as inactivate proteinase K. After adding 2 µL of S1 nuclease buffer (30 mM CH3COONa, pH 4.6, 280 mM NaCl, 1 mM ZnSO4) and 100 units of S1 nuclease, the mixture (20 µL) was incubated at 37 °C for 4 h. The solution was subsequently added with 4 µL of alkaline phosphatase buffer (50 mM Tris-HCl, 10 mM MgCl2, pH 9.0), 0.0008 units of venom phosphodiesterase I, 6 units of alkaline phosphatase, and 14.7 µL of H2O. And then the incubation was continued at 37 °C for an additional 1 h followed by adding 160 µL of sterilized water and extraction with an equal volume of phenol/chloroform (v/v, 1:1) and chloroform once. The resulting aqueous layer was collected and lyophilized to dryness, and then reconstituted in 100 µL water for subsequent LC-ESI-MS/MS analysis. Data acquisition and processing were performed as our previous report.48 The contents of modified nucleoside were calculated using the following expressions: 5-mdC % = M5-mdC/MdG × 100% where M5-mdC is the molar quantity of 5-mdC, while MdG is the molar quantity of dG determined in samples. Statistical analyses were performed using SPSS 16.0 software (SPSS Inc.). Student's unpaired t test was used to assess the differences of DNA methylation between retrieved CTCs from breast cancer patients and the corresponding blood cells from healthy controls. P value was two-sided, and p < 0.05 was considered to be statistically significant.

RESULTS AND DISCUSSION


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Fabrication of flowerlike HZnPNS. A considerable number of studies showed that nanostructured substrates facilitated cell attachment relying on the enhanced local topographical interaction.29-38 Furthermore, fractal nanostructures have been found on the cell surface and the fractal dimensionality of cancerous cells is higher than that for normal cells.49,50 In this work, we aim to fabricate a novel hierarchical nanostructured substrate with similar fractal dimensions to the surface of cancer cells. ZnO NW substrates (ZnO NWS) prepared under mild aqueous conditions (Figure 1a),46 were selected as the initial material to obtain transparent hierarchical nanostructured substrates. Previous study showed that ZnO could be substituted by bivalent magnesium (Mg) metals, causing dramatic structure changes.51 Additionally, ZnO is sensitive towards phosphate ions even at neutral pH, and the interaction between ZnO and phosphate ions could dissolve and transform ZnO into zinc phosphate.52 Our results show that stable flowerlike hierarchical nanosubstrates with fractal dimensions (Figure 1b and S1a) are generated by immerging ZnO NWS in physiological PBS containing 10 mM Mg2+ at 37 °C. However, incubation of ZnO NWS in PBS without Mg2+ produces a relatively smooth substrate without more complex details (Figure S1b). Additionally, in the presence of Mg2+ without phosphate ions, ZnO NWS does not have visible surface change (Figure S1c). These results indicate that Mg2+ and phosphate ions are two critical factors responsive for generation of flowerlike hierarchical nanosubstrates.


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Figure 1. Characterization of HZnPNS. (a, b) SEM images of ZnO NWS and HZnPNS. Inset: height of ZnO NW and HZnPNS. (c) XRD spectra of ZnO NWS (black line) and HZnPNS (red line). Red numbers represent orthorhombic hopeite. Black numbers represent wurtzite ZnO. (d) EDX analysis of HZnPNS. (e) The element mapping of HZnPNS. The scanning area is labelled by a yellow dot line in figure (b). To confirm whether there was a crystalline transformation of ZnO NW in the copresence of phosphate ions and Mg2+, x-ray diffraction (XRD) analysis was used to characterize as-prepared hierarchical nanostructures. XRD spectra show that wurtzite ZnO (PDF#36-1451) and orthorhombic hopeite (Zn3 (PO4)2·4H2O) (PDF#33-1474) mainly coexist in the substrates (Figure 1c). Thus, we denote this zinc phosphate-based hierarchical nanosubstrate different from ZnO NWS as HZnPNS. For substrates formed in PBS without Mg2+, triclinic parahopeite (Zn3 (PO4)2·4H2O) (PDF#39-1352), another polymorphs of zinc phosphate occurring as tetrahydrate,53 is found on the spectrum (Figure S1d). According to previous study,52 these


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transformation products are similar to that of ZnO nanoparticles dissolved by phosphate. As another control, in the presence of Mg2+ without phosphate ions, there is not any change in the crystalline phase of ZnO NW (Figure S1e). To make a further understanding about the composition of hierarchical nanostructures, energy dispersive x-ray spectroscopy (EDXS) was used to analyze the elements. In addition to Zn and O elements, both P and Mg elements are measured on HZnPNS (Figure 1d, S1f and S1g). Figure 1e shows that these elements are homogeneously distributed on HZnPNS. Taken together, ZnO NW could be partially transformed into zinc phosphate by phosphate ions, and Mg2+ plays an important role in fabricating HZnPNS with multi-scale hierarchical architecture. As an added benefit, as-prepared HZnPNS is transparent ranging from 400-800 nm as shown in the UV-Vis spectrum (Figure S1h). The absorption peak of ZnO (around 368 nm) appears on the spectrum. The maximum transmittance is up to 65%, and we could clearly see the cells on transparent HZnPNS from microscopy (Figure S1i). Cell-capture capability of anti-EpCAM/HZnPNS to cancer cells. Before conducting the cellcapture experiment, we investigated the biocompatibility of as-prepared HZnPNS for cancer cells. As shown in Figure S2, MCF-7 cells spread well on HZnPNS with fractal dimensions after 24 h cultivation. However, the majority of cells on ZnO NWS remain spherical although cells are viable, which is consistent with previous report about cell spreading on ZnO NWS.54 Therefore, compared to ZnO NWS, HZnPNS is more biocompatible and better facilitates cell adhesion possibly owing to its fractal dimensions complementary with fractal behaviour of cancer cells. To achieve specific cell-capture performance, HZnPNS was further bioconjugated with cancercell-affinitive anti-EpCAM since EpCAM is the cell surface marker commonly used for epithelial CTC isolation. Here, silane coupling agent containing carboxylate groups was


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introduced to modify the substrates. Then, via SA-biotin interaction, anti-EpCAM was functionalized on the substrates using our previously reported method (Figure S3).55 To assess the cell-capture performance of anti-EpCAM/HZnPNS, we selected MCF-7 breast cancer cells as model CTCs, and EpCAM-negative Hela human cervix cancer cells were used as control cells.11,34,35 Figure S4 displays that a significantly large number of MCF-7 cells are captured on transparent anti-EpCAM/HZnPNS, compared to control cells. Subsequently, we quantitatively verified the specific capture of anti-EpCAM/HZnPNS for MCF-7 cells. As shown in Figure 2a, solely relying on the topographical interaction between HZnPNS and structures of the cellular surface, 27 ± 8% of target cells are non-specifically adhered to the substrates. However, after being modified with anti-EpCAM, the capture efficiency of anti-EpCAM/HZnPNS for MCF-7 cells comes to 90 ± 1%, which is ascribed to the synergistic effect of affinitive and topographic interaction.34,35 Comparatively, anti-EpCAM/ZnO NWS shows much lower capture efficiency (57 ± 6%). This indicates that compared to regular-shaped ZnONW, flowerlike HZnPNS with multi-scale hierarchical architecture better match the fractal dimensionality of cancerous cells, resulting in the enhanced topographical interaction and cell-capture efficiency. As another control, Hela cells display low capture efficiency on anti-EpCAM/HZnPNS (28 ± 10%).


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Figure 2. Specific cell-capture performance of anti-EpCAM/HZnPNS. (a) Quantitative capture efficiency of anti-EpCAM/HZnPNS for MCF-7 cells. Error bars represent standard deviation (n = 3). (b, c) SEM images of topographic interaction between structures of cellular surface and HZnPNS (b) or flat glass slide (c). SEM was used to further study the topographical interaction between cancer cells and HZnPNS. As shown in Figure 2b, model target cells exhibit more and longer outspread filopodia on flowerlike HZnPNS after 45 min incubation, leading to an enhanced topographical interaction. However, cells with few stretched-out filopodia are observed on the smooth glass substrates under the same conditions (Figure 2c). Therefore, anti-EpCAM/HZnPNS, which simultaneously possesses affinitive and topographic interaction with the cell surface, could capture EpCAMpositive cancer cells with high specificity and sensitivity. Benefited from the vastly enhanced enrichment of EpCAM-positive cancer cells, we explored the potential clinical application of anti-EpCAM/HZnPNS. We prepared artificial clinical blood


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samples by spiking MCF-7 cells into lysed blood samples (blood with red blood cells lysed) at different concentrations. As shown in Figure 3a, as few as 10 cells are isolated from lysed blood with more than 70% recovery rate. To determine the purity of captured MCF-7 cells, isolated cells were fixed and immunostained by a common three-color immunocytochemistry (ICC) method. DAPI nuclear stain, FITC-labeled anti-CK 19, and PE-labeled anti-CD45 were used to distinguish MCF-7 cells from white blood cells (WBCs). Figure 3c shows that MCF-7 cells are DAPI+/CK+/CD45-, and WBCs are DAPI+/ CK-/ CD45+. Benefited from synergistically affinitive and topographical interaction, the purity of isolated MCF-7 cells comes to 63 ± 4% (Figure 3b). These results imply that anti-EpCAM/HZnPNS could be potentially applied to CTC isolation from whole blood samples.

Figure 3. Potential clinical application of anti-EpCAM/HZnPNS. (a) Recovery rate of MCF-7 cells spiked into lysed blood samples with different concentrations. Error bars represent standard deviation (n = 3). (b) Quantitative results of cancer cell capture and purity from lysed blood samples. (c) Immunostaining-based analysis of captured cells. DAPI nuclear staining, FITClabelled anti-CK19 and PE-labelled anti-CD45 were applied to distinguish cancer cells from WBCs.


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Cancer cell release and viability assay. To achieve the full availability of CTCs for downstream cellular and molecular analysis, it is necessary to gently release cancer cells and ensure their viability. Here, a simple method of ligand-assisted substrate dissolution is introduced to break down the interaction between anti-EpCAM/HZnPNS and cancer cells. Citrate acid, a common coordinating ligand with biological significance and a widely used anticoagulant to stabilize blood, could form strong complexes with metal ions such as Ca2+, Zn2+ and Mg2+.56 In our experiment, we find that biocompatible sodium citrate can dissolve HZnPNS at room temperature, allowing release of captured cells from hierarchical nanostructured substrates. Before using sodium citrate for releasing captured cells, we first investigated the effect of sodium citrate on the cell viability by methylthiazoletetrazolium (MTT) assay. As shown in Figure S5, cell viability slightly decreases after incubation in sodium citrate with increasing concentrations for 30 min. When the concentration of sodium citrate comes to 2%, cancer cells still exhibit good viability (85 ± 13%). To rapidly dissolve the substrate and gently release cancer cells, pH 6.5 of physiological PBS containing 1% sodium citrate is finally employed for cell release, which does not obviously affect cell viability (Figure S5). Then, we evaluated the dissolution rate of FITC-labelled HZnPNS under this condition (via SA and biotin interaction, SA/HZnPNS was modified with FITC-labelled biotin). Figure S6 shows that as-prepared HZnPNS could be almost completely dissolved within 20 min at room temperature. In addition, the dissolution product of HZnPNS treated by 1% sodium citrate in water (pH 6.5) for 20 min was filtered and analyzed by ICP-AES. Zn, P and Mg elements exist in the solution of dissolved zinc phosphate-based hierarchical architectures (Figure 4a), indicating that citrate anions indeed cause the dissolution of HZnPNS. However, we find pure zinc phosphate is hardly soluble in 1% sodium citrate (pH 6.5). Considering that ZnO could be


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dissolved in the presence of citrate ions.56 Thus, we think ZnO existing in HZnPNS may first react with citrate followed by damaging the hierarchical architecture. Finally, the optimized conditions for substrate degradation were applied to release captured cancer cells. Figure 4b shows that few captured cells remain on the flat glass substrates after the dissolution of flowerlike hierarchical nanostructure. The release efficiency (as a percentage of captured cells) is 88 ± 4% (Figure 4c).

Figure 4. Ligand-assisted substrate dissolution for cell release. (a) ICP-AES analysis of the dissolution product. HZnPNS was treated by 1% sodium citrate in water (pH 6.5) for 20 min. (b) Microscopic images of captured cells before and after being released from transparent antiEpCAM/HZnPNS. Cells were pre-stained with SYTO@13 nucleic acid dye. (c) Quantitative evaluation of the release efficiency and viability of released cancer cells. Error bars represent standard deviation (n = 3). To further evaluate the viability of retrieved cancer cells, annexin V-FITC and PI were employed to stain released cancer cells. Early-apoptotic cells were labelled by annexin V-FITC, whereas late-apoptotic cells were labelled by PI. As shown in Figure S7a, neither FITC nor PI staining is


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observed for the majority of retrieved cancer cells. Quantitatively, 92 ± 1% of released cells exhibits good viability after treatment with 1% sodium citrate (pH 6.5) at room temperature for 20 min (Figure 4b). Subsequently, released cells were collected and further cultured. Figure S7b shows that retrieved cancer cells spread well and only few cells are stained by PI after cultivation for 48 h. These results indicate that the approach of sodium citrate-assisted cell release from degradable HZnPNS is efficient and cell-friendly. Isolation and DNA methylation analysis of CTCs from cancer patient blood samples. Having investigated the versatility of anti-EpCAM/HZnPNS for cancer cell capture and release, we applied this substrate to isolate CTCs from whole blood samples. In each assay, a 1.0 mL blood sample was introduced onto anti-EpCAM/HZnPNS followed by immunostaining to identify CTCs and WBCs. We score as CTCs that are DAPI+/CK+/CD45- (30 µm > cell sizes > 10 µm), and WBCs that are DAPI+/CK-/CD45+ (cell sizes < 15 µm) (Figure 5a). CTC-capture results from 13 cancer patient blood samples are summarized in Figure 5b, Table S1 and S2. Additionally, we compared the CTC-capture performance of anti-EpCAM/HZnPNS with the gold standard, FDA-cleared CellSearch. Our method detect 6-75 CTCs/mL from 4 of 5 metastatic cancer patient blood samples, whereas CellSearch assay isolates 1-125 CTCs/7.5 mL from 4 of 5 blood samples (Figure 5c and Table S2). As such, benefitting from synergistically affinitive

and

topographical

interaction

between

substrate

and

cancer

cells,

anti-

EpCAM/HZnPNS could detect more CTCs than CellSearch from same blood volumes. Some representative images (single CTC or CTC clusters isolated by anti-EpCAM/HZnPNS from each patient) are displayed in Figure S8. Here, intact CTC clusters from one of colorectal patients' blood are observed on substrate. However, only one CTC is detected by CellSearch, possibly


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because blood processing breaks the integrity of CTC clusters. These results indicate that antiEpCAM/HZnPNS could be reliably applied to whole blood samples for CTC isolation.

Figure 5. Quantitative assay of CTCs in cancer patient blood samples. (a) Microscopic images of CTCs isolated from a breast cancer patient's blood sample. (b) Quantitative results of CTCs enumerated from 13 cancer patient blood samples. (c) Comparison of anti-EpCAM/HZnPNS with CellSearch for CTC isolation from 5 metastatic cancer patient blood samples. (d) The measured contents of 5-mdC in CTCs of breast cancer patients and corresponding blood cells of healthy controls (n=11). To demonstrate downstream manipulation and molecular analysis feasibility, CTCs enriched from 11 breast cancer patients were released from anti-EpCAM/HZnPNS and their global DNA


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methylation was analyzed by mass spectrometry. Released CTCs were identified by three-color ICC method (Figure S9), and retrieved for cell lysis, nucleic acids digestion, and nucleosides extraction

using

our

previously

reported

strategy.48

Results

from

liquid

chromatography−electrospray ionization−tandem mass spectrometry (LC-ESI-MS/MS) show that the contents of 5-methyl-2'-deoxycytidine (5-mdC) in CTCs of breast cancer patients are lower than that in blood cells of healthy controls with matched ages and genders (P=0.002) (Figure 5d and Table S3). This is consistent with our previous result48 and matches well with literatures that global DNA methylation declined in breast cancer tissues.57-59 Taken together, it is clear that retrieved CTCs from degradable anti- EpCAM/HZnPNS can be successfully used for downstream molecular analysis, which will benefit personalized cancer therapy.

CONCLUSION

In summary, we have demonstrated a zinc phosphate-based hierarchical nanosubstrate for effective CTC isolation and cell-friendly CTC release. As-prepared HZnPNS is transparent, biocompatible and degradable. The fabrication process is simple, repeatable and easily manipulated. Compared with regular-shaped nanosubstrate ZnO NWS, anti-EpCAM modified HZnPNS with fractal dimensions better facilitates the topographic interaction and greatly enhances capture efficiency. Furthermore, HZnPNS could be rapidly dissolved by biocompatible sodium citrate at room temperature, resulting in gentle release of captured cells from nanostructured substrates. This allows downstream molecular analysis and demonstrates DNA hypomethylation in tumor cells from whole blood. It is conceivable that this versatile hierarchical nanosubstrate we developed will hold great promise for reliable and sensitive


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detection of CTCs from clinical blood samples. The ability to capture CTCs from cancer patient blood and to release them for culture and downstream molecular assay will facilitate detailed analysis of their role in cancer progression and metastasis. ASSOCIATED CONTENT Supporting Information. Figure S1 Characterization of substrates formed in control conditions. Figure S2 Biocompatibility of HZnPNS. Figure S3 Modification of HZnPNS. Figure S4 Microscopic images of captured cancer cells on HZnPNS. Figure S5 MTT assay. Figure S6 Microscopic images of HZnPNS dissolution. Figure S7 Viability of released cancer cells. Figure S8 Representative images of CTCs from metastatic cancer patients' blood. Figure S9 Images of retrieved CTCs and WBCs. Table S1 and S2 Quantitative results of CTC enumeration from cancer patients' blood. Table S3 Contents of 5-mdC in retrieved CTCs from breast cancer patients and blood cells of healthy controls. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] * E-mail: [email protected] Author Contributions ◊

These authors contributed equally.

Notes The authors declare no competing financial interest.


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ACKNOWLEDGMENT This work was supported by the National Basic Research Program of China (973 Program, Nos. 2012CB720600, 2012CB720603, 2012CB720604, 2012CB720605), and the National Natural Science Foundation of China (Nos. 21432008, 91413109, 21575110). REFERENCES (1) Alix-Panabières, C.; Pantel, K. Challenges in Circulating Tumour Cell Research. Nat. Rev. Cancer 2014, 14, 623-631. (2) De Mattos-Arruda, L.; Cortes, J.; Santarpia, L.; Vivancos, A.; Tabernero, J.; Reis-Filho, J. S.; Seoane, J. Circulating Tumour Cells and Cell-Free DNA as Tools for Managing Breast Cancer. Nat. Rev. Clin. Oncol. 2013, 10, 377-389. (3) Yu, M.; Bardia, A.; Aceto, N.; Bersani, F.; Madden, M. W.; Donaldson, M. C.; Desai, R.; Zhu, H.; Comaills, V.; Zheng, Z.; Wittner, B. S.; Stojanov, P.; Brachtel, E.; Sgroi, D.; Kapur, R.; Shioda, T.; Ting, D. T.; Ramaswamy, S.; Getz, G.; Iafrate, A. J.; Benes, C.; Toner, M.; Maheswaran, S.; Haber, D. A. Ex Vivo Culture of Circulating Breast Tumor Cells for Individualized Testing of Drug Susceptibility. Science 2014, 345, 216-220. (4) Miyamoto, D. T.; Zheng, Y.; Wittner, B. S.; Lee, R. J.; Zhu, H.; Broderick, K. T.; Desai, R.; Fox, D. B.; Brannigan, B. W.; Trautwein, J.; Arora, K. S.; Desai, N.; Dahl, D. M.; Sequist, L. V.; Smith, M. R.; Kapur, R.; Wu, C.-L.; Shioda, T.; Ramaswamy, S.; Ting, D. T.; Toner, M.; Maheswaran, S.; Haber D. A. RNA-Seq of Single Prostate CTCs Implicates Noncanonical Wnt Signaling in Antiandrogen Resistance. Science 2015, 349, 1351-1356. (5) Cristofanilli, M.; Budd, G. T.; Ellis, M. J.; Stopeck, A.; Matera, J.; Miller, M. C.; Reuben, J. M.; Doyle, G. V.; Allard, W. J.; Terstappen, L. W. Circulating Tumor Cells, Disease Progression, and Survival in Metastatic Breast Cancer. N. Engl. J. Med. 2004, 351, 781-791.


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Degradable Zinc-Phosphate-Based Hierarchical ... - ACS Publications

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