Construction of Epidermal Growth Factor Receptor Peptide Magnetic

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Construction of Epidermal Growth Factor Receptor Peptide Magnetic Nanovesicles with Lipid Bilayers for Enhanced Capture of Liver Cancer Circulating Tumor Cells Jian Ding,*,†,‡ Kai Wang,†,§ Wen-Jie Tang,*,∥ Dan Li,⊥ You-Zhen Wei,∥ Ying Lu,∥ Zong-Hai Li,§ and Xiao-Fei Liang*,§ ‡

Digestive Department, The First Affiliated Hospital of Fujian Medical University, 20 Chazhong Road, Fuzhou 350005, China State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, No.25/Ln2200 Xie Tu Road, Shanghai 200032, China ∥ Research Centre for Translational Medicine, East Hospital, Tongji University School of Medicine, 150 Jimo Road, Shanghai 200120, China ⊥ Digestive Department, Union Hospital of Fujian Medical University, Fuzhou 350001, China §

S Supporting Information *

ABSTRACT: Highly effective targeted tumor recognition via vectors is crucial for cancer detection. In contrast to antibodies and proteins, peptides are direct targeting ligands with a low molecular weight. In the present study, a peptide magnetic nanovector platform containing a lipid bilayer was designed using a peptide amphiphile (PA) as a skeleton material in a controlled manner without surface modification. Fluorescein isothiocyanate-labeled epidermal growth factor receptor (EGFR) peptide nanoparticles (NPs) could specifically bind to EGFRpositive liver tumor cells. EGFR peptide magnetic vesicles (EPMVs) could efficiently recognize and separate hepatoma carcinoma cells from cell solutions and treated blood samples (ratio of magnetic EPMVs versus anti-EpCAM NPs: 3.5 ± 0.29). Analysis of the circulating tumor cell (CTC) count in blood samples from 32 patients with liver cancer showed that EPMVs could be effectively applied for CTC capture. Thus, this nanoscale, targeted cargo-packaging technology may be useful for designing cancer diagnostic systems.

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However, detection of CTCs has proven to be technically challenging because of the extremely low abundance of CTCs among the large numbers of hematologic cells in the blood.23−26 In contrast to antiepithelial cell adhesion molecule (EpCAM) antibodies,27 peptides can be used to construct NPs with a low molecular weight, which can improve the CTC capture efficiency. Our primary purpose was to introduce a stable, peptide magnetic-targeted nano/microvesicle platform with a lipid bilayer and high capacity for identification of tumor cells. EPMVs with high affinity for EGFR-expressing cancer cells may be potential tools for CTC capture.

ighly efficient recognition and separation of circulating tumor cells (CTCs) from the peripheral blood or other body fluids is a major challenge in cancer detection and individualized treatment.1−5 Targeted identification by vectors can overcome key difficulties, such as the limited stability of diagnostic reagents, lack of selectivity, and low tumor-cell affinity.6 Ligands that interact with membrane-anchored receptors can promote enhanced recognition through receptor-mediated internalization pathways.6−9 Herein, we developed epidermal growth factor receptor (EGFR) peptide magnetic nanovesicles (EPMVs) using a peptide derivative; these vesicles had the advantages of a lipid bilayer structure,10 controlled surface peptide content, and high ligand-targeting ability.11−13 In contrast to our previous studies,14−17 here we used a peptide amphiphile (PA) to construct nanoparticles (NPs) that can directly search for CTCs in a peptide-targeted manner. This approach could overcome limitations of traditional peptide nanocarriers, such as immune magnetic beads and magnetic liposomes. Notably, isolation of CTCs from the blood of patients with metastatic cancer using immune magnetic particles18−20 is an important application requiring targeted recognition of functional immune magnetic nanovectors.21,22 © XXXX American Chemical Society

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EXPERIMENTAL SECTION Materials. GE11 peptide of YC21 (NH2-YHWYGYTPQNVI-GGGSGGGS-Cys-COOH) and AChE gene (NG 007474.1) were synthesized by Cancer Institute of Shanghai Jiaotong University. Absolute ethanol, epoxy chloropropane, isopropanol, chloroform, and ethylenediaminetetraacetic acid Received: April 13, 2016 Accepted: August 25, 2016

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Analytical Chemistry (EDTA), N,N-dimethylhexadecylamine were obtained from Sinopharm Chemical Reagent (China) or Feixiang Group (China), respectively. 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, 60010), fluorescein isothiocyanate isomer I (FITC), N,N-dimethyl tetradecylamine, were purchased from Sigma (St. Louis, U.S.A.). Cholesterol (lot number: C8667; ≥ 99%) was obtained from Sigma (St. Louis, MO, U.S.A.). SMMC-7721 human hepatoma cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in an atmosphere containing 5% CO2. Preparation of Peptide Precursors. Initially, epoxy chloropropane and N,N-dimethyltetradecylamine were used to synthesize glycidyl hexadecyl dimethylammonium chloride (GHDC). Next, GHDC was incubated with GE11 solution, which was prepared by dissolving 50 mg of GE11 peptide in 10 mL of water saturated with isopropanol for 24 h at 4 °C to finally obtain the hexadecyl quaternized peptide (GE11conjugated GHDC [GE11-GHDC]). Samples were then dialyzed (1000 Da cutoff) for 24 h against deionized water and lyophilized. GE11-conjugated GHDCs with different GE11 contents were synthesized by changing the ratio of GHDC to GE11 in the reaction. Quaternization of CMCs with long carbon chains (tetradecyl) was conducted as previously described.15 To obtain the FITC-labeled GE11 peptide, FITC (1.0 mg) and YC21 (5.0 mg) were dissolved in sodium carbonate and sodium bicarbonate buffer (0.1 M, pH = 9.0). After stirring at 4 °C for 3 h, the mixture was dialyzed (1000 Da cutoff) at 4 °C for 8 h to remove free FITC. The FITC-labeled GE11 peptide was used to prepare FITC-labeled GE11-GHDC (FITC-GE11GHDC). Characterization of GE11 and GE11-GHDC. FT-IR spectra were recorded on a Bio-Rad FTS 6000 spectrometer with KBr pellets at room temperature. The samples were thoroughly milled with KBr and pressed into pellets. 1H NMR spectra were recorded on a UNITY Plus-400 spectrometer at 25 °C. The samples were dissolved in D2O solution to a concentration of 30 mg/mL. Evaluation of Lipid Bilayers of Peptide Nanovesicles (PVs). FITC-labeled PVs were constructed using the following method. First, FITC-GE11-GHDC and cholesterol were mixed in dichloromethane and incubated for 5−10 min. The mixture was then evaporated to obtain a thin peptide film. Next, blank FITC-labeled PVs were obtained by dispersing the peptide lipid film in deionized water by vortexing. For qualitative analysis of FITC-GE11-GHDC in PVs, different PV solutions were dropped on filter paper and the optimal cutting temperature (OCT) medium was added immediately. Pieces of the filter paper were frozen and a cryostat microtome (Leica CM 1850) was used to prepare fivemicron sections of the frozen pieces. FITC-associated green fluorescence was detected using a confocal laser-scanning microscope with an excitation filter of 488 nm and emission filter of 525 nm. Detection of EGFR Expression by Cell Uptake Quantification Analysis. The human hepatoma cell line SMMC-7721 was selected for evaluation of EGFR expression on the cell membrane in NP cell uptake experiments. Each cell suspension (1 mL, 1 × 106 cells/mL) was seeded in a 24-well plate. Twenty-4 h later, the cells were incubated with 20 μL of FITC-labeled CL, 20 μL of FITC-labeled GPL, and 20 μL of

FITC-labeled GPL with added GE11(YC21) for 1.0 h. Subsequently, phosphate-buffered saline (PBS) was added, and the cells were incubated at room temperature for 30 min. The cells were then trypsinized, washed with PBS, and filtered through a 200-nylon mesh to prepare single-cell suspensions. Next, the FITC fluorescence of 10000 cells was determined using a BD FACSCalibur flow cytometer. To avoid interference from autofluorescence, blank cells without any added FITClabeled NPs were analyzed, and their fluorescence intensity was designated as the threshold value. Only fluorescence that exceeded the threshold value was considered as the uptake signal. Preparation of EPMVs. EPMVs were prepared using the reverse-phase evaporation (REV) method, as follows. GE11GHDC and cholesterol (weight ratio of 1:0.81, total lipid: 30 mg) were dissolved in 4 mL of chloroform at room temperature to obtain the organic phase. Then, 6 mL of deionized water was prepared to obtain the aqueous phase. In total, 8 mg of hydrophobic magnetic nanoparticles (BM) was added to the organic phase with continuous stirring. The aqueous solution was then added to this organic phase and sonicated for approximately 2.0 min to obtain an emulsion. Next, rotary evaporation was used to rapidly remove the organic solvent from the emulsion at room temperature. After three cycles of magnetic separation, the subsequent gel-like, highly concentrated EPMV suspension could be diluted with a suitable aqueous buffer solution. Magnetic cationic particles (MCPs) with the same weight ratio of TQCMC/cholesterol used for GE11-GHDC/cholesterol were obtained using the same method. EpCAM immunomagnetic NPs (EIPs) with the same weight ratio of EpCAM-GHDC/cholesterol used for GE11-GHDC/cholesterol were obtained using the same method. Cell Isolation from DMEM Samples. SMMC-7721 cells were isolated from cultures in DMEM supplemented with 10% fetal bovine serum using 50 μL of MCPs and EPMVs. Magnetic Dynabeads were incubated with DMEM on a low-speed rotating device for 30 min at room temperature, after which the labeled cells were separated using an external magnetic field. The cell/magnetic NP complexes were washed three times with PBS, and the retained cells were collected. After staining with DAPI, the separated cells were observed with a fluorescence microscope (Olympus BX-51). Localization and Distribution of EPMVs in SMMC7721 Cells. Cells were cultured in FITC-labeled EPMVcontaining medium in a glass-bottomed dish (35 mm dish with 14 mm bottom wells) for 30 min, followed by treatment with 4′,6-diamidino-2-phenylindole (DAPI) for 5−10 min. Stained cells were then examined by confocal microscopy. Blood Specimen Collection and Processing for CTC Capture. Blood samples were obtained from patients with liver cancer by our collaborators at the Chinese and Western Medicine Clinical Center at Fudan University Shanghai Cancer Center (FUSC) with FUSC IRB approval. According to a standard protocol, all blood specimens were collected in EDTA-containing vacuum tubes and were processed within 24 h. After centrifugation at 800g for 20 min, the supernatant was removed, and the cell pellet was resuspended in an equivalent volume of PBS (0.1 M, pH 7.4). In total, 50 μL of a solution of EPMVs (1.0 mg/mL in PBS with 1.0% [w/v] bovine serum albumin [BSA] and 0.1% [w/v] sodium azide) was pipetted into the supernatant of each blood specimen. After incubation for 30 min, the magnetic bead/cell complexes were removed by B

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Analytical Chemistry magnetic separation. DAPI dye was applied to the magnetic bead/cell complexes for nuclear staining (15 min) and then removed by washing with 500 μL of PBS. The supernatant was cleared by magnetic separation. The cancer cells collected by the separation were stained for CK19 and CD45 for 15 min, and excess dye was removed by washing with 500 μL of PBS. The supernatant was cleared by magnetic separation, and ddH2O was added to the pellet. CTCs captured on slides were defined based on cell staining (green: CK19+, blue: DAPI+, red: CD45-) for appropriate phenotypic morphological characteristics. Cellular and morphometric characteristics, including cell size, cell shape, and nucleus size, were recorded and employed to identify CTCs with a fluorescence microscope (Olympus BX-61).

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RESULTS AND DISCUSSION Construction of the Peptide Derivative and EPMVs. The preparation method and material components of EPMVs

Figure 2. FT-IR (a) and 1H NMR (b) spectra of GE11 and GE11GHDC. GE11 and GE11-GHDC were dissolved in D2O for the 1H NMR spectrum.

Figure 1. Schematic diagram showing the preparation of EPMVs. (a) The molecular structure and preparation process for GE11-GHDC. (b) Preparation process for EPMVs.

are shown in Figure 1. EPMVs, which had properties of both peptides and liposomes, were prepared using the modified peptide and cholesterol. Multifunctional PV-supported bilayers synergistically combined the properties of lipid vesicles and PA micelles, facilitating their application as nanocarriers. In contrast to conventional liposomes composed of lecithin and cholesterol, the peptide surfactant GE11-GHDC, rather than lecithin, was used as the skeletal material for PVs, and cholesterol was used as the stabilizing agent.6,15,16 Fourier transform infrared (FT-IR) spectroscopy and 1H NMR were used to characterize the prepared oligopeptide surfactant, and the relative content of the oligopeptide in GE11-GHDC was calculated from the weight ratio of the peptide to the total modified conjugate. The FT-IR and 1H NMR spectra of GE11 and GE11-GHDC are shown in Figure 2. In the GE11-GHDC FT-IR spectra, new peaks appeared at approximately 2850−2915, 1466, and 721 cm−1; these peaks were attributed to the long carbon chain and methyl groups on the quaternary ammonium salt, representing the existence of GHDC on the GE11 peptide. In the 1H NMR spectrum, the most intense signals were at δ 0.88, 1.19−1.65, and 3.30. The

Figure 3. Physical and microstructural characteristics of GE11 PVs encapsulating different types of cargo. (a) Large PVs showing the external peptide lipid bilayer (∼2.52 μm thick) of a large unilamellar vesicle. (b) Large PVs encapsulating hydrophobic QDs, showing the external peptide lipid bilayer (∼2.00 μm thick) of large unilamellar vesicles.

protons at δ 6.7 ppm were assigned to the aromatic group of GE11 (YC21), and the methyl protons at δ 0.5−1.0 ppm were assigned to GE11. High molar content of GHDC in GE11GHDC was also confirmed.9 The solubility of the peptide derivative in water and organic solvents is shown in Supporting Information, Table S1. The C

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Figure 5. (a) Uptake efficiency of FITC-labeled EPMVs. SMMC-7721 cells were incubated with targeted EPMVs or nontargeted MVs for 15 min at the same NP concentration (20 μg/mL). NPs were labeled with FITC. 1, control; 2, free FITC; 3, FITC-MVs (GHDC/cholesterol); 4, FITC-EPMVs (GE11-GHDC/cholesterol). (b) Flow cytometric analysis of the targeted delivery of different cargoes to SMMC-7721. Cells were incubated with FITC-labeled NP-containing medium (QDs in EPMVs) in 24-well plates for 0.5 h.

Figure 4. (a) AFM image of EPMVs. (b) Size distribution of EPMVs in the solution according to fluorescence intensity. (c) Magnetization curves of EPMVs at 300.0 K.

peptide showed better affinity for organic solvents than the original peptide. This difference could be explained by the extent of grafting of GHDC, which provided higher solubility in organic solvent as a result of the introduction of the hydrophobic side chains of the long-chain alkyl. The peptidelipid GE11-GHDC also exhibited good water solubility. Material Characteristics of EPMVs. All GE11-GHDC samples could be dissolved in distilled water and chloroform (CHCl3). To verify the microstructure of EPMVs, including the lipid bilayer, the FITC-labeled peptide was prepared with the constructed PVs by loading hydrophobic quantum dots (QDs; yellow fluorescence); these components were then combined to assemble large vesicles. As shown in Figure 3, hydrophobic QD NPs could be encapsulated in large EGFR peptidecontaining vesicles to form different unilamellar or multilamellar microspheres. Hydrophobic QD NPs were encapsulated in the peptide lipid hydrophobic bilayer with a thickness of 1.0−3.0 μm. The GE11 peptide with intense green

Figure 6. (a) Fluorescence microscopic images of SMMC-7721 cells captured by MVs (a1) and EPMVs (a2, a3). Nuclei are labeled with DAPI (blue). (b) Capture efficiency of different magnetic nanoparticles: 1, MV-captured cells in PBS; 2, EPMV (3 days after preparation)-captured cells in PBS; 3, EPMV (1 month after preparation)-captured cells in PBS; 4, EPMV (3 months after preparation)-captured cells in PBS; 5, EPMV (3 months after preparation)-captured cells in whole blood.

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Figure 7. Localization and distribution of EPMVs in hepatoma cancer cells. SMMC-7721 cells were cultured in FITC-labeled EPMV-containing medium in glass-bottomed dishes for 0, 5, 10, 30, and 60 min. Cells were treated with DAPI and Dil for 5−10 min and then examined by confocal microscopy. (a1) Cells showing DAPI-stained (blue) nuclei. (a2) Cells showing distribution of FITC-derived fluorescence (green). (a3) Cells showing distribution of Dil-derived fluorescence (red). (a4) Overlaid images.

fluorescence was mainly distributed on the surface of the external lipid bilayer. Atomic force microscopy (AFM) imaging of the EPMVs revealed that the magnetic PVs were irregular, ∼200 nm spheres with a narrow size distribution and coarse surfaces (Figure 4a). The average diameter of the EPMVs in aqueous solution was 219.1 nm, with a corresponding polydispersity index (PDI) of 0.169 (Figure 4b). Room temperature magnetization curves of the Fe3O4 ferrofluid and the EPMVs are shown in Figure 4c. Dry EPMVs showed typical superparamagnetic behavior at room temperature, without any hysteresis loop. The saturation magnetization value of the EPMVs was 42.3 emu/g at 300 K, which was approximately 76.4% of the magnetization value of the Fe3O4 ferrofluid. Cell-Targeted Recognition Ability of EPMVs. SMMC7721 liver cancer cells were selected for analysis of EGFR expression14,17 at the cell membrane and to determine the uptake efficiency of FITC-labeled EPMVs (Figure 5a). The uptake efficiencies of SMMC-7721 cells for EPMVs and EGFRnontargeting MVs were semiquantitatively determined by flow cytometry. The EPMVs bound to and entered SMMC-7721

cells to a greater degree than the non-EGFR-specific MVs. QDloaded FITC-EPMVs exhibited greater affinity for EGFRoverexpressing SMMC-7721 cells, showing 2-fold higher fluorescence signals than MVs (Q2 7.78% for EPMVs versus 3.23% for MVs; Figure 5b). This result indicates that EPMVs with EGFR peptide guidance could deliver more magnetic NPs into SMMC-7721 cells. A simple capture experiment using SMMC-7721 cells was designed to verify the cell recognition capability of EPMVs. Notably, we observed a significant difference in the number of cells captured by these magnetic NPs incubated with the cell suspension for 0.5 h (38 for EPMVs versus 8 for MCPs; P < 0.001). Moreover, SMMC7721 cells were fully covered by EPMVs, as shown in the highmagnification image (Figure 6a). To study the recognition behavior in cancer cell capture by magnetic NPs, we characterized the cell morphology and magnetic NP distribution after different contact times (5, 10, 30, and 60 min) by laser confocal microscopy in substrateimmobilized cells on adhesive microscope slides (Figure 7). When a sample containing CTCs was mixed with EPMVs, the functional magnetic PVs automatically searched for CTCs and E

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overlapped well with green fluorescence representing the FITC-labeled EPMVs at both 30 and 60 min, indicating that the immunomagnetic lipid vesicles were distributed around the cell membrane (Figure 6a3). In addition, the prepared EPMVs were stable and able to capture cancer cells for at least 3 months after preparation (Figure 6b). Clinical Application Analysis in Liver Cancer CTC Separation. Peripheral blood samples were obtained from patients with liver cancer to test the tumor recognition ability of EPMVs. We then validated the performance of the EPMV CTC-capture platform by performing side-by-side comparisons with the commonly used EIPs. The CTC separation function of EIPs was compared with that of EpCAM immune-magnetic beads in the CellSearch assay (Figure S1). As shown in Figure 8a, both EIPs and EPMVs exhibited higher CTC capture for hepatic carcinoma cells than CellSearch. There was a significant difference in the number of CTCs captured by different magnetic NPs (EPMVs versus EIPs, n = 25; P < 0.0001; Figure 8b). Moreover, analysis of the CTC count in blood samples from 25 patients with cancer showed that EPMVs could be effectively applied for CTC separation. As shown in Figure 8c, CTCs separated by EPMVs exhibited strong expression of cytokeratin (CK) and negligible signals for CD45 with positive DAPI staining (DAPI+/CK +/CD45−, 40 μm > cell size > 8 μm). Even though some very small magnetic NPs were present on the cell surface, all captured CTCs exhibited clear liver cell morphology. These results indicate that magnetic EGFR-PVs have the ability to capture EGFR-overexpressing tumor cells and that the peptide NP construction method is also applicable to antibodies.

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CONCLUSION The target ligand of EGFR peptide was successfully modified to form GE11-GHDC and used to produce PV-supported lipid bilayer membranes with various sizes and surface characteristics. Magnetic EGFR-targeted PVs more effectively recognized and separated hepatoma cells from cell solutions and treated blood samples than EpCAM magnetic vesicles. The resulting synergistic effects led to the high CTC capture performance observed in both spiked and clinical blood samples. The EPMVs demonstrated high affinity for EGFR-positive cancer cells and can therefore be used for efficient detection of cancer and CTCs before surgery. EPMVs coupled with chemotherapy might improve prognosis and enable the introduction of individualized therapy for patients with liver cancers. The EGFR-targeted detection may also enhance treatment with tyrosine kinase inhibitors in these patients.

Figure 8. (a) Side-by-side representation of CTC enumeration results obtained from our integrated CTC-capture technology and a CellSearch assay in matched samples. (b) Graph showing the total number of CTCs collected from each tumor-bearing patient with the two different methods: EIP (EpCAM immunomagnetic separation) and EPMV separation. (c) Fluorescent micrographs of CTCs captured from blood samples from a patient with liver cancer. Three-color immunocytochemistry based on FITC-labeled anticytokeratin 19, PElabeled anti-CD45, and DAPI nuclear staining was applied to identify and enumerate CTCs from the captured cells by magnetic immunological recognition.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b01443. Table S1: Solubility and zeta potential of GE11-GHDC with different degrees of quaternary substitution (DS). Figure S1: Typical fluorescence images of EGFR+ CTCs identified using EPMVs (a), EpCAM magnetic nanobeads in CellSearch system (b), and EpCAM magnetic NPs made by the same method as that used for EPMVs (c) in the CellSearch system. CTCs are defined as cells staining positively for CK and DAPI and negatively for CD45. Circulating tumor microemboli (CTM) are

adhere to the cell surface. Increased numbers of EPMVs gradually assembled around the CTCs, starting as early as 15 min after application. After 30 min, the cell surface was surrounded by a sufficient number of magnetic NPs, and the magnetic NPs did not continue to adhere to the cells as the time increased. Moreover, there was no obvious increase in the content of magnetic NPs at 60 min compared with that at 30 min. Red fluorescence representing the cell membrane dye Dil F

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(13) Ferreira, T.; Topgaard, D.; Ollila, S. O. H. Biophys. J. 2014, 106, 41a. (14) Fan, M.; Yang, D.; Liang, X.; Ao, J.; Li, Z.; Wang, H.; Shi, B. Biomed. Pharmacother. 2015, 70, 268−273. (15) Liang, X.; Li, X.; Chang, J.; Duan, Y.; Li, Z. Langmuir 2013, 29, 8683−8693. (16) Liang, X.; Wang, H.; Jiang, X.; Chang, J. J. Nanopart. Res. 2010, 12, 1723−1732. (17) Zhang, P.; Shi, B.; Gao, H.; Jiang, H.; Kong, J.; Yan, J.; Pan, X.; Li, K.; Zhang, P.; Yao, M.; Yang, S.; Gu, J.; Wang, H.; Li, Z. Cancer Immunol. Immunother. 2014, 63, 121−132. (18) Bouchlaka, M. N.; Sckisel, G. D.; Wilkins, D.; Maverakis, E.; Monjazeb, A. M.; Fung, M.; Welniak, L.; Redelman, D.; Fuchs, A.; Evrensel, C. A.; Murphy, W. J. PLoS One 2012, 7, e48049. (19) Meng, C.; Tian, J.; Li, Y.; Zheng, S. Acta Microbiol. Sin. 2010, 50, 817−821. (20) Takahashi, M.; Yoshino, T.; Takeyama, H.; Matsunaga, T. Biotechnol. Prog. 2009, 25, 219−226. (21) Ahrens, E. T.; Bulte, J. W. M. Nat. Rev. Immunol. 2013, 13, 755− 763. (22) Prinz, E. M.; Eggers, R.; Lee, H. H.; Steinfeld, U.; Hempelmann, R. J. Phys. Conf. Ser. 2010, 200, 122009. (23) Hofman, V.; Ilie, M. I.; Long, E.; Selva, E.; Bonnetaud, C.; Molina, T.; Vénissac, V.; Mouroux, J.; Vielh, P.; Hofman, P. Int. J. Cancer 2011, 129, 1651−1660. (24) Zampino, G. M.; Magni, E.; Ravenda, S. P.; Botteri, E.; Bertani, E.; Chiappa, A.; Valvo, M.; Zorzino, L.; Adamoli, L.; Nole, F.; Sandri, M. T. Ann. Oncol. 2009, 20 (Suppl 8), 121. (25) Cristofanilli, M.; Hayes, D. F.; Budd, G. T.; Ellis, M. J.; Stopeck, A.; Reuben, J. M.; Doyle, G. V.; Matera, J.; Allard, W. J.; Miller, M. C.; Fritsche, H. A.; Hortobagyi, G. N.; Terstappen, L. W. M. M. Breast Cancer Res. Tr. 2005, 23, 1420−1430. (26) Harris, L. N.; Solomon, N.; Roberts, L.; Ngo, T.; Abi Raad, R.; Gioioso, C.; Kuter, I.; Smith, B.; Iglehart, J. D.; Friedman, P.; Taghian, A. G. Breast Cancer Res. Tr. 2005, 94 (Suppl 1), 1021. (27) Konigsberg, R.; Pfeiler, G.; Obermayr, E.; Gneist, M.; Ruckser, R.; Hudec, M.; Zeillinger, R.; Dittrich, C. J. Clin. Oncol. 2010, 28, n/a.

defined as CTC clusters. Apoptotic CTCs are defined as CTCs with fragmented, condensed DAPI-stained nuclei (PDF).

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax: +8659188808826. *E-mail: [email protected]. *E-mail: xfl[email protected]. Author Contributions †

These authors contributed equally to this paper (J.D. and K.W.). Author Contributions

J.D., X.F.L., and Z.H.L. designed the experiments. K.W. and J.F.G. developed the protocols, performed all experiments, and analyzed the data. X.F.L., K.W., and Y.X.Y. wrote the manuscript. W.J.T., D.L., Y.Z.W., and Y.L. provided the blood samples and clinical data for patients with liver cancer. All authors reviewed the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by The National Natural Science Foundation of China (Nos. 81572973, 81300321, and 31271586), the Fujian Provincial Natural Science Fund (No. 2013J01369), the Biological Medicine Industry-UniversityResearch Project of Shanghai (No. 12DZ1941702), and the Fujian Provincial Natural Science Fund (No. 2014J01419). We are also thankful to Mr. Junping Ao for flow cytometry analysis at the Flow Cytometry and Cellular Imaging Core Facility at State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine.

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