Biotechnol Lett DOI 10.1007/s10529-015-1896-z

ORIGINAL RESEARCH PAPER

A CD44 specific peptide developed by phage display for targeting gastric cancer Dan Zhang . Huan Jia . Yan Wang . Wei-Ming Li . Ying-Chun Hou . Shi-Wei Yin . Thomas D. Wang . Shui-Xiang He . Shao-Ying Lu

Received: 27 March 2015 / Accepted: 23 June 2015 Ó Springer Science+Business Media Dordrecht 2015

Abstract Objective To develop a peptide probe that could be used for gastric cancer detection via binding to CD44 protein with specificity and affinity. Results A 12-mer phage peptide library was screened against immobilized CD44 protein. Bound phage counts using ELISA were performed to identify phage clones carrying the most highly selective peptide, which termed RP-1. Immunofluorescence and flow cytometry analysis indicated that the consensus peptide RP-1 could bind to CD44-positive gastric cancer cells with mean fluorescence intensities significantly higher than that of CD44-negative cells.

CD44 knockdown led to decreased binding activity of RP-1 to the same cell line. Tissue array technique was used to identify the relationship (r = 0.556) between peptide binding and CD44 detection on gastric cancer tissues. Further, the hyaluronan-binding domain of CD44 was docked with RP-1 using computer modeling/docking approaches, revealing a RP-1/CD44 interaction with geometrical and energy match (-8.6 kcal/mol). Conclusions The RP-1 peptide we screened exhibits affinity and specificity to CD44 on cells and has the potential to be used as a candidate probe for gastric cancer cell targeting.

D. Zhang  S.-X. He Department of Gastroenterology, The First Affiliated Hospital of Medical School, Xian Jiaotong University, Xi’an 710061, China e-mail: [email protected]

W.-M. Li  S.-Y. Lu (&) Department of General Surgery, The First Affiliated Hospital of Medical School, Xian Jiaotong University, Xi’an 710061, Shaanxi, China e-mail: [email protected]

S.-X. He e-mail: [email protected]

W.-M. Li e-mail: [email protected]

H. Jia Department of General Surgery, The First Affiliated Hospital of Xi’an Medical University, Xi’an 710077, Shaanxi, China e-mail: [email protected]

Y.-C. Hou College of Life Science, Shaanxi Normal University, Xi’an 710119, Shaanxi, China e-mail: [email protected]

Y. Wang Institute for Biomedical Sciences of Pain, Tangdu Hospital, The Fourth Military Medical University, Xi’an 710038, Shaanxi, China e-mail: [email protected]

S.-W. Yin College of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710119, Shaanxi, China e-mail: [email protected]

123

Biotechnol Lett

Keywords CD44  Gastric cancer  Molecular docking  Molecular imaging  Peptide probe  Phage display  Tissue array

Introduction Gastric cancer is a severe malignancy, accounting for 10 % of all cancer-related deaths (Jemal et al. 2011). Current methods are not sensitive and specific enough to detect the disease at early stage. However, molecular imaging, which can identify both architectural changes and functional alterations, can improve early diagnosis and overall survival (Goetz and Wang 2010). Probes are agents with selective affinity for a given target; they can be antibodies, antibody fragments, peptides or aptamers (Seaman et al. 2010). Peptide probes have been widely used because of their high affinity, diversity, rapid clearance and minimal immunogenicity (Atreya and Goetz 2013). Peptide probes have proven particularly useful for the detection of early stage lesions within the gastrointestinal tract because they can be more easily delivered to the location of the carcinoma and can penetrate into the lesion with rapid binding kinetics (Miller et al. 2011). Phage display is a powerful method for peptide discovery that uses recombinant DNA technology to generate genetically modified phages, which present randomized peptides or proteins on their surfaces (Noren and Noren 2001). A phage library often expresses up to 107–109 unique sequences (Noren and Noren 2001) and can be screened on various targets, including proteins, cells and organs (Costantini et al. 2012; Wang et al. 2013; Xiao et al. 2011). CD44 is a membrane glycoprotein and the principle receptor of hyaluronic acid, takes part in tumor invasion, metastasis and proliferation (Ponta et al. 2003). CD44 is absent in normal epithelium but higher expressed in gastric cancer tissues (Wang et al. 2011; Washington et al. 1994); its expression also correlates with clinical outcome (Winder et al. 2011). CD44-positive gastric cancer cells exhibit spheroid colony formation and tumorigenic ability—both properties of cancer stem cells (Chen et al. 2013). As such, CD44 may be a biomarker T. D. Wang Division of Gastroenterology and Hepatology, Department of Medicine, School of Medicine, University of Michigan Ann Arbor, Ann Arbor, MI 48109, USA e-mail: [email protected]

123

for gastric neoplasia diagnosis and prognosis, as well as an indicator of cancer stem cells. However, to the best of our knowledge, peptides have never been screened against CD44 for the diagnosis of gastric carcinoma. Thus, we designed this study to screen and identify a CD44 specific peptide that can target gastric cancer.

Materials and methods Cell lines BGC-823, SGC-7901, and MKN-28, all gastric cancer cell lines, were cultured in RPMI 1640 medium with 10 % (v/v) fetal bovine serum (FBS) and maintained in a 5 % CO2 incubator at 37 °C. Each cell line was purchased from the Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. Selection of CD44 specific phages Screening was carried out according to the manufacturer’s instructions of the Ph.D.-12 Phage Display Peptide Library (New England BioLabs). Briefly, 5 lg CD44 which generated form human HEK 293 cells (OriGene) in 100 ll 0.1 M NaHCO3 (pH 8.6) was immobilized on an ELISA plate at 4 °C overnight. Approx. 1011 plaque-forming units (pfu) of phages from the library were incubated with BSA for 1 h at 25 °C; following this, unbound phages were added to the well coated by CD44 for 1 h at room temperature to allow phage-protein binding. After the fourth round of screening, 30 clones were randomly picked and analyzed. The recovery rate of each cycle was calculated according to the following formula: Recovery rate ¼ output phages=input phages Enzyme-linked immunosorbent assay CD44 (1 lg/well) and BSA (negative control) were immobilized on an ELISA plate and incubated with candidate phages and unrelated phages randomly selected from the original library (1010/well). The bound phages were incubated with goat anti-M13 major coat protein polyclone antibody (1:200 v/v, Santa Cruz) and horseradish peroxidase (HRP) labeled rabbit anti goat antibody (1:5000 v/v) sequentially. 3,30 ,5,50 -Tetramethylbenzidine (TMB) buffer was applied to a color-

Biotechnol Lett

developing reaction which detected by a microplate reader at 450 nm. Selectivity was determined as (A1CD44–A2CD44)/(A1BSA–A2BSA), where A1CD44 and A1BSA indicated the A values obtained when candidate phages bound to CD44 and BSA separately. A2CD44 and A2BSA indicated background absorption values for CD44 and BSA, obtained in wells in which phages were substituted with TBS. Western Blot Whole cell protein was obtained using RIPA lysis buffer and separated on 10 % SDS-PAGE. Nitrocellulose membranes with transferred protein were blocked in 3 % (w/v) BSA and then incubated with either anti-CD44 monoclonal antibody or antiGAPDH monoclonal antibody at 1:200 and 1:500 dilutions (v/v), respectively. Membranes were then incubated with HRP-conjugated goat anti mouse antibody diluted 1:5000 (v/v) in TBST. This was followed by chemiluminescence to visualize the bands using ECL Plus Western Blotting Substrate (Pierce). Bound phage counts SGC-7901, BGC-823 and MKN-28 cells were seeded on 6-well plate (2 9 105/well) separately and grown overnight. Candidate phages, 1011, and unrelated phage (URps) were incubated with all the cells for 1 h at 4 °C. Bound phages were eluted and titered. Peptide synthesis The RP-1 peptide (WHPWSYLWTQQA) and a scrambled peptide (DSEPTYMRLGWV) were synthesized by ChinaPeptides Co., LTD and purified to greater than 95 % purity. All peptides were labeled with fluorescein isothiocyanate (FITC) and biotin at the N-terminus. Competitive binding assay CD44 (1 lg/well) was immobilized on an ELISA plate and treated with either candidate peptide or control peptide at 0.01, 0.1, 1, 10, 100, and 1000 lM for 1 h at 37 °C. This was followed by incubation with candidate phages. Bound phages were estimated by ELISA as described above. The rate of inhibition = 100 - 100 % 9 (A value of test—A value of blank control)/(A value of inhibition control—A value of blank

control). Inhibition control represented the reaction between phages and antibody without a peptide competitor, and the A value of the blank control was obtained from the control group lacking the anti-M13 antibody. Binding affinity of RP-1 peptide Non-competitive enzyme immunoassay was used for peptide bind affinity measurement. Immobilized CD44 and BSA (negative control) were probed by biotin labeled RP-1 at 16,000, 4000, 1000, 250, 62.5, 15.62, 3.91, 0.98 nM. Bound peptide was incubated with streptavidin-HRP then reacted with TMB. Measurements were made from the absorption (A) values and fitting curves were regressed using Origin 6.0. The equilibrium dissociation constants were calculated with the formula Kd = 2(n [L]0 - [L])/(n - 1). [L] and [L]0 represented the concentrations of peptide at which OD was half of the maximum A value when CD44 was not diluted or diluted n times, separately. Immunofluorescence BGC823, SGC7901, and MKN28 cells were grown on coverslips overnight and fixed with 4 % (w/v) paraformaldehyde at room temperature for 15 min. Samples were blocked in 3 % BSA (w/v) in PBS for 30 min at 37 °C. Cells were then incubated with 5 lM FITC-labeled peptides for 10 min at 37 °C and washed with 0.1 % (v/v) Tween-20 in TBS three times. This was followed by nuclear staining with 4,6diamidino-2-phenylindole (DAPI). A fluorescence microscope was used to visualize the slides. CD44 knockdown A double-stranded small interfering RNA (siRNA) for CD44 (50 -GAACGAAUCCUGAAGACAUCU-30 , sense strand) and a scrambled siRNA (50 -UUCUCCGAACGUGUCACGUUU-30 , sense strand) were synthesized by Biomics Biotech Co., LTD and transfected into cells using Lipofectamine 2000 reagent (Invitrogen) based on the manufacturer’s instructions. Flow cytometry Gastric cancer cells were digested with 0.25 % (w/v) trypsin, suspended in a staining buffer containing 1 % (v/ v) FBS at 2 9 107/ml. After blocked in 5 % (v/v) FBS,

123

Biotechnol Lett

Fig. 1 Selection and identification of phage clones. a Recovery rate increased with each cycle of panning. b Binding of phage clones to CD44 (black column) and BSA (white column) was evaluated by ELISA. TBS and unrelated phage were used as blank and negative controls, respectively. For each data point, measurements were made four times. Single asterisk denotes a selectivity ratio greater than 2.1. c CD44 is expressed at significant levels in both SGC-7901 and BGC-823 cells but not expressed in MKN-28 cells. GAPDH was used as an internal

control. d Bound phage count performed increasing binding of RP-1 (black column) and RP-3 (striped column) phages compared with unrelated phages (white column) on CD44 positive SGC-7901 and BGC-823 cells (P \ 0.05). No such binding affinity of candidate phages was seen on MKN-28 cells. For each data point, measurements were made there times. Single asterisk denotes statistically significant difference (P \ 0.05). All data were presented as mean ± SD

the cells were incubated with 1.5 lM FITC-labeled candidate peptide, control peptide, PBS, or 5 lg FITClabeled rat anti-CD44 monoclonal antibody (eBioscience)/ml; all incubations were performed for 30 min at room temperature. The binding ability of peptide probes was then analyzed using a flow cytometer.

accompanied by the corresponding pericarcinous tissue. Samples were from patients with primary gastric adenocarcinoma who had no prior treatment; all patients provided signed informed consent. The Medical Ethics Committee of Taizhou Hospital of Zhejiang province approved all studies. Sections were deparaffinized using xylene and then rehydrated, submerged in TE buffer (pH 9.0) containing 10 mM Tris/HCl and 1 mM EDTA, and heated in a microwave oven for antigen retrieval. While sections were cooled at room temperature, 3 % (v/v) H2O2 in

Tissue array and immunohistochemistry The tissue array, purchased from Shanghai Outdo Biotech Co., LTD., contained 31 gastric cancer cases

123

Biotechnol Lett Table 1 Amino acid sequences of biopanned peptides Peptide no.

AA sequences

Frequencya

RP-1

WHPWSYLWTQQAb

12

RP-2

EYVSPYAGPTHQ

3

RP-3 RP-4

TLWHPWHYPAMRb WHYNSWYRWPVM

8 2

RP-5

NNGHAQIYMVHK

3

RP-6

SYHHTPHFAGPP

1

RP-7

HWWSWYTPFNHT

1

a

The number of isolated individual phage clones with the same peptide sequence

b

The sequences with the hexapeptide motif WHPWXY, where X represents a variable amino acid

methanol was applied to eliminate the activity of endogenous peroxidase. After blocked with 5 % (v/v) FBS, sections were incubated with either 10 lM biotin-labeled peptides at 37 °C for 30 min, or mouse anti-CD44 monoclonal antibody at a 1:100 dilution (v/ v) for 1 h followed by incubation with biotin-labeled goat anti-mouse antibody at a dilution of 1:200 (v/v) for 1 h. Unbound peptides and antibody were washed away. Next, streptavidin-HRP was added to form a complex with biotin. Staining was performed with diaminobenzidine (DAB). All sections were counterstained with hematoxylin. Staining strength was determined by product of scores of the percentage of stained cells and the staining intensity. The percentage of stained cells was characterized as follows: 0, 0 %; 1, 0–10 %; 2, 10–50 %; 3, 50–80 %, and 4, [80 %. Staining intensity was scored as: 1 weak; 2 moderate; and 3 intense. For each specimen, 5 views were randomly taken, and the mean value was calculated. All scoring was blinded.

Autodock4.2 (The Scripps Research Institute) with Autodocktools1.5.6.

Molecular docking

Statistical analysis

The RP-1 peptide was constructed using SwissPdbViewer4.1 (ExPasy); energy was minimized using Scalable Molecular Dynamics (NAMD). The threedimensional structure of full length human CD44 has not been solved; thus, the hyaluronan binding domain of CD44 (PDB code: 1UUH) was used instead. The molecular docking process was performed using

Statistical analysis was performed using SPSS 11.0 for Windows. Data were expressed as mean ± SD. Student’s t test was used for analyzing differences between groups of quantitative data. Association between CD44 and probe staining was determined by Pearson correlation coefficient. P \ 0.05 was considered statistically significant.

Fig. 2 Binding of RP-1 to CD44 protein. a Competitive inhibition of phage binding to CD44 by corresponding peptides. As the concentration of pre-incubated RP-1 peptide increased, the rate of inhibition also gradually increased. A scrambled peptide served as negative controls. Triplicate detections were performed independently, and data are shown as mean ± SD. b Fitting curves of RP-1 peptide binding reaction. Non-linear fitting of A values to peptide concentrations was showed and used to determine binding affinity of RP-1 to CD44

123

Biotechnol Lett

Fig. 3 Binding of RP-1 peptide to gastric cancer cells. a FITClabeled RP-1 peptide stained CD44-positive gastric cancer cells SGC-7901 and BGC-823 but not CD44-negative cells MKN-28. FITC-labeled control peptide did not bind to all the three cell lines. Cells were visualized using a fluorescence microscope.

Scale bar 100 lm. b Flow cytometry analysis of RP-1 binding to CD44 positive and negative gastric cancer cells. SGC-7901, BGC-823 and MKN-28 cells were incubated with PBS (black), FITC-labeled control peptide (blue), FITC-labeled RP-1 (red) or FITC-labeled anti-CD44 antibody (green)

Results and discussion

1.65 9 10-3, indicating an obvious enrichment of phages with high affinity for CD44 (Fig. 1a). After four rounds panning, 30 phage clones were randomly selected and analyzed. Seven different peptide sequences were obtained, as shown in Table 1. In the CD44-binding group, the absorption (A) values of all candidate phages were statistically significantly different compared to those of unrelated phages (P \ 0.05). However, there was no such difference in the BSA-binding group (P [ 0.05). RP-1, RP-2, RP-3, RP-4, and RP-5 had selectivity ratios greater than 2.1. In particular, RP-1 had the

In vitro panning of CD44 protein and selection of phage clones A 12-mer phage display library was used to probe recombinant CD44 protein that was generated in a eukaryotic expression host (human HEK 293 cells) to allow for more biologically relevant expression. The number of recovered phages in the last round had risen 1140–fold compared to the number in the first round. The recovery rate increased from 1.84 9 10-6 to

123

Biotechnol Lett

significant levels in BGC-823 and SGC-7901 cells but was absent in MKN-28 cells. RP-1, RP-3 were tested on phage bound count. About 3.8 9 107, 5.9 9 106 and 6 9 105 RP-1 phages were eluted from SGC-7901, BGC-823 and MKN-28 cells separately. The numbers of bound phages were 4.3 9 106 (SGC-7901), 3 9 106 (BGC-823) and 3.3 9 105(MNK-28) in the RP-3 group. As shown in Fig. 1d, more RP-1 and RP-3 phages, especially PR-1 phages, performed high affinity to CD44 positive cells compared with CD44 negative cells (P \ 0.05). The numbers of unrelated phages which bound to SGC7901 and BGC-823 cells were only 1.1 9 106 and 1.3 9 106, significantly different from those obtained from candidate phage groups (P \ 0.05). RP-1 showed the highest frequency and selectivity, as well as bound phage on CD44 positive cells. As such, RP-1 was selected for further study. Binding of RP-1 to CD44 protein

Fig. 4 Binding activity of RP-1 peptide to CD44 knocked down SGC-7901 cells. a Detection of CD44 in SGC-7901 cells transfected with CD44 siRNA (lane 3), non-target siRNA (lane 2) or non-transfected cells (lane 1). GAPDH served as a loading control. b FITC-labeled RP-1 peptide stained non-transfected SGC-7901 cells (control) or SGC-7901 cells transfected with non-target siRNA (NT siRNA) more strongly than c those transfected with CD44 siRNA. Cells were visualized using a fluorescence microscope. Scale bar = 100 lm

highest selectivity ratio at 8.1; RP-1 also had the highest frequency (Fig. 1b; Table 1). With the same hexapeptide motif of RP-1, RP-3 showed the second highest selectivity and frequency. Although screening on recombinant protein maximizes the specificity of candidate peptides, this approach requires validation in cell-based systems. Three human gastric cancer cell lines were used in this study,two CD44-positive cell lines (BGC-823 and SGC-7901) (Xue et al. 2012; Zhang et al. 2013) and one CD44-negative cell line (MKN-28) (Yokozaki 2000). As shown in Fig. 1c, CD44 was expressed at

To determine if phages and synthesized peptide shared the same binding site, we competed the binding of RP1 phages to CD44 protein with increasing concentrations of RP-1 and control peptide. For RP-1, inhibition occurred in a dose-dependent manner demonstrating that the phages interacted with CD44 via the displayed peptide rather than via the M13 coat protein (Fig. 2a). In contrast to RP-1 peptide, the binding of phages could not be competed by the control peptide. A values, as measurements of RP-1 bound to CD44 protein, increased with concentration of incubated peptide until saturation reached (Fig. 2b). Concentrations of peptide at which the A value was half of the maximum A value were calculated by regression of the data. With the dissociation constants of Kd = 97.1 ± 6.89 nM, RP-1 could bind to CD44 with high affinity. Binding of RP-1 to gastric cancer cell lines Based on the results of immunofluorescence analysis, specific fluorescent staining, located at the cell membrane and cytoplasm, was observed in CD44-positive BGC-823 and SGC-7901 cells after targeting with FITC labeled RP-1. In contrast, no significant fluorescence was observed in FITC labeled RP-1 treated MKN-28 cells, which do not express any CD44 protein (Fig. 3a). Only background staining was

123

Biotechnol Lett

Fig. 5 Immunohistochemical staining of RP-1 and correlation with CD44 expression in gastric tissues. a Biotin labeled RP-1 peptide and anti-CD44 antibody bound to serial sections from the same gastric cancer and adjacent normal tissues. Scale bar 100 lm. b Statistical analysis of staining strength score between gastric adenocarcimoma (striped box) and adjacent non-

tumorous gastric tissue (white box) in the RP-1 and CD44 stained groups were shown. Single asterisk denotes statistically significant difference (P \ 0.05). c Correlation between RP-1 staining and CD44 expression was examined in 31 gastric cancer tissues. A linear positive correlation (r = 0.556 P \ 0.05) was observed using Pearson correlation analysis

observed when any of the three cell lines was incubated with FITC labeled control peptide. Flow cytometry was performed to validate the RP1/CD44 interaction quantitatively. Comparisons were made among RP-1, control peptide, PBS, and antiCD44 antibody. Following incubation with FITC labeled anti-CD44 antibody, the mean fluorescence intensities were 179 ± 4.4 (BGC-823) and 231 ± 7.7

(SGC-7901), while those in RP-1-stained groups were 47.3 ± 3.6 and 133 ± 2.6, respectively, following a trend as CD44 expression. Mean fluorescence intensities in the control peptide group were only 7.18 ± 0.23 (BGC-823) and 9.27 ± 0.78 (SGC7901), which were significantly different from RP-1 (P \ 0.05) (Fig. 3b). Following incubation with either RP-1 or control peptide, CD44-negative MKN-28

123

Biotechnol Lett Fig. 6 Docked structure of RP-1 on the hyaluronan binding domain of CD44. a The confirmation of RP-1 (gray) bound to the hyaluronan-binding domain of CD44 (green) with the lowest energy. b The interaction between RP-1 and the hyaluronan-binding domain of CD44 (PDB: 1UUH). The receptor is shown as a green ribbon, and the stick model represents residues near RP1. The ligand is shown as a line model. The carbon, oxygen, and nitrogen atoms are colored gray, red, and blue, respectively. Wireframe balls denote hydrogen bonds

cells showed fluorescent intensities of only 20 ± 0.12 and 5.8 ± 0.12, respectively, both of which were significantly lower than those obtained in CD44positive BGC-823 and SGC-7901 cells (P \ 0.05) (Fig. 3b). The expression of CD44 was knocked down in SGC-7901 cells with siRNA, and the results were validated via western blot (Fig. 4a). Compared to the fluorescent staining of RP-1 on wild type SGC-7901 cells and those transfected with a non-target siRNA, staining was weaker on the same cell line whose expression of CD44 was knocked down when probed by FITC labeled RP-1 (Fig. 4b). These results suggest that RP-1 binds to gastric cancer cells via CD44. Immunohistochemical staining of RP-1 and correlation with CD44 expression in gastric cancers Both CD44 and biotin labeled RP-1 staining were localized at the cell membrane and cytoplasm

(Fig. 5a). The average score of RP-1 staining in adjacent non-tumor gastric tissues was 3.72 ± 1.81; the score rose to 5.47 ± 2.93 in the gastric cancer group. The staining strength grade was stratified as low (\6 score) and high (C6 score). Among the 31 cases of gastric cancer, 15 (48.4 %) and 19 (61.3 %) cases were high grade for RP-1 and CD44, respectively; 12 (38.7 %) cases scored high for both. Staining strength of both the peptide probe and CD44 antibody on gastric cancer tissues was significantly higher than that in adjacent normal tissue (P \ 0.05) (Fig. 5b). By Pearson correlation analysis, we observed a linear positive correlation between RP1 and CD44 (r = 0.556 P \ 0.05) (Fig. 5c), indicating that RP-1 might target gastric carcinomas by binding to CD44. Molecular docking The structure of the hyaluronan binding domain of human CD44 (PDB code: 1UUH) had been

123

Biotechnol Lett

determined from X-ray crystallography and NMR spectroscopy (Teriete et al. 2004). The docking assay revealed nine different docked confirmations. Of these, -8.6 kcal/mol was the lowest estimated binding free energy (Fig. 6). RP-1 formed hydrogen bonds with residues Glu37 and Asn94 of the CD44 hyaluronan binding domain. Through this interaction, RP-1 may influence CD44 biological function and its role in oncogenesis. However, because the three-dimensional structure of full-length CD44 has not yet been solved, we cannot rule out the possibility that RP-1 may also interact with other domains of the CD44 protein. In conclusion a novel peptide specific to CD44 is reported. The in vitro binding assay provides strong evidence of affinity and specificity of RP-1 towards CD44-positive gastric cancer cells and tissues. Thus, RP-1 can be used as a probe for the detection and monitoring of CD44 positive stomach tumors. Further studies should focus on in vivo tumor imaging and pharmacokinetics, as well as the application of RP-1 for the treatment of CD44 associated tumor growth and metastasis. Such efforts will make RP-1 more applicable for cancer diagnosis and treatment. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (No. 81172359 and 81472747). Compliance with Ethical Standards Conflict of interest

The authors declare no conflict of interest.

References Atreya R, Goetz M (2013) Molecular imaging in gastroenterology. Nat Rev Gastroenterol Hepatol 10:704–712 Chen W, Zhang X, Chu C, Cheung WL, Ng L, Lam S, Chow A, Lau T, Chen M, Li Y et al (2013) Identification of CD44 ? cancer stem cells in human gastric cancer. Hepatogastroenterology 60:949–954 Costantini TW, Eliceiri BP, Putnam JG, Bansal V, Baird A, Coimbra R (2012) Intravenous phage display identifies peptide sequences that target the burn-injured intestine. Peptides 38:94–99 Goetz M, Wang TD (2010) Molecular imaging in gastrointestinal endoscopy. Gastroenterology 138(828–833):e821

123

Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D (2011) Global cancer statistics. CA Cancer J Clin 61:69–90 Miller SJ, Joshi BP, Feng Y, Gaustad A, Fearon ER, Wang TD (2011) In vivo fluorescence-based endoscopic detection of colon dysplasia in the mouse using a novel peptide probe. PLoS ONE 6:e17384 Noren KA, Noren CJ (2001) Construction of high-complexity combinatorial phage display peptide libraries. Methods 23:169–178 Ponta H, Sherman L, Herrlich PA (2003) CD44: from adhesion molecules to signalling regulators. Nat Rev Mol Cell Biol 4:33–45 Seaman ME, Contino G, Bardeesy N, Kelly KA (2010) Molecular imaging agents: impact on diagnosis and therapeutics in oncology. Expert Rev Mol Med 12:e20 Teriete P, Banerji S, Noble M, Blundell CD, Wright AJ, Pickford AR, Lowe E, Mahoney DJ, Tammi MI, Kahmann JD et al (2004) Structure of the regulatory hyaluronan binding domain in the inflammatory leukocyte homing receptor CD44. Mol Cell 13:483–496 Wang T, Ong CW, Shi J, Srivastava S, Yan B, Cheng CL, Yong WP, Chan SL, Yeoh KG, Iacopetta B et al (2011) Sequential expression of putative stem cell markers in gastric carcinogenesis. Br J Cancer 105:658–665 Wang H, Ma C, Li R, Guo Y, He Y, Wang X, Chen Y, Hou Y (2013) Selection and characterization of colorectal cancer cell-specific peptides. Biotechnol Lett 35:671–677 Washington K, Gottfried MR, Telen MJ (1994) Expression of the cell adhesion molecule CD44 in gastric adenocarcinomas. Hum Pathol 25:1043–1049 Winder T, Ning Y, Yang D, Zhang W, Power DG, Bohanes P, Gerger A, Wilson PM, Lurje G, Tang LH et al (2011) Germline polymorphisms in genes involved in the CD44 signaling pathway are associated with clinical outcome in localized gastric adenocarcinoma. Int J Cancer 129:1096–1104 Xiao N, Cheng D, Wang Y, Chen L, Liu X, Dou S, Liu G, Liang M, Hnatowich DJ, Rusckowski M (2011) Identification of a high affinity TAG-72 binding peptide by phage display selection. Cancer Biol Ther 11:22–31 Xue Z, Yan H, Li J, Liang S, Cai X, Chen X, Wu Q, Gao L, Wu K, Nie Y et al (2012) Identification of cancer stem cells in vincristine preconditioned SGC7901 gastric cancer cell line. J Cell Biochem 113:302–312 Yokozaki H (2000) Molecular characteristics of eight gastric cancer cell lines established in Japan. Pathol Int 50:767–777 Zhang H, Xi H, Cai A, Xia Q, Wang XX, Lu C, Zhang Y, Song Z, Wang H, Li Q et al (2013) Not all side population cells contain cancer stem-like cells in human gastric cancer cell lines. Dig Dis Sci 58:132–139