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Screening of a specific peptide binding to esophageal squamous carcinoma cells from phage displayed peptide library
Molecular and Cellular Probes xxx (2015) 1e8
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Screening of a specific peptide binding to esophageal squamous carcinoma cells from phage displayed peptide library Caixia Ma a, Chunyan Li a, Dongliang Jiang b, Xiaojie Gao c, Juanjuan Han a, Nan Xu a, Qiong Wu a, Guochao Nie d, Wei Chen e, Fenghuei Lin f, Yingchun Hou a, * a
Co-Innovation Center for Qinba Region's Sustainable Development, Shaanxi Normal University, Xi'an, Shaanxi 710062, China Shaanxi Railway Institute, Weinan, Shaanxi 714000, China College of Life and Geography Science, Qinghai Normal University, Xining, Qinghai 810008, China d Center of Medical Nanomaterial, Yulin Normal College, Yulin, Guangxi 537000, China e Department of Physics and the SAVANT Center, The University of Texas at Arlington, Arlington, TX 76019, USA f Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 January 2015 Received in revised form 4 April 2015 Accepted 6 April 2015 Available online xxx
To select a specifically binding peptide for imaging detection of human esophageal squamous cell carcinoma (ESCC), a phage-displayed 12-mer peptide library was used to screen the peptide that bind to ESCC cells specifically. After four rounds of bio-panning, the phage recovery rate gradually increased, and specific phage clones were effectively enriched. The 60 randomly selected phage clones were tested using cellular enzyme-linked immunosorbent assay (ELISA), and 41 phage clones were identified as positive clones with the over 2.10 ratio of absorbance higher than other clones, IRP and PBS controls. From the sequencing results of the positive clones, 14 peptide sequences were obtained and ESCP9 consensus sequence was identified as the peptide with best affinity to ESCC cells via competitive inhibition, fluorescence microscopy, and flow cytometry. The results indicate that the peptide ESCP9 can bind to ESCC cells specifically and sensitively, and it is a potential candidate to be developed as an useful molecule to the imaging detection and targeting therapy for ESCC. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Esophageal squamous cell carcinoma Peptide Phage display peptide library Molecular imaging and targeting of cancer
1. Introduction Esophageal cancer (EC) is the second most common cause of death from gastrointestinal cancers over the past decades [1]. Esophageal squamous cell carcinoma (ESCC) is the predominant histological type of EC in East Asian countries, and one of the most malignant tumors [2]. Despite of the development of various therapeutic strategies, the prognosis of ESCC remains poor. The 5years survival rate of ESCC was 20e30% depending on the clinical stage at the time of diagnosis [3]. Thus, novel methods for the early detection of ESCC are urgently needed. Molecular imaging is an emerging technology that enables minimally-invasive visualization of disease-specific functional tissue alterations, provide a wide field of view, and highlight
* Corresponding author. Department of Cell Biology, School of Life Sciences, Shaanxi Normal University, 199 South Chang'an Road, Xi'an, Shaanxi 710062, China. Tel.: þ86 29 85310266; fax: þ86 29 85310564. E-mail address: [email protected] (Y. Hou).
abnormalities at the cellular even at molecular levels [4]. The early detection of cancers can be visualized with use of exogenous probes that target unique protein expression patterns [5,6]. Peptides have the advantages as detection probes because of their high clonal diversity, small size, rapid binding kinetics, and low immunogenicity [7,8]. Therefore, exploring novel peptides that bind to ESCC cells with high specificity and sensitivity is extremely important for the early detection and treatment in ESCC patients. With the development of phage display technology, more and more peptide-based probes with high specificity and affinity have been identified for the imaging detection and targeting therapy of cancers. Phage display is a powerful technique that allows the presentation of multiple different peptides on the surface of filamentous phage particles for various applications, providing a means to improve peptide affinity and generate unique peptides that bind any given target [9]. Recently, the bio-panning of phage display peptide library on intact cells has proven successful for selecting peptides [10,11]. In this study, we have tried to find out novel peptides targeting ESCC cells from a 12-mer phage display peptide library. One of 14
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consensus sequences, ESCP9, was identified as the peptide with best affinity to ESCC cells via competitive inhibition, fluorescence microscopy, and flow cytometry. The results demonstrated that the peptide ESCP9 can bind to ESCC cells specifically and sensitively, and it is a potential candidate to be developed as an useful molecule to the imaging detection and targeting therapy for ESCC.
10e30 min, and the reaction was then terminated by adding 2M H2SO4. The 96-well plates were then measured at 450 nm using an ELISA reader (Bio-Tek ELX800, USA). Irrelevant phage clone (IRP, an amplified phage randomly selected from the original phage peptide library) and PBS were used as control groups. 2.4. DNA sequencing of the positive phage clones
2. Materials and methods 2.1. Cell lines and cell culture Human Eca-109 and TE-1 (esophageal squamous carcinoma cells), and HEK293 (human embryonic kidney cells) were cultured in either RPMI-1640 or DMEM media, respectively, supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin (Amresco, USA). All cell lines were grown as adherent monolayer cultures at 37 C with 5% CO2 in a humidified incubator, and purchased from the Tissue Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). 2.2. Subtractive bio-panning in vitro The Ph.D.-12™ phage display peptide library kit consisting of M13 bacteriophage that expresses ~109 unique 12-amino acid sequences, Escherichia coli host strain ER2738, and a bio-panning protocol based on a subtractive whole cell approach was purchased from New England BioLabs (Beverly, MA, USA). Eca-109 and HEK293 cells were used as positive target cells and negative absorber cells, respectively. When Eca-109 and HEK293 cells were grown to log phase, the HEK293 cells were washed 3 times with phosphate-buffered saline (PBS) and blocked with 3% bovine serum albumin (BSA) for 2 h at 37 C. Approximately 1e2 1011 pfu phages were incubated with HEK293 cells at 37 C for 1 h with gentle agitation. After incubation, the supernatant containing unbound phages was incubated with the blocked Eca-109 cells at 37 C for 2 h with gentle agitation. The Eca-109 cells were washed 3 times with 0.1% PBST (PBS/0.1% Tween-20, v/v) to remove the unbound phages. The cell membrane-bound phages were then eluted with 260 ml of 0.2 M glycine (pH 2.2) for 10 min on ice and immediately neutralized with 40 ml of 1 M TriseHCL (pH 9.1). The eluted phages were amplified, purified, and tittered according to the manufacturer's instructions. Subsequently, 1e2 1011 pfu phages were subjected to the next round of bio-panning. After the fourth round of bio-panning, 60 phage clones were randomly picked out from titered phage plaques for enzyme-linked immunosorbent assay (ELISA). 2.3. ELISA assay Eca-109 and HEK293 cells were seeded into 96-well plates (1 104 cells/well). The cells were washed twice and fixed with 4% paraformaldehyde for 30 min at room temperature. A solution of H2O2 (3%, 100 ml/well) was added, and the plates were placed at room temperature for 30 min to inhibit the activity of endogenous peroxidase. The cells were then blocked with 3% BSA at 37 C for 2 h. The phages were added to Eca-109 and HEK293 cells (1 1010 pfu, 100 ml/well) and incubated at 37 C for 1 h. After washing with 0.1% PBST 3 times, 100 ml of goat anti-M13 polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was incubated with the cells at 37 C for 1 h. After washing 3 times with 0.1% PBST, 100 ml of horseradish peroxidase (HRP)-labeled rabbit anti-goat antibody IgG (Beijing Biosynthesis Biotechnology Co., Ltd., China) was incubated with the cells at 37 C for 1 h. After washing 3 times with 0.1% PBST, 3,30 ,5,50 -tetramethylbenzidine (TMB) (Sigma, Saint Louis, MO) was incubated with the cells at 37 C for
The selected positive phage clones were used to extract DNA for the sequencing analysis. An overnight culture of E. coli ER2738 was diluted to 1:100 in LB medium, and to a portion of 10 ml monoclonal phages were added. The mixture was shaken at 37 C for 4.5 h, and the supernatant was harvested by centrifugation at 13,000 rpm for 10 min. Then, 200 ml of PEG/NaCl was added to the supernatant to precipitate the phages. The precipitate was suspended in iodide buffer (10 mM TriseHCl (pH 8.0), 1 mM EDTA, and 4 M NaI), and followed by ethanol precipitation at room temperature for 10 min. The single-stranded DNA (ssDNA) was recovered and dissolved in TE buffer consisting of 10 mM Tris-HCL (pH 8.0), and 1 mM EDTA. DNA sequencing of the selected phages was carried out by GENEWIZ (Suzhou, China). The primer used for sequencing was -96gIII 50 -CCCTCATAGTTAGCG TAACG-30 . Homology analysis and multiple sequence alignment were performed according to the National Center of Biotechnology Information BLAST and Clustal W programs to determine groups of related peptides. 2.5. Cell immunofluorescence assay of positive phage clones Eca-109 and HEK293 cells were cultured on coverslips overnight and then fixed with 4% paraformaldehyde for 30 min at room temperature. Blocking of non-specific binding was performed by the addition of 1% BSA at 37 C for 30 min. Approximately 1 1010 pfu phages diluted in PBS were added and incubated with the cells at 37 C for 1 h. IRP and PBS were used as control groups. After washing 3 times with 0.1% PBST, the cells were incubated with goat anti-M13 polyclonal antibody at 4 C overnight. After washing 3 times with 0.1% PBST, the cells were then incubated with fluorescein isothiocyanate (FITC)-labeled rabbit anti-goat antibody IgG (Beijing Biosynthesis Biotechnology Co., Ltd., China) at 37 C for 40 min. After washing 3 times with 0.1% PBST, 40 ,6-diamidino-2phenylindole (DAPI) (SigmaeAldrich, St. Louis, MO, CA) was used to stain the nucleus. Fluorescence images were observed using laser scanning confocal microscope (LSCM, Leica, Germany). 2.6. Peptide synthesis The candidate and control peptide were synthesized using standard solid-phase fluorenylmethoxycarbonyl chemistry. An Aminocaproic Acid linker was added to the N-terminus for FITC labeling. The compounds were purified to 95% by high performance liquid chromatography (HPLC), isolated by lyophilization, and stored at 20 C. The sequence of each peptide was analyzed using mass spectrometry. 2.7. Competitive inhibition assay Eca-109 cells were seeded into 96-well plates (1 104 cells/well). The cells were washed twice and fixed with 4% paraformaldehyde for 30 min at room temperature. Synthetic peptides at concentrations of 0, 100, 200, 300, 400, 500, and 600 mM were incubated with Eca-109 Cells at 37 C for 1 h, and the random peptide was used as a control. Subsequently, the homologous positive phage clones (1e2 1011 pfu, 100 ml/well) was incubated with the cells at 37 C for 1 h. The antibody was used as described in the section “ELISA assay”. The rate of competitive inhibition was calculated according to the following
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2.8. Fluorescence microscopy and flow cytometry
Table 1 The recovery rate of each round bio-panning. Round
Input (pfu/ml)a
1 2 3 4
1.50 1.04 1.21 1.03
a b c
1011 1011 1011 1011
Output (pfu/ml)b 2.37 2.66 1.13 1.59
3
105 105 107 107
Recovery rate (%)c 1.58 2.56 9.34 1.54
106 106 105 104
Number of phages used at the initiation of a bio-panning round. Number of phages obtained at the end of a bio-panning round. Recovery rate ¼ output/input.
formula: inhibition ratio ¼ (ODcontrol ODphage)/ODcontrol 100%, where ODcontrol and ODphage represent the OD450 values of PBS and phage binding groups, respectively.
LSCM was used to observe the binding of synthetic ESCP9 peptide to ESCC (Eca-109, TE-1) and HEK293 cells. The cells were cultured on coverslips overnight, washed 3 times with PBS, and fixed with 4% paraformaldehyde at room temperature for 20 min. After washing 3 times with PBS, Dil (Beyotime, China) was used to stain the cell membrane at 37 C for 20 min. The cells were then washed 3 times with PBS and blocked with 1% BSA for 30 min. The FITC-labeled peptide ESCP9 was incubated with the cells at 37 C for 25 min. After washing 3 times with PBS, DAPI was used to stain the nucleus, and the slides were observed using LSCM. The binding ability of ESCP9 was further verified by flow cytometry (Guava easyCyte 8HT, Millipore, USA).
Fig. 1. Evaluation of the binding selectivity of 60 phage clones by cellular ELISA. (A) ELISA result of phage clones E1-E32. (B) ELISA result of phage clones E33-E60, IRP and PBS. Binding was plotted as a ratio of cancer/normal cell binding. Error bars represent the standard deviation of three replicates.
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3.2. Affinity confirmation of positive phage clones by cellular ELISA
Table 2 Peptide sequences of positive phage clones. Namesa
Phage clones
Peptide sequences
Frequencyb
ESCP1 ESCP2 ESCP3 ESCP4 ESCP5 ESCP6 ESCP7 ESCP8 ESCP9 ESCP10 ESCP11 ESCP12 ESCP13 ESCP14
E1/E14 E3/E10/E19 E7/E32 E8/E27/E28/E35/E38 E12/E15/E29 E2/E13/E20/E31/E33 E26 E21-25/E34/E41/E47 E30/E37/E39/E43/E49/E51/E60 E40 E42 E46 E48 E53
MHDASPQLYRGR QKESSTHFMAIH MEDNDTHWARMA QTHHTHLPIQRA SFPTMTENFYPR TINSNTLNSTPP TMYEPTTTRSPA LPADEDHHFYWT LLQLTTTKRPIT SVTAGMPNRSNR HLSVAKKPLHRP WPGLLGTTSPNI VHWPASQPVQIP HDHNTNDMTLNA
2 3 2 5 3 5 1 8 7 1 1 1 1 1
a The 41 positive phage clones were selected for DNA sequencing. 14 peptide sequences were obtained and named as ESCP1 to ESCP14. b The frequency represents the number of the peptide sequences appeared in the whole selected phage clones.
2.9. Statistical analysis Each experiment was repeated at least 3 times. Statistical analysis was performed using SPSS 20 (IBM, Armonk, NY, USA). All data are expressed as the mean ± S.D. Independent-Samples t-test was used to analyze the differences between the means. *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant.
After the fourth round of bio-panning, 60 independent phage clones were randomly selected and analyzed for the ability to selectively bind Eca-109 cells and not HEK293 cells. A cellular ELISA was performed to determine the affinity of the 60 phage clones for Eca-109 cells and exclude false positive clones. The phage affinity and specificity were analyzed by comparing target to non-target binding ratios. As shown in Fig. 1, 41 phage clones were identified as positive clones with the over 2.10 ratio of absorbance higher than other clones, IRP and PBS controls. The results demonstrated that 41 of 60 phage clones were identified as positive clones. 3.3. DNA sequencing of the positive phage clones The peptide sequences displayed on the 41 positive phage clones were deduced from the DNA sequences. As shown in Table 2, these amino acid sequences were analyzed and classified, 14 peptide sequences were obtained and designated ESCP1 to ESCP14. The ESCP4, ESCP6, ESCP8, and ESCP9 appeared five, five, eight, and seven times, respectively. And the corresponding phage clones E28, E13, E22, and E37 were further investigated. Similar sequences were identified by Clustal W and National Center of Biotechnology Information BLAST analysis between the peptides displayed by the phages and known proteins (Table 3). The results indicated that no homologous protein was occurred. 3.4. ESCC affinity analysis of the phage clones with consensus sequences
3. Results 3.1. Screening of Eca-109 cell specifically binding phage clones Phages that specifically bound to Eca-109 cells were identified through four rounds of in vitro subtractive bio-panning with Eca109 and HEK293 cells. In each round, a series of phages binding to the Eca-109 cells instead of control cells were rescued and amplified for the subsequent panning, whereas the unbound phages were removed by washing with different concentrations of PBST. As shown in Table 1, the recovery rate of phages after each round bio-panning was used to determine the enrichment efficiency, which increased from 1.58 106 to 1.54 104. The results revealed that phages that were capable of specifically binding to Eca-109 cells were significantly enriched.
Based on the results of ELISA and DNA sequencing, the positive phage clones E28, E13, E22, and E37 that specifically bound to Eca109 cells were used for further identification. Cell immunofluorescence assay was performed to detect the specific binding of the four phage clones to Eca-109 cells. As shown in Fig. 2A, the four phage clones all successfully discriminated Eca-109 cells from HEK293 cells (P < 0.001), but the phage E37 bound preferably to Eca-109 cells than the other phage clones. In contrast, the IRP bound similarly to the two types of cells. As the four phage clones showed different intensities of immunofluorescence staining, the intensity of green fluorescence signal was calculated using Image pro-Plus software 7.0, and the values are shown in Fig. 2B. The results confirmed that the four phage clones differ in their ability to interact with Eca-109 cells. Among the four phage clones, E37 bound most effectively to Eca-109 cells.
Table 3 Homology analysis of 14 peptides using protein data bank. Peptides
Homologous proteinsa
Identities
Homology (%)
ESCP1 ESCP2 ESCP3 ESCP4 ESCP5 ESCP6 ESCP7 ESCP8 ESCP9 ESCP10 ESCP11 ESCP12 ESCP13 ESCP14
Immunoglobulin epsilon heavy chain variable region Aftiphilin protein, isoform CRA-d Unnamed protein Protein FAM27D1 HCG2002731, isoform CRA Phosphatidylinositol 5-phosphate 4-kinase type-2 alpha ABLIM1 protein DNA polymerase alpha catalytic subunit CD99L2 protein GRB2-associated-binding protein 1 Transforming, acidic coiled-coil containing protein 2 Sodium-dependent vitamin C transporter 1 MAP kinase-interacting kinase EF-hand calcium-binding domain-containing protein 5
9/15 8/14 8/18 8/10 8/11 7/8 7/9 9/17 7/8 8/10 7/9 9/16 7/9 6/8
60% 57% 44% 80% 73% 88% 78% 53% 88% 80% 78% 56% 78% 75%
a
All peptides were analyzed with the homologous proteins from Homo sapiens.
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Fig. 3. Competitive inhibition of phage E37 to Eca-109 cells with synthetic peptide ESCP9. Binding of phage E37 to Eca-109 cells is reduced by competition with increasing concentrations of ESCP9 in a dose-dependent manner. The addition of the control peptide revealed no competitive inhibition.
interact with additional cancer cells (Fig. 2D). These results suggested that E37 was identified as the best positive clone with specific affinity to ESCC cells. 3.5. Peptide synthesis The candidate peptide (LLQLTTTKRPIT) displayed on positive phage clone E37 was synthesized using standard solid-phase Fmoc chemistry and named as ESCP9, and the control peptide (TIYEEDTTRSPA) displayed on IRP was also synthesized. An Aminocaproic Acid linker was added to the N-terminus for FITC labeling. The purity of ESCP9 on HPLC was >95%. On mass spectrometry, the experimental mass-to-charge (m/z) ratio measured for ESCP9 agreed with that expected. 3.6. Competitive inhibition assay
Fig. 2. Specific binding of the selected positive phage clones to Eca-109 and other control cells. (A) Immunofluorescence staining of Eca-109 and HEK293 cells with phage clones E28, E13, E22, and E37. The cell nuclei were shown in blue (DAPI) and the phages were shown in green. Scale bar 25 mm. (B) Fluorescence signal intensity analysis. The four phage clones had a significantly higher affinity for Eca-109 than IRP and PBS controls. (C) Immunofluorescence staining of additional cancer cells with phage E37. The cell lines include human esophageal (TE-1), gastric (SGC-7901), colorectal (Caco2), hepatic (SMMC-7721), breast (MCF7), and cervical (SiHa) cancers. (D) Fluorescence signal intensity analysis. The phage E37 bound TE-1 and SGC-7901 cells, but with a significantly lower affinity for other cancer cells. IOD/Area were calculated as the average green fluorescence intensity. Three fields of view per well were measured for each experiment. Values were shown as the mean ± standard deviation. ***P < 0.001; *P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
To further assess specificity, the top one phage clone E37 were screened for binding with a panel of cultured cells, including human esophageal (TE-1), gastric (SGC-7901), colorectal (Caco2), hepatic (SMMC-7721), breast (MCF7), cervical (SiHa) cancers. Fig. 2C demonstrated that the phage E37 bound TE-1 and SGC-7901 cells (P < 0.05), but with a lower affinity for Caco2, SMMC-7721, MCF7, and SiHa cells (P < 0.001). The fluorescent signal intensity analysis suggested that the phage E37 differ in their ability to
The competitive inhibition assay was performed to determine whether the peptide ESCP9 and the corresponding positive phage clone E37 could compete for the same binding site. With an increase in the concentration of ESCP9, the inhibition ratio increased gradually. When the peptide concentration was increased above 600 mM, the inhibition ratio was approximately 41.56% (Fig. 3). The control peptide had no significant effect on the binding of phage E37 to Eca-109 cells. The results demonstrated that the ESCP9 can compete with the corresponding phage E37 for binding to the Eca109 plasma membrane. 3.7. ESCC affinity of synthesized peptide To directly observe the specific binding of ESCP9 to ESCC cells, a fluorescence microscopy assay using FITC-labeled peptide ESCP9 was performed. After ESCC cells were incubated with ESCP9, specific fluorescence was observed on the membrane (Fig. 4A, B). In contrast, there was no significant green fluorescence in the control HEK293 cells (Fig. 4C), which might have resulted from a lack of ESCP9 receptors. The control peptide showed minimal binding to ESCC (Fig. 4D, E) and HEK293 cells (Fig. 4F). The results indicated that ESCP9 can bind to the membrane of ESCC cells specifically and sensitively.
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Fig. 4. Fluorescent imaging of ESCP9 binding to ESCC cells. The FITC-labeled peptide ESCP9 bound to Eca-109 (A) and TE-1 cells (B) but not to HEK293 cells (C). At the same time, the FITC-labeled control peptide bound similarly and with low affinity to Eca-109 (D), TE-1 (E), and HEK293 cells (F). The cell nuclei were shown in blue (DAPI), the plasma membrane was shown in red (Dil), and the peptide was shown in green (FITC). Scale bar 5 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
The specific binding activity of ESCP9 to ESCC cells was further validated by fluorescence-activated cell sorting (FACS) analysis, as shown in Fig. 5. The mean fluorescence value for binding of FITClabeled peptide ESCP9 to Eca-109 cells was 22.47 compared with 11.65 and 10.65 for the FITC-labeled control peptide and for PBS, respectively. Furthermore, the mean fluorescence value for binding of FITC-labeled peptide ESCP9 to TE-1 cells was 17.94 compared with 10.79 and 11.85 for the control peptide and PBS, respectively. In addition, the mean fluorescence values for binding to HEK293 cells were 12.19, 10.37, and 11.44 for the candidate peptide, the
control peptide, and PBS, respectively. The results suggested that the FITC-labeled peptide ESCP9 has specific binding activity to the ESCC cells and not to the control HEK293 cells. 4. Discussion ESCC, a predominant gastrointestinal malignancy, is increasing in incidence and associated with a poor prognosis [12]. Despite the development of various therapeutic strategies for ESCC, including chemotherapy, radiotherapy, surgical resection, and combination
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Fig. 5. FACS analysis of ESCP9 binding to ESCC cells. The FITC-labeled peptide ESCP9 (green line) and the FITC-labeled control peptide (red line) were incubated with Eca-109 (A), TE-1 (B), and control HEK293 cells (C), respectively. PBS was used as a blank control (black line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
therapy, the prognosis for ESCC patients remains unfavorable. Therefore, an innovative approach for early detection of ESCC are urgently needed. Phage display technology, a highly efficient tool to identify small peptides with affinity to a target, has been investigated in recent years for use in the clinical diagnosis and treatment of various types of cancers [13]. Hence, the discovery of peptides functionally targeting ESCC cells has received great attention to introduce a new concept of imaging for more competent diagnostics and be applied to a potent target therapy. In the present study, we used a subtractive whole cell biopanning strategy of the 12-mer phage display peptide library to identify peptides that bind specifically to the membrane of Eca-109 cells. To decrease non-specific binding in each round, the original phage display peptide library was panned against HEK293 cells before screening with Eca-109 cells. Since it is difficult to isolate the normal tissue cells, the human HEK293 embryonic kidney cells was often chosen as control cells [14,15]. After four rounds of biopanning, the phage recovery rate increased gradually, and specific phage clones were effectively enriched. We randomly selected 60 independent phage clones and individually amplified for further identification. Preferential binding of the 60 phage clones to Eca-109 cells was validated by ELISA. The results showed that 41 phage clones were identified as positive clones with the over 2.10 ratio of absorbance higher than other clones, IRP and PBS controls. Previous work has demonstrated that analyzing phage affinity and specificity by comparing target to non-target binding ratios is a successful predictor of tumor homing ability [16]. After the 41 phage clones were sequenced, 14 different peptide sequences were obtained and designated ESCP1 to ESCP14. A multiple sequence alignment showed that the peptide sequence of ESCP9 did not exhibit complete homology to the sequences of any characterized proteins in various protein databases. The peptide was found to have partial homology (7 of 8 amino acids) to the CD99L2 protein. This finding demonstrated that ESCP9 was a novel peptide. The competitive inhibition assay demonstrated that E37 and ESCP9 compete for the same binding sites and the binding was determined by the specific sequence of the peptide rather than by the phage coat proteins. Although the ESCP9 binds specifically to Eca-109 cells, future studies should be performed to determine whether the peptide can interact with one or more membrane receptors in ESCC. The fluorescence microscopy and flow cytometry assay were performed to directly observe the binding of ESCP9 to ESCC cells. These results indicated that ESCP9 bound specifically to ESCC cells and not to the control HEK293 cells. Therefore, ESCP9 may be an effective targeting probe for the diagnosis and therapy of ESCC. According to the previously reported studies, a novel peptide has been considered to be useful for a small molecule probe
leading to multifunctional properties for both imaging detection and targeting therapy [17,18]. Peptides could be conjugated to apply as an imaging probe with various molecules such as radionuclides [19,20], iron oxide [21], and fluorescent agents [22]. Peptides could be utilized to provide a feasibility of therapeutic targets for the development of potential therapeutics [23]. Peptides also could be tethered to nanoparticles for the development of chemotherapeutic drug delivery systems [24,25]. Further studies will aim to identify whether ESCP9 can specifically deliver chemotherapeutic drugs to tumors, bind to certain receptors with high affinity, act as an antagonist to inhibit cancer migration and growth, and bind to ESCC in vivo. In conclusion, it is warranted that the peptide ESCP9 specifically targeting ESCC cells provides substantive possibilities for imaging detection and targeting therapy.
Acknowledgments The study was supported by National Natural Science Foundation of China (No. 81172359).
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[13] Brown KC. New approaches for cell-specific targeting. Identification of cellselective peptides from combinatorial libraries. Curr Opin Chem Biol 2000;4:16e21. [14] Wang YQ, Cheng XX, Wang P, Wang L, Fan JP, Wang XB, et al. Investigating migration inhibition and apoptotic effects of Fomitopsis pinicola Chloroform extract on human colorectal Cancer SW-480 cells. PLoS One 2014;9. [15] Guo Y, Ma C, Li C, Wu J, Zhang D, Han J, et al. Screening and identification of a specific peptide binding to hepatocellular carcinoma cells from a phage display peptide library. J Pept Sci 2014;20:196e202. [16] Newton JR, Kelly KA, Mahmood U, Weissleder R, Deutscher SL. In vivo selection of phage for the optical imaging of PC-3 human prostate carcinoma in mice. Neoplasia 2006;8:772e80. [17] Park HY, Kim J, Cho JH, Moon JY, Lee SJ, Yoon MY. Phage display screen for peptides that bind Bcl-2 protein. J Biomol Screen 2011;16:82e9. [18] Lee KJ, Lee JH, Chung HK, Choi J, Park J, Park SS, et al. Novel peptides functionally targeting in vivo human lung cancer discovered by in vivo peptide displayed phage screening. Amino Acids 2015;47(2):281e9. [19] Liu Y, Lan X, Wu T, Lang J, Jin X, Sun X, et al. (99m)Tc-labeled SWL specific peptide for targeting EphA2 receptor. Nucl Med Biol 2014;41:450e6.
[20] Askoxylakis V, Marr A, Altmann A, Markert A, Mier W, Debus J, et al. Peptidebased targeting of the platelet-derived growth factor receptor beta. Mol Imaging Biol 2013;15:212e21. [21] Ghosh D, Lee Y, Thomas S, Kohli AG, Yun DS, Belcher AM, et al. M13-templated magnetic nanoparticles for targeted in vivo imaging of prostate cancer. Nat Nanotechnol 2012;7:677e82. [22] Sturm MB, Joshi BP, Lu S, Piraka C, Khondee S, Elmunzer BJ, et al. Targeted imaging of esophageal neoplasia with a fluorescently labeled peptide: first-inhuman results. Sci Transl Med 2013;5:184ra161. [23] Fei LK, Yap LP, Conti PS, Shen WC, Zaro JL. Tumor targeting of a cell penetrating peptide by fusing with a pH-sensitive histidine-glutamate co-oligopeptide. Biomaterials 2014;35:4082e7. [24] Wang S, Placzek WJ, Stebbins JL, Mitra S, Noberini R, Koolpe M, et al. Novel targeted system to deliver chemotherapeutic drugs to EphA2-expressing cancer cells. J Med Chem 2012;55:2427e36. [25] Han CY, Yue LL, Tai LY, Zhou L, Li XY, Xing GH, et al. A novel small peptide as an epidermal growth factor receptor targeting ligand for nanodelivery in vitro. Int J Nanomed 2013;8:1541e9.
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Screening of a specific peptide binding to esophageal squamous carcinoma cells from phage displayed peptide library Caixia Ma a, Chunyan Li a, Dongliang Jiang b, Xiaojie Gao c, Juanjuan Han a, Nan Xu a, Qiong Wu a, Guochao Nie d, Wei Chen e, Fenghuei Lin f, Yingchun Hou a, * a
Co-Innovation Center for Qinba Region's Sustainable Development, Shaanxi Normal University, Xi'an, Shaanxi 710062, China Shaanxi Railway Institute, Weinan, Shaanxi 714000, China College of Life and Geography Science, Qinghai Normal University, Xining, Qinghai 810008, China d Center of Medical Nanomaterial, Yulin Normal College, Yulin, Guangxi 537000, China e Department of Physics and the SAVANT Center, The University of Texas at Arlington, Arlington, TX 76019, USA f Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 January 2015 Received in revised form 4 April 2015 Accepted 6 April 2015 Available online xxx
To select a specifically binding peptide for imaging detection of human esophageal squamous cell carcinoma (ESCC), a phage-displayed 12-mer peptide library was used to screen the peptide that bind to ESCC cells specifically. After four rounds of bio-panning, the phage recovery rate gradually increased, and specific phage clones were effectively enriched. The 60 randomly selected phage clones were tested using cellular enzyme-linked immunosorbent assay (ELISA), and 41 phage clones were identified as positive clones with the over 2.10 ratio of absorbance higher than other clones, IRP and PBS controls. From the sequencing results of the positive clones, 14 peptide sequences were obtained and ESCP9 consensus sequence was identified as the peptide with best affinity to ESCC cells via competitive inhibition, fluorescence microscopy, and flow cytometry. The results indicate that the peptide ESCP9 can bind to ESCC cells specifically and sensitively, and it is a potential candidate to be developed as an useful molecule to the imaging detection and targeting therapy for ESCC. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Esophageal squamous cell carcinoma Peptide Phage display peptide library Molecular imaging and targeting of cancer
1. Introduction Esophageal cancer (EC) is the second most common cause of death from gastrointestinal cancers over the past decades [1]. Esophageal squamous cell carcinoma (ESCC) is the predominant histological type of EC in East Asian countries, and one of the most malignant tumors [2]. Despite of the development of various therapeutic strategies, the prognosis of ESCC remains poor. The 5years survival rate of ESCC was 20e30% depending on the clinical stage at the time of diagnosis [3]. Thus, novel methods for the early detection of ESCC are urgently needed. Molecular imaging is an emerging technology that enables minimally-invasive visualization of disease-specific functional tissue alterations, provide a wide field of view, and highlight
* Corresponding author. Department of Cell Biology, School of Life Sciences, Shaanxi Normal University, 199 South Chang'an Road, Xi'an, Shaanxi 710062, China. Tel.: þ86 29 85310266; fax: þ86 29 85310564. E-mail address: [email protected] (Y. Hou).
abnormalities at the cellular even at molecular levels [4]. The early detection of cancers can be visualized with use of exogenous probes that target unique protein expression patterns [5,6]. Peptides have the advantages as detection probes because of their high clonal diversity, small size, rapid binding kinetics, and low immunogenicity [7,8]. Therefore, exploring novel peptides that bind to ESCC cells with high specificity and sensitivity is extremely important for the early detection and treatment in ESCC patients. With the development of phage display technology, more and more peptide-based probes with high specificity and affinity have been identified for the imaging detection and targeting therapy of cancers. Phage display is a powerful technique that allows the presentation of multiple different peptides on the surface of filamentous phage particles for various applications, providing a means to improve peptide affinity and generate unique peptides that bind any given target [9]. Recently, the bio-panning of phage display peptide library on intact cells has proven successful for selecting peptides [10,11]. In this study, we have tried to find out novel peptides targeting ESCC cells from a 12-mer phage display peptide library. One of 14
http://dx.doi.org/10.1016/j.mcp.2015.04.001 0890-8508/© 2015 Elsevier Ltd. All rights reserved.
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consensus sequences, ESCP9, was identified as the peptide with best affinity to ESCC cells via competitive inhibition, fluorescence microscopy, and flow cytometry. The results demonstrated that the peptide ESCP9 can bind to ESCC cells specifically and sensitively, and it is a potential candidate to be developed as an useful molecule to the imaging detection and targeting therapy for ESCC.
10e30 min, and the reaction was then terminated by adding 2M H2SO4. The 96-well plates were then measured at 450 nm using an ELISA reader (Bio-Tek ELX800, USA). Irrelevant phage clone (IRP, an amplified phage randomly selected from the original phage peptide library) and PBS were used as control groups. 2.4. DNA sequencing of the positive phage clones
2. Materials and methods 2.1. Cell lines and cell culture Human Eca-109 and TE-1 (esophageal squamous carcinoma cells), and HEK293 (human embryonic kidney cells) were cultured in either RPMI-1640 or DMEM media, respectively, supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin (Amresco, USA). All cell lines were grown as adherent monolayer cultures at 37 C with 5% CO2 in a humidified incubator, and purchased from the Tissue Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). 2.2. Subtractive bio-panning in vitro The Ph.D.-12™ phage display peptide library kit consisting of M13 bacteriophage that expresses ~109 unique 12-amino acid sequences, Escherichia coli host strain ER2738, and a bio-panning protocol based on a subtractive whole cell approach was purchased from New England BioLabs (Beverly, MA, USA). Eca-109 and HEK293 cells were used as positive target cells and negative absorber cells, respectively. When Eca-109 and HEK293 cells were grown to log phase, the HEK293 cells were washed 3 times with phosphate-buffered saline (PBS) and blocked with 3% bovine serum albumin (BSA) for 2 h at 37 C. Approximately 1e2 1011 pfu phages were incubated with HEK293 cells at 37 C for 1 h with gentle agitation. After incubation, the supernatant containing unbound phages was incubated with the blocked Eca-109 cells at 37 C for 2 h with gentle agitation. The Eca-109 cells were washed 3 times with 0.1% PBST (PBS/0.1% Tween-20, v/v) to remove the unbound phages. The cell membrane-bound phages were then eluted with 260 ml of 0.2 M glycine (pH 2.2) for 10 min on ice and immediately neutralized with 40 ml of 1 M TriseHCL (pH 9.1). The eluted phages were amplified, purified, and tittered according to the manufacturer's instructions. Subsequently, 1e2 1011 pfu phages were subjected to the next round of bio-panning. After the fourth round of bio-panning, 60 phage clones were randomly picked out from titered phage plaques for enzyme-linked immunosorbent assay (ELISA). 2.3. ELISA assay Eca-109 and HEK293 cells were seeded into 96-well plates (1 104 cells/well). The cells were washed twice and fixed with 4% paraformaldehyde for 30 min at room temperature. A solution of H2O2 (3%, 100 ml/well) was added, and the plates were placed at room temperature for 30 min to inhibit the activity of endogenous peroxidase. The cells were then blocked with 3% BSA at 37 C for 2 h. The phages were added to Eca-109 and HEK293 cells (1 1010 pfu, 100 ml/well) and incubated at 37 C for 1 h. After washing with 0.1% PBST 3 times, 100 ml of goat anti-M13 polyclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was incubated with the cells at 37 C for 1 h. After washing 3 times with 0.1% PBST, 100 ml of horseradish peroxidase (HRP)-labeled rabbit anti-goat antibody IgG (Beijing Biosynthesis Biotechnology Co., Ltd., China) was incubated with the cells at 37 C for 1 h. After washing 3 times with 0.1% PBST, 3,30 ,5,50 -tetramethylbenzidine (TMB) (Sigma, Saint Louis, MO) was incubated with the cells at 37 C for
The selected positive phage clones were used to extract DNA for the sequencing analysis. An overnight culture of E. coli ER2738 was diluted to 1:100 in LB medium, and to a portion of 10 ml monoclonal phages were added. The mixture was shaken at 37 C for 4.5 h, and the supernatant was harvested by centrifugation at 13,000 rpm for 10 min. Then, 200 ml of PEG/NaCl was added to the supernatant to precipitate the phages. The precipitate was suspended in iodide buffer (10 mM TriseHCl (pH 8.0), 1 mM EDTA, and 4 M NaI), and followed by ethanol precipitation at room temperature for 10 min. The single-stranded DNA (ssDNA) was recovered and dissolved in TE buffer consisting of 10 mM Tris-HCL (pH 8.0), and 1 mM EDTA. DNA sequencing of the selected phages was carried out by GENEWIZ (Suzhou, China). The primer used for sequencing was -96gIII 50 -CCCTCATAGTTAGCG TAACG-30 . Homology analysis and multiple sequence alignment were performed according to the National Center of Biotechnology Information BLAST and Clustal W programs to determine groups of related peptides. 2.5. Cell immunofluorescence assay of positive phage clones Eca-109 and HEK293 cells were cultured on coverslips overnight and then fixed with 4% paraformaldehyde for 30 min at room temperature. Blocking of non-specific binding was performed by the addition of 1% BSA at 37 C for 30 min. Approximately 1 1010 pfu phages diluted in PBS were added and incubated with the cells at 37 C for 1 h. IRP and PBS were used as control groups. After washing 3 times with 0.1% PBST, the cells were incubated with goat anti-M13 polyclonal antibody at 4 C overnight. After washing 3 times with 0.1% PBST, the cells were then incubated with fluorescein isothiocyanate (FITC)-labeled rabbit anti-goat antibody IgG (Beijing Biosynthesis Biotechnology Co., Ltd., China) at 37 C for 40 min. After washing 3 times with 0.1% PBST, 40 ,6-diamidino-2phenylindole (DAPI) (SigmaeAldrich, St. Louis, MO, CA) was used to stain the nucleus. Fluorescence images were observed using laser scanning confocal microscope (LSCM, Leica, Germany). 2.6. Peptide synthesis The candidate and control peptide were synthesized using standard solid-phase fluorenylmethoxycarbonyl chemistry. An Aminocaproic Acid linker was added to the N-terminus for FITC labeling. The compounds were purified to 95% by high performance liquid chromatography (HPLC), isolated by lyophilization, and stored at 20 C. The sequence of each peptide was analyzed using mass spectrometry. 2.7. Competitive inhibition assay Eca-109 cells were seeded into 96-well plates (1 104 cells/well). The cells were washed twice and fixed with 4% paraformaldehyde for 30 min at room temperature. Synthetic peptides at concentrations of 0, 100, 200, 300, 400, 500, and 600 mM were incubated with Eca-109 Cells at 37 C for 1 h, and the random peptide was used as a control. Subsequently, the homologous positive phage clones (1e2 1011 pfu, 100 ml/well) was incubated with the cells at 37 C for 1 h. The antibody was used as described in the section “ELISA assay”. The rate of competitive inhibition was calculated according to the following
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2.8. Fluorescence microscopy and flow cytometry
Table 1 The recovery rate of each round bio-panning. Round
Input (pfu/ml)a
1 2 3 4
1.50 1.04 1.21 1.03
a b c
1011 1011 1011 1011
Output (pfu/ml)b 2.37 2.66 1.13 1.59
3
105 105 107 107
Recovery rate (%)c 1.58 2.56 9.34 1.54
106 106 105 104
Number of phages used at the initiation of a bio-panning round. Number of phages obtained at the end of a bio-panning round. Recovery rate ¼ output/input.
formula: inhibition ratio ¼ (ODcontrol ODphage)/ODcontrol 100%, where ODcontrol and ODphage represent the OD450 values of PBS and phage binding groups, respectively.
LSCM was used to observe the binding of synthetic ESCP9 peptide to ESCC (Eca-109, TE-1) and HEK293 cells. The cells were cultured on coverslips overnight, washed 3 times with PBS, and fixed with 4% paraformaldehyde at room temperature for 20 min. After washing 3 times with PBS, Dil (Beyotime, China) was used to stain the cell membrane at 37 C for 20 min. The cells were then washed 3 times with PBS and blocked with 1% BSA for 30 min. The FITC-labeled peptide ESCP9 was incubated with the cells at 37 C for 25 min. After washing 3 times with PBS, DAPI was used to stain the nucleus, and the slides were observed using LSCM. The binding ability of ESCP9 was further verified by flow cytometry (Guava easyCyte 8HT, Millipore, USA).
Fig. 1. Evaluation of the binding selectivity of 60 phage clones by cellular ELISA. (A) ELISA result of phage clones E1-E32. (B) ELISA result of phage clones E33-E60, IRP and PBS. Binding was plotted as a ratio of cancer/normal cell binding. Error bars represent the standard deviation of three replicates.
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3.2. Affinity confirmation of positive phage clones by cellular ELISA
Table 2 Peptide sequences of positive phage clones. Namesa
Phage clones
Peptide sequences
Frequencyb
ESCP1 ESCP2 ESCP3 ESCP4 ESCP5 ESCP6 ESCP7 ESCP8 ESCP9 ESCP10 ESCP11 ESCP12 ESCP13 ESCP14
E1/E14 E3/E10/E19 E7/E32 E8/E27/E28/E35/E38 E12/E15/E29 E2/E13/E20/E31/E33 E26 E21-25/E34/E41/E47 E30/E37/E39/E43/E49/E51/E60 E40 E42 E46 E48 E53
MHDASPQLYRGR QKESSTHFMAIH MEDNDTHWARMA QTHHTHLPIQRA SFPTMTENFYPR TINSNTLNSTPP TMYEPTTTRSPA LPADEDHHFYWT LLQLTTTKRPIT SVTAGMPNRSNR HLSVAKKPLHRP WPGLLGTTSPNI VHWPASQPVQIP HDHNTNDMTLNA
2 3 2 5 3 5 1 8 7 1 1 1 1 1
a The 41 positive phage clones were selected for DNA sequencing. 14 peptide sequences were obtained and named as ESCP1 to ESCP14. b The frequency represents the number of the peptide sequences appeared in the whole selected phage clones.
2.9. Statistical analysis Each experiment was repeated at least 3 times. Statistical analysis was performed using SPSS 20 (IBM, Armonk, NY, USA). All data are expressed as the mean ± S.D. Independent-Samples t-test was used to analyze the differences between the means. *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant.
After the fourth round of bio-panning, 60 independent phage clones were randomly selected and analyzed for the ability to selectively bind Eca-109 cells and not HEK293 cells. A cellular ELISA was performed to determine the affinity of the 60 phage clones for Eca-109 cells and exclude false positive clones. The phage affinity and specificity were analyzed by comparing target to non-target binding ratios. As shown in Fig. 1, 41 phage clones were identified as positive clones with the over 2.10 ratio of absorbance higher than other clones, IRP and PBS controls. The results demonstrated that 41 of 60 phage clones were identified as positive clones. 3.3. DNA sequencing of the positive phage clones The peptide sequences displayed on the 41 positive phage clones were deduced from the DNA sequences. As shown in Table 2, these amino acid sequences were analyzed and classified, 14 peptide sequences were obtained and designated ESCP1 to ESCP14. The ESCP4, ESCP6, ESCP8, and ESCP9 appeared five, five, eight, and seven times, respectively. And the corresponding phage clones E28, E13, E22, and E37 were further investigated. Similar sequences were identified by Clustal W and National Center of Biotechnology Information BLAST analysis between the peptides displayed by the phages and known proteins (Table 3). The results indicated that no homologous protein was occurred. 3.4. ESCC affinity analysis of the phage clones with consensus sequences
3. Results 3.1. Screening of Eca-109 cell specifically binding phage clones Phages that specifically bound to Eca-109 cells were identified through four rounds of in vitro subtractive bio-panning with Eca109 and HEK293 cells. In each round, a series of phages binding to the Eca-109 cells instead of control cells were rescued and amplified for the subsequent panning, whereas the unbound phages were removed by washing with different concentrations of PBST. As shown in Table 1, the recovery rate of phages after each round bio-panning was used to determine the enrichment efficiency, which increased from 1.58 106 to 1.54 104. The results revealed that phages that were capable of specifically binding to Eca-109 cells were significantly enriched.
Based on the results of ELISA and DNA sequencing, the positive phage clones E28, E13, E22, and E37 that specifically bound to Eca109 cells were used for further identification. Cell immunofluorescence assay was performed to detect the specific binding of the four phage clones to Eca-109 cells. As shown in Fig. 2A, the four phage clones all successfully discriminated Eca-109 cells from HEK293 cells (P < 0.001), but the phage E37 bound preferably to Eca-109 cells than the other phage clones. In contrast, the IRP bound similarly to the two types of cells. As the four phage clones showed different intensities of immunofluorescence staining, the intensity of green fluorescence signal was calculated using Image pro-Plus software 7.0, and the values are shown in Fig. 2B. The results confirmed that the four phage clones differ in their ability to interact with Eca-109 cells. Among the four phage clones, E37 bound most effectively to Eca-109 cells.
Table 3 Homology analysis of 14 peptides using protein data bank. Peptides
Homologous proteinsa
Identities
Homology (%)
ESCP1 ESCP2 ESCP3 ESCP4 ESCP5 ESCP6 ESCP7 ESCP8 ESCP9 ESCP10 ESCP11 ESCP12 ESCP13 ESCP14
Immunoglobulin epsilon heavy chain variable region Aftiphilin protein, isoform CRA-d Unnamed protein Protein FAM27D1 HCG2002731, isoform CRA Phosphatidylinositol 5-phosphate 4-kinase type-2 alpha ABLIM1 protein DNA polymerase alpha catalytic subunit CD99L2 protein GRB2-associated-binding protein 1 Transforming, acidic coiled-coil containing protein 2 Sodium-dependent vitamin C transporter 1 MAP kinase-interacting kinase EF-hand calcium-binding domain-containing protein 5
9/15 8/14 8/18 8/10 8/11 7/8 7/9 9/17 7/8 8/10 7/9 9/16 7/9 6/8
60% 57% 44% 80% 73% 88% 78% 53% 88% 80% 78% 56% 78% 75%
a
All peptides were analyzed with the homologous proteins from Homo sapiens.
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Fig. 3. Competitive inhibition of phage E37 to Eca-109 cells with synthetic peptide ESCP9. Binding of phage E37 to Eca-109 cells is reduced by competition with increasing concentrations of ESCP9 in a dose-dependent manner. The addition of the control peptide revealed no competitive inhibition.
interact with additional cancer cells (Fig. 2D). These results suggested that E37 was identified as the best positive clone with specific affinity to ESCC cells. 3.5. Peptide synthesis The candidate peptide (LLQLTTTKRPIT) displayed on positive phage clone E37 was synthesized using standard solid-phase Fmoc chemistry and named as ESCP9, and the control peptide (TIYEEDTTRSPA) displayed on IRP was also synthesized. An Aminocaproic Acid linker was added to the N-terminus for FITC labeling. The purity of ESCP9 on HPLC was >95%. On mass spectrometry, the experimental mass-to-charge (m/z) ratio measured for ESCP9 agreed with that expected. 3.6. Competitive inhibition assay
Fig. 2. Specific binding of the selected positive phage clones to Eca-109 and other control cells. (A) Immunofluorescence staining of Eca-109 and HEK293 cells with phage clones E28, E13, E22, and E37. The cell nuclei were shown in blue (DAPI) and the phages were shown in green. Scale bar 25 mm. (B) Fluorescence signal intensity analysis. The four phage clones had a significantly higher affinity for Eca-109 than IRP and PBS controls. (C) Immunofluorescence staining of additional cancer cells with phage E37. The cell lines include human esophageal (TE-1), gastric (SGC-7901), colorectal (Caco2), hepatic (SMMC-7721), breast (MCF7), and cervical (SiHa) cancers. (D) Fluorescence signal intensity analysis. The phage E37 bound TE-1 and SGC-7901 cells, but with a significantly lower affinity for other cancer cells. IOD/Area were calculated as the average green fluorescence intensity. Three fields of view per well were measured for each experiment. Values were shown as the mean ± standard deviation. ***P < 0.001; *P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
To further assess specificity, the top one phage clone E37 were screened for binding with a panel of cultured cells, including human esophageal (TE-1), gastric (SGC-7901), colorectal (Caco2), hepatic (SMMC-7721), breast (MCF7), cervical (SiHa) cancers. Fig. 2C demonstrated that the phage E37 bound TE-1 and SGC-7901 cells (P < 0.05), but with a lower affinity for Caco2, SMMC-7721, MCF7, and SiHa cells (P < 0.001). The fluorescent signal intensity analysis suggested that the phage E37 differ in their ability to
The competitive inhibition assay was performed to determine whether the peptide ESCP9 and the corresponding positive phage clone E37 could compete for the same binding site. With an increase in the concentration of ESCP9, the inhibition ratio increased gradually. When the peptide concentration was increased above 600 mM, the inhibition ratio was approximately 41.56% (Fig. 3). The control peptide had no significant effect on the binding of phage E37 to Eca-109 cells. The results demonstrated that the ESCP9 can compete with the corresponding phage E37 for binding to the Eca109 plasma membrane. 3.7. ESCC affinity of synthesized peptide To directly observe the specific binding of ESCP9 to ESCC cells, a fluorescence microscopy assay using FITC-labeled peptide ESCP9 was performed. After ESCC cells were incubated with ESCP9, specific fluorescence was observed on the membrane (Fig. 4A, B). In contrast, there was no significant green fluorescence in the control HEK293 cells (Fig. 4C), which might have resulted from a lack of ESCP9 receptors. The control peptide showed minimal binding to ESCC (Fig. 4D, E) and HEK293 cells (Fig. 4F). The results indicated that ESCP9 can bind to the membrane of ESCC cells specifically and sensitively.
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Fig. 4. Fluorescent imaging of ESCP9 binding to ESCC cells. The FITC-labeled peptide ESCP9 bound to Eca-109 (A) and TE-1 cells (B) but not to HEK293 cells (C). At the same time, the FITC-labeled control peptide bound similarly and with low affinity to Eca-109 (D), TE-1 (E), and HEK293 cells (F). The cell nuclei were shown in blue (DAPI), the plasma membrane was shown in red (Dil), and the peptide was shown in green (FITC). Scale bar 5 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
The specific binding activity of ESCP9 to ESCC cells was further validated by fluorescence-activated cell sorting (FACS) analysis, as shown in Fig. 5. The mean fluorescence value for binding of FITClabeled peptide ESCP9 to Eca-109 cells was 22.47 compared with 11.65 and 10.65 for the FITC-labeled control peptide and for PBS, respectively. Furthermore, the mean fluorescence value for binding of FITC-labeled peptide ESCP9 to TE-1 cells was 17.94 compared with 10.79 and 11.85 for the control peptide and PBS, respectively. In addition, the mean fluorescence values for binding to HEK293 cells were 12.19, 10.37, and 11.44 for the candidate peptide, the
control peptide, and PBS, respectively. The results suggested that the FITC-labeled peptide ESCP9 has specific binding activity to the ESCC cells and not to the control HEK293 cells. 4. Discussion ESCC, a predominant gastrointestinal malignancy, is increasing in incidence and associated with a poor prognosis [12]. Despite the development of various therapeutic strategies for ESCC, including chemotherapy, radiotherapy, surgical resection, and combination
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Fig. 5. FACS analysis of ESCP9 binding to ESCC cells. The FITC-labeled peptide ESCP9 (green line) and the FITC-labeled control peptide (red line) were incubated with Eca-109 (A), TE-1 (B), and control HEK293 cells (C), respectively. PBS was used as a blank control (black line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
therapy, the prognosis for ESCC patients remains unfavorable. Therefore, an innovative approach for early detection of ESCC are urgently needed. Phage display technology, a highly efficient tool to identify small peptides with affinity to a target, has been investigated in recent years for use in the clinical diagnosis and treatment of various types of cancers [13]. Hence, the discovery of peptides functionally targeting ESCC cells has received great attention to introduce a new concept of imaging for more competent diagnostics and be applied to a potent target therapy. In the present study, we used a subtractive whole cell biopanning strategy of the 12-mer phage display peptide library to identify peptides that bind specifically to the membrane of Eca-109 cells. To decrease non-specific binding in each round, the original phage display peptide library was panned against HEK293 cells before screening with Eca-109 cells. Since it is difficult to isolate the normal tissue cells, the human HEK293 embryonic kidney cells was often chosen as control cells [14,15]. After four rounds of biopanning, the phage recovery rate increased gradually, and specific phage clones were effectively enriched. We randomly selected 60 independent phage clones and individually amplified for further identification. Preferential binding of the 60 phage clones to Eca-109 cells was validated by ELISA. The results showed that 41 phage clones were identified as positive clones with the over 2.10 ratio of absorbance higher than other clones, IRP and PBS controls. Previous work has demonstrated that analyzing phage affinity and specificity by comparing target to non-target binding ratios is a successful predictor of tumor homing ability [16]. After the 41 phage clones were sequenced, 14 different peptide sequences were obtained and designated ESCP1 to ESCP14. A multiple sequence alignment showed that the peptide sequence of ESCP9 did not exhibit complete homology to the sequences of any characterized proteins in various protein databases. The peptide was found to have partial homology (7 of 8 amino acids) to the CD99L2 protein. This finding demonstrated that ESCP9 was a novel peptide. The competitive inhibition assay demonstrated that E37 and ESCP9 compete for the same binding sites and the binding was determined by the specific sequence of the peptide rather than by the phage coat proteins. Although the ESCP9 binds specifically to Eca-109 cells, future studies should be performed to determine whether the peptide can interact with one or more membrane receptors in ESCC. The fluorescence microscopy and flow cytometry assay were performed to directly observe the binding of ESCP9 to ESCC cells. These results indicated that ESCP9 bound specifically to ESCC cells and not to the control HEK293 cells. Therefore, ESCP9 may be an effective targeting probe for the diagnosis and therapy of ESCC. According to the previously reported studies, a novel peptide has been considered to be useful for a small molecule probe
leading to multifunctional properties for both imaging detection and targeting therapy [17,18]. Peptides could be conjugated to apply as an imaging probe with various molecules such as radionuclides [19,20], iron oxide [21], and fluorescent agents [22]. Peptides could be utilized to provide a feasibility of therapeutic targets for the development of potential therapeutics [23]. Peptides also could be tethered to nanoparticles for the development of chemotherapeutic drug delivery systems [24,25]. Further studies will aim to identify whether ESCP9 can specifically deliver chemotherapeutic drugs to tumors, bind to certain receptors with high affinity, act as an antagonist to inhibit cancer migration and growth, and bind to ESCC in vivo. In conclusion, it is warranted that the peptide ESCP9 specifically targeting ESCC cells provides substantive possibilities for imaging detection and targeting therapy.
Acknowledgments The study was supported by National Natural Science Foundation of China (No. 81172359).
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Please cite this article in press as: Ma C, et al., Screening of a specific peptide binding to esophageal squamous carcinoma cells from phage displayed peptide library, Molecular and Cellular Probes (2015), http://dx.doi.org/10.1016/j.mcp.2015.04.001