Screening and Identification of a peptide specifically targeted to NCI-H1299 from a phage display peptide library

Screening and Identification of a peptide specifically targeted to NCI-H1299 from a phage display peptide library

Cancer Letters 281 (2009) 64–70 Contents lists available at ScienceDirect Cancer Letters journal homepage: www.elsevier.com/locate/canlet Screening...

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Cancer Letters 281 (2009) 64–70

Contents lists available at ScienceDirect

Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Screening and Identification of a peptide specifically targeted to NCI-H1299 from a phage display peptide library Linquan Zang a,*, Lei Shi a,1, Jiao Guo b, Qin Pan a, Wei Wu a, Xuediao Pan a, Junye Wang c a b c

Department of Pharmacology, Novel Drug Screening Center, School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, Guangdong, PR China The Institute of Traditional Chinese Medicine of Science, Guangdong Pharmaceutical University, Guangzhou 510006, Guangdong, PR China Sun Yat-sen University Cancer Center, Guangzhou 510060, Guangdong, PR China

a r t i c l e

i n f o

Article history: Received 5 January 2009 Received in revised form 10 February 2009 Accepted 13 February 2009

Keywords: Lung cancer Peptide Phage display Biomarker NCI-H1299 Phage M13

a b s t r a c t In this study, a NCI-H1299 (Non-Small Cell Lung Cancer, NSCLC) and a normal lung cell line (Small Airway Epithelial Cells, SAEC) were used for the subtractive screening in vitro with a phage display-12 peptide library. After three rounds of panning, there was an obvious enrichment for the phages specifically binding to the NCI-H1299 cells, and the output/ input ratio of phages increased about 875-fold (from 0.4  104 to 3.5  106). A group of peptides being capable of binding specifically to the NCI-H1299 cells were obtained, and the affinity of these peptides to bind to the targeted cells and tissues was studied. Through a cell-based ELISA, immunocytochemical staining, immunohistochemical staining, and immunofluorescence, a M13 phage isolated and identified from the above screenings, and a synthetic peptide ZS-1 (sequence EHMALTYPFRPP) corresponded to the sequence of the surface protein of the M13 phage were demonstrated to be capable of binding to the tumor cell surfaces of NCI-H1299 and A549 cell lines and biopsy specimens, but not to normal lungs tissue samples, other different cancer cells, or nontumor surrounding lung tissues. In conclusion, the peptide ZS-1 may be a potential candidate of biomarker ligands used for targeted drug delivery in therapy of lung cancer. Ó 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Genetic and proteomic changes occur during carcinogenesis, resulting in an alteration of cell surface features [1]. Difference in the cell surface profile between cancerous cells and their nonmalignant counterparts could be served as a molecular address for targeting reagents to deliver a molecule of choice to the desired cell type. Identification of biomarkers for cancerous cells is a critical step in terms of developing new targeting reagents for cell-specific delivery of chemotherapeutics or imaging reagents. Mass spectrometric-based proteomic approaches have been used to identify biomarkers being useful for disease diag-

* Corresponding author. Tel.: +86 0 1372 5197 120; fax: +86 20 3447 7158. E-mail address: [email protected] (L. Zang). 1 Contributed equally to this work. 0304-3835/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2009.02.021

nosis and prognosis, or drug targets [2,3]. However, it is difficult to detect proteins in low levels using conventional methods, and such classes of proteins are underrepresented, especially for membrane proteins which are usually used as biomarkers. Those biomarkers could be used for targeted drug delivery. Phage display biopanning on viable cells has been proved to be a powerful approach used to identify such cell-specific membrane proteins that are capable of binding to a targeted tumor type even in the absence of knowledge of the targeted cellular receptors [4– 7]. Thus, the peptides are recognizing distinct cell surface receptors that may possess a clinical value. Hence, targeting reagents can be identified using biopanning and the corresponded peptides can be used for discovery of unique cell surface biomarkers. The peptides are able to deliver a chemotherapeutic agent, resulting in the death of the target cell [8]. Our results suggest that such a peptide may have a value as a diagnostic and cell-targeting reagent.

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In the present study, we identified a specific novel peptide that was able to bind to the cell surface of NCI-H1299 cells lines generated in this laboratory by using in vitro phage-displayed random peptide libraries. Our results demonstrate that this biopanning strategy can be used to identify tumor-specific targeting peptides. One of our selected peptides is most effective in the targeting cells and tissues, indicating its potential for a use in early diagnosis or a therapy of lung cancer. 2. Materials and methods 2.1. Materials NCI-H1299 and A549 cell lines, a normal lung cell line – Small Airway Epithelial Cells (SAEC), a human cervical cancer cell line (Hela) and nude mice with a body weight from 15 to 25 g were all obtained from Medical Academy of China. Fetal calf serum (FCS) and DMEM medium were purchased from GiBCO (USA). Phage DNA sequencing was performed by Shanghai Sangon Corp. (Shanghai, PR China). Peptide ZS-1 (EHMALTYPFRPP) and nonspecific control peptide (EAFSILQWPFAH) were synthesized and labeled with fluorescein isothiocyanate (FITC) by Shanghai Bioengineering Ltd. Mass analysis of the peptides was confirmed by a matrix-assisted laser desorption/ionization time-offlight mass spectrometry, and all peptides are >90% pure as determined by a reverse-phase HPLC. Peptide stock solutions were prepared in PBS (pH 7.4). Horseradish peroxidase-conjugated sheep anti-rabbit antibody and rabbit anti-M13 bacteriophage antibody were purchased from Pharmacia (Peapack, NJ, USA). Trizol reagent was purchased from Gibco BRL (Gaithersburg, MD, USA) and the reverse transcriptase polymerase chain reaction (RT-PCR) system kit was purchased from Promega (Madison, WI, USA). All experiments were repeated for a minimum of three times in duplicate. The Ph.D.-12 phage display peptide library kit (New England Biolabs, Berverly, MA, USA) was used to screen specific peptides binding to NCI-H1299 cells. The phage display library contains random peptides constructed at the N terminus of the minor coat protein (cpIII) of M13 phage. The titer of the library is 2.3  1013 pfu (plaque-forming units). The library contains a mixture of 3.1  109 individual clones, representing an entire obtainable repertoire of 12-mer peptide sequences, which expresses random 12amino-acid sequences. Extensively sequencing the naive library has revealed a wide diversity of sequences with no obvious positional biases. The Escherichia coli host strain ER2738 (A robust F+ strain with a rapid growth rate, New England Biolabs) was used for M13 phage propagation. The NCI-H1299 and Small Airway Epithelial Cells (SAEC) cell lines were cultured in a DMEM medium supplemented with penicillin, streptomycin, and 10% fetal bovine serum. Cells were harvested at subconfluent, and the total number of cells were counted using a hemocytometer. 2.2. In vitro panning NCI-H1299 cells were taken as the target cells, and the normal lung cell line (SAEC) as the absorber cells for a

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whole-cell subtractive screening from a phage display 12-peptide library. The panning protocol was described as reported [9]. 2.3. Sequence analysis of selected phages and peptide synthesis After three rounds of in vitro panning, 60 blue plaques were randomly selected and their sequences were analyzed with an ABI Automatic DNA Analyzer (Shanghai Sangon Corp.). A primer used for sequencing was 50 -CCC TCA TAG TTA GCG TAA CG-30 (–96 gIII sequencing primer, provided in the Ph.D.-12 Phage display peptide library kit, New England Biolabs). Homologous analysis and multiple sequence alignment were done using BLAST and Clustal W programs to determine the groups of related peptides. 2.4. Cell-based ELISA with phage NCI-H1299 and SAEC were cultured in DMEM medium with 10% FCS at 37 °C in a humidified atmosphere containing 5% CO2, and the cells were seeded into 96-well plates (1  105 cells/well) over night. Cells were then fixed on 96-well plates by 4% paraformaldehyde for 15 min at room temperature until these cells were attached to the plates. Triplicate determinations were done at each data point. The Elisa performed procedure and selectivity was determined using a formula as follows [10]: Selectivity = ODM13  ODC1/ODS2  ODC2. Here, ODM13 and ODc1 represent the OD values from the selected phages and control phages binding to NCI-H1299 cells, respectively, and ODs2 and ODc2 represent the OD values from the selected phage and control phage binding to the control cell line SAEC, respectively. 2.5. Immunocytochemical staining and immunohistochemical staining of phage M13 Before staining of phage M13 [11], the cells in the different groups (NCI-H1299, A549 and SAEC, Hela) were cultured on coverslips and fixed with acetone at 4 °C for 20 min. Then, about 1  1011 pfu of phage M13 diluted in PBS were added onto the coverslips and incubated at 4 °C overnight. Coverslips were then washed for five times with TBST. The coverslips were blocked by H2O2 (3% in PBS) at room temperature for 510 min. After being washed by PBS for 5 min at 37 °C, the coverslips were incubated with normal sheep serum for 20 min at 37 °C. Subsequently, the coverslips were incubated overnight at 4 °C with a mouse anti-M13 phage antibody with a work dilution of 1:5000. The next day, the coverslips were rinsed for three times (10 min for each rinse) in PBS and incubated with a secondary antibody for 1 h at room temperature. Afterward, the coverslips were rinsed for three times (5 min for each rinse) in PBS. The bound antibody was visualized using TMB. The coverslips were rinsed for three times (5 min for each rinse) using running tap water before staining by hematoxylineosin. Finally, the coverslips were rinsed for 10 min with running tap water before dehydration and mounting. Frozen sections of human lung tissues with and without tumors (provided by the pathology

L. Zang et al. / Cancer Letters 281 (2009) 64–70

department of Sun Yat-sen University Cancer Center) were also prepared. The steps of immunohistochemical staining were similar to those of immunocytochemical staining described above. Instead of the selected phage clone M13, PBS and nonspecific control phage with same titers were used as negative controls in this study. 2.6. Peptide synthesis and labeling The ZS-1 peptide (EHMALTYPFRPP; translated from the selected M13 phage DNA sequence) and nonspecific control peptide (AFSIKQWFKSF) were synthesized and purified by Shanghai Bioengineering Ltd. Fluorescein isothiocyanate (FITC)-conjugated peptides were also produced by the same company. 2.7. Peptide competitive inhibition assay for characterization of specific phage clones The in vitro blue-plaque forming assay was performed to observe the competitive inhibition effect of ZS-1 peptide with its phage counterparts (M13). NCI-H1299 cells were cultured in a 12-well plate overnight and then preincubated with blocking buffer to block nonspecific binding at 4 °C for 30 min. The synthetic peptide (0, 0.0001, 0.001, 0.01, 0.1, 1 or 10 lM) was diluted in PBS and incubated with cells at 4 °C for 1 h, then incubated with 1  1011 pfu of phage M13 at 4 °C for 1 h. The bound phages were recovered and tittered in ER2738 culture. The phages binding to NCI-H1299 cells were evaluated by blue plaque-forming assay, and the rate of inhibition was calculated by the following formula: rate of inhibition = (number of blue plaques in NCI-H1299 incubated with PBS – number of blue plaques in NCI-H1299 with ZS-1 peptide)/number of blue plaques in NCI-H1299 incubated with PBS  100%. Nonspecific control phages (a synthetic peptide corresponding to an unrelated phage picked randomly from the original phage peptide library) were used as negative controls. 2.8. Immunofluorescence microscopy and image analysis Immunofluorescence microscopy was used to study the affinity of synthetic peptide (ZS-1) binding to NCI-H1299. NCI-H1299 and SAEC were digested with 0.25% trypsin and plated on coverslips overnight, respectively. Cells were washed for three times with PBS and fixed with acetone at 4 °C for 20 min before analysis. ZS-1 peptide labeled with FITC was incubated with cells. PBS and control peptides labeled with FITC were used as negative controls. After being washed for three times with PBS, the slips were observed using a fluorescence microscope. 2.9. Analysis of the tissue distribution of M13 phage in an animal model

in 15 days after the cells inoculated. Then, 2  1011 pfu of the M13 phage were blocked with 500 lL BF for 30 min at 37 °C. The blocked phages were injected intravenously in 1 mL PBS. Mice were killed in 10 min later. Before the animals were sacrificed, these mice were perfused with PBS to facilitate the phage elimination from the vasculature. Organs such as lung, heart, and brain and nontumor lung tissue and tumor samples were removed, weighed, and washed using a plain DMEM medium with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 20 lg/mL aprotinin, and 1 lg/mL leupeptin, DMEM-PI). The organ and tumor samples were homogenized with a Homogenate Apparatus (IKA, Gaungzhou Germany). The phages were tittered on agar plates with E. coli in the presence of 1 mg/Lisopropyl1-thio-b-D-galactopyranoside/5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside. 3. Results 3.1. Specific enrichment of NCI-H1299 cell-bound phages Phages specifically binding to human NCI-H1299 cells were identified through three rounds of the in vitro panning. In each round, the bound phages were rescued and amplified in E. coli for the following round of panning, while the unbound phages were removed by washing with TBST. After the third round of the in vitro selection, the number of phages recovered from NCI-H1299 cells was increased for 875-folds (from 0.4  104 to 3.5  106) as shown in Fig. 1. However, there was a decrease in the number of phages recovered from SAEC control cells. The output/ input ratio of phages recovered after each round of the panning was used to determine the phage recovery efficiency. These results indicated an obvious enrichment of phages specifically binding to NCI-H1299 in this study. 3.2. Homology analysis of exogenous sequences of the selected phage clones After the in vitro selection, 60 individual clones were picked up, amplified and sequenced. Each of the clones and the corresponding sequence was given a sequential name from P1 to P60. Seven of these clones (P29, P31, P42, P43, P44, P52, P54) were found to lack the exogenous sequences. The rest clones were identified correctly by DNA sequencing. After analysis of the peptide sequences deduced from P1–3/P8–10/P15/ P21/P27-28/P30/P32-33/P51/P53/P55, P4-7/P11-12/P17–19/P40–41/ P45-46/ P56–57, P13/P16/ P34–P 39/P59–60, P22–26/P48–49/P58, P14/ P47, P50, and P20, seven different phage clones or peptide sequences were obtained (Table 1). Sixteen of the 60 clones were shown to contain the peptide sequence of ZS-1.

Titers of recovered phages (pfu)

66

4000000

HCI-H1299

3500000

SAEC

3000000 2500000 2000000 1500000 1000000 500000 0 T1

T2

T3

Panning round A NCI-H1299 model in Nude mouse was used to analyze the binding activity of M13 phage. We injected 1  107 NCIH1299 cells subcutaneously in the flank of mice. Solid tumors were formed from the inoculated human lung cancer cells (NCI-H1299) with sizes at about 1 cm  2 cm  1 cm

Fig. 1. A specific enrichment of HCI-H1299 cell bound phages as seen when the third round of selection was finished. The titers of the recovered phages from each round were evaluated by a blue plaque-forming assay on agar plates containing tetracycline. The alteration of the titers on the different cells in three rounds was shown above.

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L. Zang et al. / Cancer Letters 281 (2009) 64–70 Table 1 Amino acid sequences of the seven peptides and a multiple sequence alignment. Phage clones

Phage no.

Peptide no.

Sequence(N  C)

Frequency

P1–3/P8–10/P15/P21/P27–28/P30/P32–33/P51/P53/P55 P4–7/P11–12/P17–19/P40–41/P45–46/P56–57 P13/P16/P34–39/P59–60 P22–26/P48–49/P58 P14/P47 P50 P20

P1 P4 P13 P22 P14 P50 P20

ZS-1 ZS-2 ZS-3 ZS-4 ZS-5 ZS-6 ZS-7

EHMALTYPFRPP AYYPQNHKSKAE APNHIPRPPGLT YPHYSLPGSSTL AHRHPISFLSTL SILSTMSPHGAT ITMSSNAEHSRI

16 15 10 8 2 1 1

3.3. Verification of in vitro specific binding by immunocytochemistry

3.5. Competitive inhibition assay

A cellular ELISA was used to identify the affinities for the seven selected phages binding to NCI-H1299. To figure out the selectivity, the affinities of each phage binding to NCI-H1299 cells and to the control normal lung cell line SAEC were compared. The results indicated that these phage clones were demonstrated to bind effectively to NCI-H1299 cells when compared with PBS and SAEC control groups. Furthermore, clone ZS-1 appeared to bind most effectively to NCI-H1299 cells in this study (see Fig. 2). Therefore, we further analyzed the phage M13 and its displaying peptide ZS-1 (EHMALTYPFRPP).

A peptide-competitive inhibition assay was performed to discover whether the synthetic peptide ZS-1 and the corresponded phage clone competed for the same binding site. The results showed that when the synthetic peptide ZS-1 (EHMALTYPFRPP) was pre-incubated with NCIH1299 cells, the capability of the phage ZS-1 to bind to NCI-H1299 cells was decreased in a dose-dependent manner (Fig. 5). With an increase of the concentration of the peptide ZS-1(EHMALTYPFRPP), the titer of phages recovered from NCI-H1299 cells was decreased and the inhibition was increased gradually. When the concentration of peptide ZS-1 was increased above 5 lM, the inhibition reached a flat phase. The control peptide (AFSIKQWFKSF) had no effect on the binding of the phage ZS-1 to NCI-H1299 cells.

3.4. Affinity of the phage M13 to NCI-H1299 cells and lung cancer tissues To confirm the binding ability of the selected phage to target NCIH1299 cells and lung cancer tissues, the phage clone M13 (clone ZS-1) was isolated, amplified and purified for immunochemical assay. SAEC cell line, human nontumor lung tissues were also tested for clone ZS-1 as negative controls. The interaction of the M13 phage and target cells (NCIH1299) was evaluated by immunocytochemical staining as shown in Fig. 3. The surfaces of cells were showed brown after stained, indicating NCI-H1299 cells bound by the phage M13. In contrast, no positive staining was observed in control SAEC cells. The negative results were also obtained when NCI-H1299 cells bound with unrelated phage clone. Subsequently, immunohistochemical staining was performed to observe the specific binding of the phage clone ZS-1 to human lung cancer tissues as shown in Fig. 4. The cells in NCI-H1299 tumor tissue sections when bound with phage clone ZS-1 were stained brown distinctly. The NCIH1299 tumor tissue sections when bound with unrelated phage clone or the nontumor lung tissue sections when bound with phage clone ZS1 showed negative staining. It is thus clear that the phage clone ZS-1 was able to bind specifically to NCI-H1299 cells.

3.6. In vivo binding assay Nude mice bearing NCI-H1299 xenografts were injected with 1.8  1010 pfu of M13 wild type (control) and ZS-1 clone phages through the tail vein, respectively. The distribution and specificity of the phages binding to different tissues were further verified by the titers of bound phages in the tumor tissues compared to nontumor tissues. ZS-1 clone phages were found in tumor tissues at concentrations of 1.66- to 5.42fold higher than in nontumor organs such as heart, liver, brain, kidney, lung and nontumor lung tissue. Meanwhile the wild type phage did not show any targeted binding to tumor tissues (Fig. 6).

4. Discussion To target specific ligands binding on specific tumor antigens is an efficient way to increase the selectivity of

1.2

Selectivity (OD 450nm)

Selectivity (OD 450nm)

6.00 1 0.8 0.6 0.4 0.2 0

5.00 4.00 3.00 2.00 1.00

-7 Zs

Zs -6

5 Zs -

Zs -4

Zs -3

-2 ZS

ZS

-1

0.00 ZS-1

ZS-2

Zs-3

Zs-4

Zs-5

Zs-6

Zs-7

Phage Clone No. Phage Clone No.

A

B 5

Fig. 2. Evaluation of the binding selectivity for the seven phage clones using a cell-ELISA. 1  10 cells/well were seeded and grown in 96-well plates overnight. Approximate 1  1010 pfu phages were added to each well at 37 °C for 1 h. A mouse anti-M13 bacteriophage antibody and a HRP-conjugated sheep anti-mouse Ig were added in turn. An OD450 was obtained after the reaction blocking. The selectivity values for these phage clones calculated by the formula mentioned in the text wee 5.58, 3.75, 2.76, 4.63, 4.03, 4.63 and 3.27, respectively. Therefore, clone ZS-1 appeared to bind most effectively.

L. Zang et al. / Cancer Letters 281 (2009) 64–70

Inhibition rate (%)

68

100 90 80 70 60 50 40 30 20 10 0

Control

0

0.0001

0.001

ZS-1 n=3

0.01

1

10

Concentration of synthetic peptide (uM) Fig. 5. Competitive inhibition of binding of the phage ZS1 to NCI-N1299 cells by the synthetic peptide EHMALTYPFRPP. The average inhibition rates at different concentrations of the peptide are shown. When the concentration of the peptide EHMALTYPFRPP reached more than 0.001 lM, a significant inhibition occurred. The independent experiments were repeated for three times.

Fig. 3. Immunocytochemical staining of NCI-N1299 and control cells when bound with phage ZS-1 (by ABC means, 200). Cell-bound phages were detected using anti-M13 phage monoclonal antibody, secondary antibody, and ABC complex. Then the cells were stained with diaminobenzidine (DAB). Section (A) shows control cell; Section (B) shows immunocytochemical staining of NCI-N1299 cells when bound with phages without exogenous sequences (wild type phage); (C) shows immunocytochemical staining of NCI-N1299 cells when bound with unrelated phage; (D) shows immunocytochemical staining of NCI-N1299 cells when bound with phage ZS-1.

therapeutic targets in clinical oncology and helpful for cancer early detection and therapy. Tumor cells often display lot of certain cell surface antigens such as tumor-associated antigens or tumor-specific antigens that are different from that on normal tissues. To develop more biomarkers for the diagnosis of lung cancer, we used the peptide phage display technology to identify potential molecular biomarkers of NCI-H1299 cancer[12,13]. After panning for

three rounds, 60 clones were selected for further characterization. (1) A cell-based ELISA assay was used to confirm the specific binding of the phage clones to NCI-H1299 cells in vitro. As a result, ZS-1 was demonstrated to be the best candidate phage clone with the highest specificity. (2) Immunocytochemical and immunohistochemical staining were performed to confirm the selectivity of the phage ZS-1 to bind to NCI-H1299 cells and the lung tumor tissues. (3) The results of the competitive inhibitory assays suggest that the peptide displayed by the phage M13-ZS-1, and not other parts of this phage, can bind to the NCI-H1299 cell surface. Under the same conditions, the normal lung cell line SAEC did not show significant fluorescence when staining with ZS-1 peptide-FITC, a finding that further confirmed the targeting of ZS-1 to NCI-H1299 cells. (4) Our analysis of the tissue distribution of the M13 phage in an animal model indicated that M13-ZS-1 phage was distributed mainly in the lung tumor masses and livers but not in brain, heart, or nontumor lung tissues. These results also support our conclusion that M13-ZS-1 phage can specifically bind to the xenograft tumor cells but not the normal lung tissue and cells. The above findings strongly suggest that the ZS-1 peptide may be specific to NCI-H1299 and therefore it would be useful for diagnosis of lung cancer or an antitumor therapy agent deliver tool. These need a further research.

Fig. 4. Immunohistochemical staining of lung cancer and nontumorous lung tissue sections when bound with ZS-1 peptide-FITC (200). To investigate if the free ZS-1 peptide maintained its binding affinity to lung cancer cells, we made a synthetic peptide ZS-1 (EHMALTYPFRPP) labeled with an FITC. Panel (A) shows immunohistochemical staining of lung cancer tissues when bound with phage ZS-1-FITC. The specific binding sites on tumor cells were seen as green fluorescence distinctly; (B) shows immunohistochemical staining of nontumorous lung tissues when bound with phage ZS-1; (C) shows a negative control section with immunohistochemical staining with random peptide-FITC in lung cancer tissues.

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12 Wild type phage

ZS-1 phage clone n=7

10 8 6 4 2

6

10 pfu phages per gram of tissues

L. Zang et al. / Cancer Letters 281 (2009) 64–70

0

Heart

Liver

Brain

Lung

Kidney

Tumor

Tissues Fig. 6. Distribution and specificity of ZS-1 phage clone in tissues of animal models. Nude mice bearing NCI-N1299 xenografts received intravenous injections of ZS-1 phage clones. Xenografts and heart, liver, brain, kidney and lung were removed for titer phages in 30 min later. The level of ZS-1 phage identified in the tumor tissue is 1.66–5.4-fold higher than that in other tissues such as the heart, liver, brain, kidney, lung and in nontumor lung tissue. However, there was fewer phages distributed in the tumor tissue in the mice treated with the nonspecific control phages. Animal number was 7 mice (4 male, 3 female).

Tumor cells may express a number of molecular markers on cell surface or these markers are released from cells at very low levels, but the structures and characteristics of these markers are unknown, making it difficult to identify these markers with conventional methods such as HPLCMS, two-dimension electrophoresis and gene differential expression by gene chip and so on. In some cases, the markers are undetectable [14,15]. The specific binding peptides might be obtained by use of a phage display library as a powerful tool through the selection of desired binding properties [16]. Strategies for panning cells in vitro or tissues in vivo with complex phage libraries have been reported to yield phages with organ- or tumor-binding specificity [17]. Compared with in vivo phage display technology, cell-based panning is simple and effective. Capillary vessels of the vascular system may act as barriers for phage passage so that with the majority of recovered phages in vivo panning is actually binding to the vascular endothelium cells and not tumor cells. The use of in vivo phage display technology has led to reports of many specifically vascular endothelium cell-binding peptides [18]. Therefore, in this study we chose intact tumor cells as targets for panning peptides specific to NCIH1299 cells. In conclusion, we report the use of phage display technology to identify peptides specific to NCI-H1299 cells. We have developed an improved panning system, and the peptides generated by this method preferably bind to NCIH1299 cells (NCI-H1299 and A549) rather than to normal lung cells (SAEC), and bind to NCI-H1299 tumor tissues rather than nontumor lung tissue. The targeting peptide ZS-1 may have significant implications for the targeting of lung cancer treatment. Further studies are needed to investigate the binding specificity of ZS-1 to human lung cancer tissues and the application of ZS-1 to diagnosis of lung cancer or target treatment in clinical oncology. Conflict of interest statement We confirm that all authors fulfill all conditions required for authorship. We also confirm that there is no po-

tential conflict of interest or financial dependence regarding this publication, as described in the Instruction for Authors. All authors have read and approved the manuscript. Acknowledgements This work was supported by National Natural Science Foundation of China (No: 30572177), the National Education Ministry Key Research Project of China (No: 208105), and the Initiation Research Project of Guangdong Pharmaceutical University, PR China (No: 2006). Middle-youth key Teacher Project of Guangdong Pharmaceutical University (No: 2008). References [1] Y. Sekido, K.M. Fong, J.D. Minna, Molecular genetics of lung cancer, Annu. Rev. Med. 54 (2003) 73–87. [2] M. Meyerson, D.P. Carbone, Genomic and proteomic profiling of lung cancers: lung cancer classification in the age of targeted therapy, J. Clin. Oncol. 23 (2005) 3219–3226. [3] J.E. Celis, P. Gromov, Proteomics in translational cancer research: towards an integrated approach, Cancer Cell 3 (2003) 9–15. [4] K.C. Brown, New approaches for cell-specific targeting: identification of cell-selective peptides from combinatorial libraries, Curr. Opin. Chem. Biol. 4 (2000) 16–21. [5] L.A. Landon, S.L. Deutscher, Combinatorial discovery of tumor targeting peptides using phage display, J. Cell Biochem. 90 (2003) 509–517. [6] M. Shadidi, M. Sioud, Selection of peptides for specific delivery of oligonucleotides into cancer cells, Methods Mol. Biol. 252 (2004) 569–580. [7] E. Koivunen, W. Arap, D. Rajotte, J. Lahdenranta, R. Pasqualini, Identification of receptor ligands with phage display peptide libraries, J. Nucl. Med. 40 (1999) 883–888. [8] X. Zhou, Y. Chang, T. Oyama, M.J. McGuire, K.C. Brown, Cell-specific delivery of a chemotherapeutic to lung cancer cells, J. Am. Chem. Soc. 129 (2004) 15656–15657. [9] Q. Pan, L. Shi, X.D. Pan, G.F. Jin, L.Q. Zang, The in vivo screening and validation of Lung cancer specific binding peptide from phage display peptide library, J. Shan Dong Med. 48 (2008) 27–28. [10] B. Du, M. Qian, Z.L. Zhou, In vitro panning of a targeting peptide to NCI-H1299 from a phage display peptide library, Biochem. Biophys. Res. Commun. 32 (2006) 956–962. [11] X.A. Yang, X.Y. Dong, H. Qiao, Immunohistochemical analysis of the expression of FATE/BJ-HCC-2 antigen in normal and malignant tissues, Lab. Invest. 85 (2005) 205–213.

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