The non-muscle-invasive bladder tumor cell line BIU-87 (Chinese human superficial bladder cancer cell line) was kindly provided by Dr Zhou Liqun (Institute of Urology, Peking University, Beijing, China) (21–23). The other bladder tumor T24, E–J and 5637 cell lines were purchased from the Shanghai Ouro Biological Science and Technology Co., Ltd. (Shanghai, China). The cell lines SMMC-7721 (human hepatoma), SiHa (human cervical cancer), KC (human kidney cancer) and MCF-7 (human breast cancer) were provided by Shanxi Medical Center of Scientific Research (Taiyuan, China). BIU-87, T24, E–J, 5637, KC and SMMC-7721 cells were cultured in RPMI-1640 medium; MCF-7 and SiHa cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM). Each medium was supplemented with 10% fetal bovine serum (FBS) containing 1% penicillin and streptomycin. Cells were grown at 37°C in 5% CO₂.

All animal experiments were conducted according to the guidelines of the Laboratory Animals Ethics Committee of Shanxi Medical University. BALB/c nu/nu nude male mice (4–6 weeks) were purchased from Hunan SJA Laboratory Animal Co. Ltd. (Hunan, China). The model was established as previously reported (24,25) with some modifications. Briefly, a mixture of BIU-87 tumor cells (1×10⁷) and basement membrane extract (Matrigel; 200 μl; BD Biosciences, San Jose, CA, USA) was subcutaneously injected into the forelimb armpits. Visible tumors were formed after ~15 days.

The Ph.D.-C7C Phage Display Peptide Library kit containing E. coli host strain ER2738 was purchased from New England Biolabs (Beverly, MA, USA). Tumor-bearing mice were intravenously injected with 2×10¹¹ plaque forming units (PFUs) of phage peptide library via the tail vein. After 15 min, mice were sacrificed and perfused by injection of 50 ml 0.9% normal saline through the heart to wash unbound phages. Then, a portion of the tumor and control tissues (liver, kidney, lung and muscle) were harvested and preserved with 4% formaldehyde. The tumors were manually homogenized and washed with 10× Tris-buffered saline containing 0.1% Tween-20 (0.1% TBST) to remove non-specifically bound phages. Cell membrane-bound phages were eluted with 1 ml of elution buffer (0.2 M glycine-HCl, pH 2.2, 1 mg/ml BSA) for 10 min on ice, and neutralized with 150 μl of 1 M Tris-HCl (pH 9.0). After centrifugation, the supernatant was collected, and the cells in the precipitate were washed once with phosphate-buffered saline (PBS)-BSA and lysed with 0.1% Triton X-100 for 2 h at room temperature. Thus, the internalized phages contained in the cell lysate were recovered. A total of 10 μl eluted phage was titrated on agar plates in the presence of IPTG/X-gel (1 mg/l) and tetracycline (40 mg/ml). The remaining phages were amplified by ER2738 bacteria at 37°C for 4.5 h, and injected into tumor-bearing mice to repeat the above procedures. The entire biopanning process was repeated three rounds. At the end of the third round, the phage solution was eluted and titrated on LB/IPTG/X-gel plates. Then, phage clones were randomly selected, and the inserted DNA sequence was determined using the primer: 5′-CCCTCATAGTTAGCGTAACG-3′ (New England Biolabs). All peptide library DNA was sequenced using a DNA sequencer following previous protocols (26).

In vivo phage localization assays were performed as previously described (27,28). Tumor and control tissues (liver, kidney, lung and muscle), extracted from tumor-bearing mice and preserved in 4% paraformaldehyde were paraffin-embedded and 4-μm sections were obtained. After deparaffinization, the sections were rinsed (3×5 min) in PBST (0.01 M PBS, 0.05% Tween-20; pH 7.4), and blocked with 3% hydrogen peroxide-methanol at room temperature for endogenous peroxidase quenching. The sections were then incubated with blocking buffer (10% normal goat serum) at room temperature for 30 min, followed by overnight incubation at 4°C with rabbit anti-M13 bacteriophage antibody diluted in PBS (1:200). Next, the sections were rinsed in PBST (3×5 min) and incubated for 30 min at 37°C with goat anti-rabbit IgG (1:50). Detection was carried out with 3,3′-diaminobenzidine (DAB) at room temperature in the dark for 5 min. Counterstaining was performed with hematoxylin. Sections were finally dehydrated, cleared and mounted with neutral resin. For the blank control group, the same steps described above were followed, but the anti-M13 bacteriophage antibody was replaced by PBS. A ScanScope digital pathology scanning system (Aperio Technologies, Vista, CA, USA) was used to analyze the binding of the phage peptide to tissues.

After the third round of in vivo screening, 30 blue single colonies were randomly picked from the phage titer plates. Each single colony was amplified, and 1×10¹⁰ PFU of each colony were stored at 4°C until enzyme-linked immunosorbent assay (ELISA) detection.

BIU-87 and iMBT cells (normal human bladder epithelial cells) were plated at a density of 1×10⁴ cells/well in a 96-well plate and incubated at 37°C in 5% CO₂ for 24 h. Subsequently, the cells were washed and fixed with 4% paraformaldehyde for 15 min at room temperature. After incubation with 3% H₂O₂ (100 μl/well) at 37°C for 30 min, the plates were washed 3 times with PBS, and the wells were blocked with 200 ml of 5% BSA for 30 min at 37°C. The phages (1×10¹⁰ PFU/well) were added to BIU-87 or iMBT cells and incubated at 37°C for 1-2 h. The plates were then washed 3 times with TBST before addition of 100 μl HRP-anti-M13 mAb (1:5,000) for 1 h at 37°C. After washing 3 times with 0.05% TBST, 3,3′,5,5′-tetramethylbenzidine (TMB) was added at room temperature for 30 min. The reaction was terminated by the addition of 50 μl of 2 M H₂SO₄. Subsequently, the plates were read on an automated ELISA plate reader at 450 nm. Unrelated phages with equal titers were added to the wells in place of selected phage clones to serve as negative controls. Selectivity was determined using the following formula: Selectivity = [BIU-87 (OD450 - OD450_{negative control})]/[normal human bladder epithelial cells (OD450 - OD450_{negative control})]. Those with affinity ≥2 were considered positive clones; those with the highest binding affinities had their amino acid sequences analyzed.

The CSSPIGRHC (NYZL1) and control CRIGSPHSC (svNYZL1) peptides were synthesized (Chinese Peptide Co., Hangzhou, China) using the standard solid-phase fluorenylmethyloxycarbonyl chloride chemistry. The products were purified to a minimum purity of 98% by high-performance liquid chromatography (HPLC). The sequence and structure of each peptide were characterized by mass spectrometry. FITC-conjugated CSSPIGRHC and FITC-conjugated CRIGSPHSC (negative control) were also synthesized in the same manner.

The non-muscle-invasive bladder tumor BIU-87 cells (n=1×10⁵) were plated in glass-bottom dishes (35 mm; GBD-35-15; NEST) and cultured for 24 h. The cells were washed 3 times with PBS and fixed with 4% paraformaldehyde at room temperature for 20 min. After cells were blocked with 2% BSA for 1 h, the FITC-NYZL1 and FITC-svNYZL1 peptides, respectively, were added for 1, 4 or 12 h at 37°C. After 3 washes, 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nuclei for 5 min at room temperature and examined for fluorescence by laser scanning confocal microscopy (Olympus FluoView FV1000; Olympus, Tokyo, Japan). Meanwhile, the other bladder tumor cell lines, i.e. T24, E–J and 5637 as well as KC, SMMC-7721, SiHa and MCF-7 cells were further assessed by fluorescence microscopy using a procedure similar to that described above. FITC-conjugated synthetic NYZL1 peptides were incubated with the cells for 12 h at 37°C. FITC-svNYZL1 peptides were used as negative controls. Mean cellular fluorescence intensity of 5 fields (up, down, left, right and middle) was used for statistical analysis. The percentage of positive (peptide bound) cells was calculated by dividing the number of green fluorescent cells with that of DAPI-stained cells in 5 high-power fields.

Urine samples were collected from 66 bladder cancer patients hospitalized in The First Hospital of Shanxi Medical University from August 2014 to January 2015. Of these patients, 9 had T_a disease, 22 T₁ disease, 21 T₂ disease and 14 T_3–4 disease as determined by pathological analyses. Meanwhile, 12 cystitis patients and 24 healthy individuals with similar ages were selected as controls. Exfoliated cells were obtained from 200 ml of second morning voiding urine from patients with BCa and healthy adults. Urine samples were processed immediately after collection. After centrifugation for 10 min (1,300 rpm), the sediment was fixed with 4% paraformaldehyde at room temperature for 30 min. Then, cells were smeared in the Laser confocal culture dish. The FITC-NYZL1 (5 μmol/l) and FITC-svNYZL1 (5 μmol/l) peptides were incubated with the cells for 1 h at 4°C. After 3 washes, DAPI was used to stain the nuclei for 5 min at room temperature and examined for fluorescence by laser scanning confocal microscopy. Data analysis was carried out as described for cultured cells above.

The non-muscle-invasive bladder tumor BIU-87 cells (n=1×10⁶) were divided into 6 tubes for the 2 groups. NYZL-1 non-tagged and NYZL 1 FITC-tagged solutions both contained the peptides at 5 mg/ml. Experimental details are listed in Table I. BIU-87 cells were treated with various probes, followed by staining in the dark for 30 min; after two PBS washes, cells were fixed with 2% PFA, resuspended and assessed on a FACS analyzer. Experiments were repeated 3 times.

Peptide-based immunofluorescent histochemistry was performed to validate peptide binding to human bladder cancer. Frozen sections of human bladder cancer and adjacent normal appearing bladder mucosa tissues were performed according to the procedures described above. Fresh frozen sections were blocked with PBS/1% BSA at 37°C for 30 min, incubated with 100 μmol/l FITC-NYZL1 or FITC-svNYZL1 (negative control) at 4°C for 1 h, and washed with PBS. After counterstaining of cells with DAPI, fluorescent images of tissue slides were acquired under a fluorescence microscope.

To test the tumor-homing ability, FITC-NYZL1 or FITC-svNYZL1 (210 μg) was injected into the tail vein of human BIU-87 tumor-bearing mice. After 24 h, mice were anesthetized and transcardially perfused with physiological saline to remove blood and unbound peptides. Tumors and control organs were excised and frozen in tissue-freezing medium (Jung Tissue Freezing Medium; Leica Microsystems, Wetzlar, Germany), cut into 6-μm sections and arrayed on slides. The slides were then fixed with 4% paraformaldehyde for 20 min at 4°C, stained with DAPI for 8 min at room temperature and examined for fluorescence by laser scanning confocal microscopy (Olympus FluoView FV1000). Quantification of imaging data was accomplished using Image-Pro Plus 7.0 (Media Cybernetics, Bethesda, MD, USA).

For whole-organ imaging (29), tumor-bearing mice were intravenously injected with 200 μl of 720 μM fluorescein-conjugated peptide (FITC-NYZL1 or FITC-svNYZL1). After 30 min, 1, 2, 4, 8 and 12 h, the mice were anesthetized and transcardially perfused with physiological saline to remove unbound peptides; tumors and organs were excised for examination. Organ imaging was performed under blue light using a multispectral fluorescence in vivo molecular imaging system (S-0010A; Science, Taiyuan, China).

All quantitative data are mean ± standard error of the mean (SEM). According to normality test results, data were compared using one-way analysis of variance (ANOVA) followed by LSD post hoc test. Two-tailed P<0.05 was considered statistically significant; adjusted P-values were used among subgroup comparisons. All statistical analyses were performed with SPSS 17.0 (IBM, Armonk, NY, USA) for Windows.

To obtain the tumor-homing peptides by in vivo phage display biopanning against human bladder tumor cells, phage recovery from xenograft tumor tissues was assessed after every round of biopanning. The numbers of phages specifically binding to the tumor gradually increased: phage recovery rates were 1.324×10⁻⁵, 2.526×10⁻⁵ and 5.738×10⁻³ PFU/g in the first, second and third rounds, respectively (Table II). After the third round of screening, the numbers of phages that bound to the tumor tissue were enriched ~4.334×10²-fold compared to the first round.

After the third round of screening, we randomly selected 30 phage clones for ELISA. The results showed 24 phage clones with selective binding to BIU-87 cells compared with normal bladder cells (affinity ≥2.0), including 10 individual phage clones with higher binding activity (affinity >6.0), which were isolated and sequenced. After translation into corresponding peptide sequences, eight phage clones displayed a predominant CSSPIGRHC motif; one displayed the CTMSNLKGC motif and another the CNNVLSQMC peptide motif. We used ExPASy/ProtParam and NCBI/BLAST databases to further analyze the CSSPIGRHC peptide. This motif had no homology with known genes and proteins. Thus, the CSSPIGRHC peptide is a new peptide sequence specifically binding to BIU-87 cells. This peptide (CSSPIGRHC) was named NYZL1 and synthesized for further testing. Since BIU-87 cells are Chinese human superficial bladder cancer cells, the present study is the first to explore the selected NYZL1 peptide with specific binding to Chinese bladder cancer.

To evaluate the specificity of phage clones for tumor homing, we compared the tissue distribution among the tumor, liver, kidney, lung, bladder and muscle tissues in each round of screening by immunohistochemistry. Phage peptides were enriched in tumor tissues and increased with each round of screening in vivo (Fig. 1B–D). The majority of phage clones preferentially bound to tumors rather than control tissues after the third screening (Fig. 1D–I). Various non-specific adsorption phages were observed in the liver and kidney tissues, perhaps since the phages were metabolized mainly by the liver and kidneys (Fig. 1G and H). Only few phages binding to bladder, lung and muscle tissues were obtained after the third screening (Fig. 1E, F and I).

To further verify the specific binding of NYZL1 to bladder cancer cells, whole cell binding assay was performed in vitro. The NYZL1 peptide was synthesized and conjugated with green fluorescent FITC at its amino terminus. Several bladder cancer cell lines (BIU-87, T24, E–J and 5637) were selected and other tumor cells (KC, SMMC-7721, SiHa and MCF-7) were used as controls. Immunofluorescence analysis demonstrated that the FITC-conjugated NYZL1 peptide strongly bound to BIU-87, T24, E–J and 5637 bladder cancer cells, compared with the control KC, SMMC-7721, SiHa and MCF-7 cells (Fig. 2; Table III). The FITC-NYZL1 peptide bound to BIU-87 cells with a similar strength compared to the other bladder cancer T24, E–J, 5637 cells (Fig. 2A–J). Meanwhile, the negative control FITC-svNYZL1 (the control peptide, CRIGSPHSC, is a scrambled variant of NYZL1, named svNYZL1) was used to determine whether peptide NYZL1 binds to BIU-87 bladder cancer cells in vitro. Fluorescein imaging revealed that NYZL1 specifically bound to BIU-87 bladder cancer cells. By contrast, no positive fluorescence was detected in the FITC-svNYZL1 peptide group (Fig. 2K). These results revealed that binding of the NYZL1 peptide to other types of tumor cells was relatively weak compared to that to bladder tumor cells. Next, binding of the NYZL1 peptide to BIU-87 cells vs. different types of tumor cells was measured by flow cytometry. Cellular fluorescence intensity in confocal images in the experimental (FITC-NYZL1) groups was measured at 20 min, 1, 4 and 12 h. Notably, the FITC-NYZL1 peptide bound to bladder cancer cells in a time-dependent manner. FITC-NYZL1 bound to BIU-87 cell cytoplasm at 20 min (Fig. 3A–C). After a 1-h incubation, fluorescein staining was not only detected in the cytoplasm, but also partially appeared in the nucleoplasm (Fig. 3D–F). After a 4-h incubation, fluorescein staining in the cytoplasm was significantly increased, with punctiform accumulation in the nucleus (Fig. 3G–I). After a 12-h incubation, fluorescein staining in the cytoplasm and nucleus gradually decreased (Fig. 3J–L).

Specificity of the NYZL1 target was assessed by a competitive assay, using FITC-NYZL1 and the NYZL1 peptide (5 mg/ml) to co-treat BIU-87 cells. Fluorescence intensity of normal untreated BIU-87 cells was 15.24±5.1; after treatment of BIU-87 cells with 2, 5 and 10 μl of FITC-NYZL1 alone, fluorescence intensities of 416.00±13.2, 966.85±20.2 and 1751.45±16.4 were obtained, respectively, indicating a dose-dependent increase in fluorescence intensity. Meanwhile, BIU-87 cells co-treated with FITC-NYZL1 and NYZL1 peptides resulted in reduced fluorescence intensities of 180.45±12.4, 696.30±16.3, 1253.24±11.5, for 2 5 and 10 μl of FITC-NYZL1, respectively, which were significantly different compared with the control group (P<0.05) (Fig. 4). These results indicated that the NYZL1 peptide blocked the binding sites of BIU-87 cells for NYZL1-FITC.

Next, we examined whether the target peptide could bind to cells in the urine from bladder cancer patients. The fluorescent NYZL1 peptide bound to exfoliated cells from urine sample of bladder cancer patients, unlike the control peptide FITC-svNYZL1 (Fig. 5A and B). Little fluorescence by peptide binding was detected with exfoliated cells from control urine samples of a cystitis patient and a healthy individual (Fig. 5C and D, respectively). The percentages of positive peptide bound cells were relatively high in all urine samples from cancer patients, with 10–48% (mean, 30%) in Ta, 33–78% (mean, 57%) in T1, 58–87% (mean, 73%) in T2, and 71–93% (mean, 85%) in T_3–4 stage tumors. In contrast, fewer positive cells were found in urine samples from patients with inflammation (0–10%; mean, 6.5%) and healthy individuals (0–5%; mean, 3%) (Fig. 5E). The binding rates of the FITC-NYZL1 peptide in urine exfoliated cells showed overt differences in various stages of cancer; the higher the stage, the higher the specific binding rate. These findings suggested that the CSNRDARRC peptide is capable of distinguishing malignant cells from cells that exfoliate from bladder tumors, inflammatory lesions or normal tissues.

As shown in Fig. 6A, the synthetic FITC-NYZL1 peptide selectively bound to the primary bladder cancer tissue. Meanwhile, the scrambled control peptide FITC-svNYZL1 did not bind significantly to the tumor tissue (Fig. 6B). In contrast, limited binding of the FITC-NYZL1 peptide to the adjacent normal bladder tissue was observed (Fig. 6C). Quantification of fluorescence intensities in 5 primary bladder tumor and three normal bladder tissue specimens showed that NYZL1 bound to tumor tissues with a greater strength compared with normal tissues (Fig. 6I). In addition, the NYZL1 peptide did not bind to breast and kidney cancer specimens (Fig. 6E and G), suggesting the tumor-type specificity of the NYZL1 peptide.

It was important to determine whether the NYZL1 peptide homes to bladder tumors, allowing in vivo detection and imaging. FITC-NYZL1 (210 μg) was injected via tail vein into mice carrying BIU-87 xenograft tumors at 0.8–1.0 cm in diameter. Tumor tissues and control organs were then excised and processed for frozen sectioning at 24 h after injection of the peptide. Immunofluorescence showed that the FITC-NYZL1 peptide homed to the bladder tumor tissue (Fig. 7A3), but not to other organs such as the bladder, kidney, heart, lung, liver and spleen (Figs. 7 and 8) of tumor-bearing rats. By contrast, the scrambled control FITC-svNYZL1 peptide was not detected in the tumor tissue (Fig. 7A4). These results confirmed that the NYZL1 peptide specifically homed to bladder tumor tissues.

To determine whether FITC-NYZL1 accumulated in tumor tissues and can be visualized at the organ level, FITC-NYZL1 or FITC-svNYZL1 was injected intravenously into mice harboring human bladder tumor xenografts.

Various tissues and organs, including the implanted tumor, heart, liver, spleen, lung, kidney, and bladder were collected at 30 min, 1, 2, 4, 8 and 12 h after peptide injection. The isolated tissues and organs were examined by fluorescence microscopy in real-time to trace the fluorescent probe. At first a portion of the FITC-NYZL1 probe was found in the gallbladder as a result of metabolism by the liver; at this time, the liver and gallbladder showed fluorescent signals. Meanwhile, another portion of the FITC-NYZL1 probe was excreted with urine via the kidneys and bladder. The remaining portion was concentrated in the targeted tumor through blood circulation. As a result, fluorescent signals were found in the bladder and gallbladder soon after probe injection, and decreased overtime with gradual excretion. Meanwhile, fluorescence intensity in tumor tissues increased gradually: it peaked at 4–6 h, before decreasing from the 8 h time point. The FITC-NYZL1 probe had almost disappeared at 12 h after injection (Fig. 9A and B). Considering the presence of fluorescence in tumor cells despite the lack of signals at the organ and tissue levels, it can be inferred that NYZL1 shuttled the fluorescent dye to the designated tumor cells and specifically bound to the target tumor. Fluorescent microscopy results showed that the fluorescent signal was sustained for over 24 h at the molecular level, even when it disappeared at the tissue level. In addition, normal tissues and other organs were negative for fluorescence. Taken together, these findings confirmed the homing and binding affinity of the FITC-NYZL1 probe in bladder tumor cells.