1 Introduction

Bladder cancer is one of the most common malignancies in developed countries (1). Growing evidence shows that chronic inflammation of bladder is related with bladder cancer, particularly transitional cell carcinoma (2). Although most of bladder tumor patients have a superficial, noninvasive cancer, they are at high risk for recurrence, and a small number of patients will have their tumors progress and become invasive (3). Patients with bladder cancer are thus regularly monitored for tumor recurrence and progression. Cystoscopy is the most efficient method but causes significant patient discomfort. Cytology is a standard noninvasive method but has poor sensitivity (4). Such limitations of current diagnosis call for the development of more efficient noninvasive tests for the detection of bladder cancer.

It is considered that pathological tissues and even their vessels are distinct from their normal counterparts at molecular levels and put their own signature on the tissue and vasculature. For example, tumor blood vessels carry distinct molecular markers such as αvβ3 integrin on endothelial cell surface (5). In vivo and in vitro screening of phage libraries has provided peptides that specifically recognize disease-specific signatures (6, 7). Phage libraries can have as many as 1010 different peptides (8). A typical example of a homing peptide is the three-amino-acid sequence RGD motif that homes to tumor vascular endothelial cells through binding to the αvβ3 integrin (9).

Selective delivery of drugs or imaging dyes to target tumors is the central challenge for improving existing therapy and diagnosis of cancers. Homing peptides can be used for targeted delivery of therapeutic and imaging agents or to decorate the surface of nanoparticles containing therapeutic agents and thus can enhance the efficacy of the treatment while reducing the side effects. In comparison to antibodies, peptides have several advantages including better tissue penetration, less chance of immune reaction, less possibility of liver and bone marrow toxicity, and lower production cost (10).

Detailed information about the phage display has been previously described (11). This chapter focuses on the procedures for phage display selection of peptides that preferentially bind to bladder tumor cells and validation of in vivo homing to tumor tissues. It also describes the application of the tumor-specific pep-tide to the detection of bladder cancer cells in the patient urine.

2 Materials

2.1 Screening of Phage Library for Tumor-Cell-Specific Peptides

  1. 1.

    A phage library based on T7 415-1b phage vector displaying CX7C (C, cysteine; X, any amino acid residue) random pep-tides is constructed according to the manufacturer's manual (T7Select® System Manual TB178; Novagen, Madison, WI). The library has a diversity of approximately 5 × 108 plaque forming unit (pfu). Store at −80 °C.

  2. 2.

    BL21 strain of E. coli (Novagen). Store at −80 °C.

  3. 3.

    LB medium: 10-g Bacto-Tryptone/5-g yeast extract/5-g NaCl per liter, autoclaved. Store at 4 °C.

  4. 4.

    LB agar plates: LB medium + 15-g agar per liter. Store at 4 °C.

  5. 5.

    Top agarose: 1-g Bacto-Tryptone/0.5-g yeast extract/0.5-g NaCl, 0.6-g agarose per liter, autoclaved. Store at 4 °C.

  6. 6.

    HT-1376 human bladder transitional cell carcinoma cell line and NRK normal rat kidney epithelial cells (American Tissue Culture Collection, Manassas, VA).

  7. 7.

    HUVEC human umbilical endothelial cells: primary cultured.

  8. 8.

    Minimum essential medium (MEM) and Dulbecco's modified Eagle's medium (DMEM) (Gibco/BRL, Besthesda, MD).

  9. 9.

    Fetal bovine serum (FBS) (Gibco/BRL, Besthesda, MD).

  10. 10.

    Magnetic beads conjugated with BerEP4 anti-human epithelial cell adhesion molecule antibody (Dynal Biotech, Brown Deer, WI).

  11. 11.

    Medimachine (DAKO, Carpinteria, CA).

2.2 PCR, DNA Sequencing, and Peptide Sequence Analysis

  1. 1.

    Primers: up primer (5′-AGCGGACCAGATTATCGCTA-3′) and down primer (5′-AACCCCTCAAGACCCGTTTA-3′).

  2. 2.

    PCR premix (Promega, Madison, WI).

  3. 3.

    96-Well reaction plates with round bottom.

2.3 In Vivo Homing Assays of Fluorescein-Labeled Synthetic Peptides

  1. 1.

    N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) (Tokyo Kasei Kogyo Co., Tokyo, Japan). Store at 4 °C.

  2. 2.

    Fluorescein-labeled peptide is synthesized by standard Fmoc method from a commercial company. The peptide is dissolved at 100 mM in DMSO and then slowly diluted with water until the concentration is 1 mM. Store in aliquots at −80 °C. Working solutions are prepared by dilution in water. Protect from light.

  3. 3.

    Mouse anticytokeratin 18 monoclonal antibody (Chemicon, Temecula, CA).

  4. 4.

    Alexa red-conjugated anti-mouse secondary antibody (Molecular Probes, Carlsbad, CA).

  5. 5.

    Mounting media with DAPI (Vector laboratories, Burlin-game, CA).

2.4 Detection of Bladder Cancer Cells in the Patient Urine Using the Tumor-Cell-Specific Peptide

  1. 1.

    ThinPrep™ \PreservCyt™ solution vial (Cytyc Co., Boxbor-ough, MA). Store at room temperature (RT).

  2. 2.

    ThinPrep™ processor (Cytyc Co.).

3 Methods

3.1 Screening of Phage Library for Tumor-Cell-Specific Peptides

  1. 1.

    To make subcutaneous tumor xenograft, 3-month-old BALB/c nu/nu female nude mice are subcutaneously injected with HT-1376 cell suspension containing 1 × 106 cells in phosphate-buffered saline (PBS). Tumors usually develop within 1 month (see Note 1). HT-1376 cells are maintained with MEM containing 10% FBS and nonessential amino acids.

  2. 2.

    A mouse bearing a subcutaneous human tumor xenograft is anesthetized by the inhalation of enflurane. The tumor tissue is removed and minced with a sterile blade. To prepare single-cell suspension, minced tissues are incubated with collagenase (0.5 mg/ml) by shaking at 37 °C for 30 min. Alternatively, tissues are homogenized by using Medimachine. Cells are collected by centrifugation and then resuspended in DMEM containing 1% bovine serum albumin (BSA).

  3. 3.

    To label tumor epithelial cells, the tumor cell suspension containing 2 × 106 cells in 1 ml of DMEM/1% BSA is incubated by rotation with 25 μl (1 × 107 beads) of magnetic beads conjugated with BerEP4 anti-human epithelial cell adhesion molecule antibody at 4 °C for 30 min.

  4. 4.

    A normal cell suspension containing 2 × 106 cells (5 × 105 cells isolated from normal mouse bladder tissues, 1 × 106 NRK cells, and 5 × 105 HUVEC cells) is prepared and combined with the tumor cell suspension (see Note 2). NRK and HUVEC cells are maintained in DMEM supplemented with 10% FBS.

  5. 5.

    A total of 4 × 106 cells are then incubated with the CX7C phage library (109 pfu) by rotation at 4 °C overnight. After incubation, magnetic bead-labeled tumor epithelial cells are isolated using a magnet and washed five times with DMEM/1%BSA.

  6. 6.

    The tumor cell-bound phages are eluted by lysing cells in 100 μl of 1% NP-40 on ice for 5 min and adding 900 μl of BL21 culture to the lysates. For the bacterial elution, overnight BL21 culture is diluted to an OD at 600 nm of 1 using LB medium.

  7. 7.

    For titering phage output, a portion (10 μl) of the phage elutes is serially diluted. Aliquots (10 μl) of the dilution are mixed with 300 μl of a BL21 culture (OD at 600 nm of 1) and 4 ml of melted (45–50 °C) top agar and plated onto LB agar plates. The plates are incubated for 3 h at 37 °C or overnight at RT.

  8. 8.

    For amplifying phage, the remaining phage elutes (990 μl) are added to 10 ml of BL21 culture (in the mid-log phase of growth, OD at 600 nm of 0.5) and incubated with shaking at 37 °C for 2 h until the culture is lysed and clarified.

  9. 9.

    The cultures are transferred into 30-ml centrifuge tubes and centrifuged at 7,000 × g for 10 min. The supernatant is filtered through a 0.2-μm filter into a sterile 15-ml conical vial and stored at 4 °C.

  10. 10.

    Typical titer for T7 culture supernatant is 107 pfu/μl. The amplified phages are used for subsequent rounds of selection (see Note 3).

3.2 PCR, DNA Sequencing, and Peptide Sequence Analysis

  1. 1.

    After screening, 96 plaques are randomly picked (see Note 4) and suspended in 10 μl of Tris-buffered saline (TBS) at each well of 96-well reaction plates. Half or all of them are subjected to DNA sequencing.

  2. 2.

    The insert coding region of selected phage clones is amplified by PCR. Two microliters of the primer pair solution containing 5 pmol/μl of each primer is prepared and added to 22 μl of PCR premix. One microliter of phage suspension is added to each of the PCR reaction mixture containing primers. The PCR reaction is run using the following condition: 35 cycles of 94 °C for 50 s, 50 °C for 1 min, 72 °C for 1 min; hold at 72 °C for 6 min; hold at 4 °C until ready to sequence.

  3. 3.

    The PCR products (approximately 250 bp) are checked by electrophoresis on 2–4% agarose gel, purified, and sequenced by automatic DNA sequencer.

  4. 4.

    The deduced amino acid sequences are aligned using CLUS-TAL W program to find out the consensus sequence. The NCBI BLAST search against the SWISSPROT database, using the option for short nearly exact matches, is conducted to find proteins with significant homology to a peptide sequence. Candidate peptides are chosen and synthesized for further study (see Note 5).

3.3 In Vivo Homing Assays of Fluorescein-Labeled Synthetic Peptides

  1. 1.

    A carcinogen-induced rat bladder tumor model is prepared using BBN. Seven-week-old female Fischer 344 rats are supplied ad libitum with tap water containing 0.05% BBN for 8 weeks and thereafter with tap water without BBN. Tumors usually develop within 20 weeks after the start of BBN administration (12) (see Note 6).

  2. 2.

    To validate in vivo tumor homing of a fluorescein-labeled synthetic peptide that is selected by previous steps, aliquots of the fluorescent peptide at the 1 mM concentration in 500-μl PBS are injected into the tail vein of an anesthetized rat. After 2 h of circulation (see Note 7), the rat is perfused with 10 ml of PBS injected into the left ventricle of the heart and then with 4% paraformaldehyde.

  3. 3.

    Bladder and other control organs are isolated out, fixed in 4% paraformaldehyde for 2 h, immersed in 28% sucrose overnight at 4 °C for cryoprotection, embedded in O.C.T. media, and then frozen.

  4. 4.

    Frozen sections are prepared in 5-μm thickness. To examine the cellular localization of the fluorescein-labeled peptide (green color), sections are stained with anticytokeratin 18 (epithelial cell marker) antibody at RT for 1 h. Alexa red-conjugated secondary antibody (red color) is then incubated at 1:200 dilutions for 1 h at RT. After mounting with the media containing DAPI for nuclear counterstaining (blue color), tissue slides are observed under a fluorescence microscope.

3.4 Detection of Bladder Cancer Cells in the Patient Urine Using the Tumor-Cell-Specific Peptide

  1. 1.

    Urinary cells are collected by centrifugation of 100–200 ml of urine samples at 300 x g for 5 min. Cells are washed with PBS, blocked with PBS/1% BSA at 4 °C for 30 min, and incubated with the 10 μM solution of a fluorescent synthetic peptide in 1-ml PBS/1%BSA at 4 °C for 1 h.

  2. 2.

    After incubation, cells are washed with PBS, transferred into a vial containing ThinPrep™ PreservCyt™ solution, and fixed overnight (see Note 8). The vial is then placed into the Thin-Prep™ processor, which generates negative pressure and in turn draws fluid through a filter that collects cellular material. The cellular material is then transferred to a glass slide (see Note 9).

  3. 3.

    Slides are washed with PBS and incubated with the mounting media containing DAPI for nuclear staining before examining under a fluorescence microscope. A diagram of the procedure is shown in Fig. 1. Parallel slides may be prepared for conventional urinary cytology using Papanicolaou staining.

  4. 4.

    The percentage of positive (blue fluorescent peptide-bound) cells is determined by the number of green fluorescent cells relative to the number of DAPI-stained cells counted from five microscopic fields at the 400 × magnification.

Fig. 1.
figure 1_20

A diagram of the urine peptide analysis. Urinary cells are collected by centrifugation of urine samples. Cells are incubated with a solution of fluorescein-labeled peptide and then fixed in the ThinPrep™ PreservCyt™ solution. Fixed cells are placed in ThinPrep™ processor and transferred onto glass slides. Slides are observed under a fluorescence microscope. A representative photo shows positive (green fluorescent peptide-bound) cells in a urine sample.

Full size image

4 Notes

  1. 1.

    Screening of phage library may be carried out on culture tumor cell lines. This approach is common and experimentally easier. We prepare tumor xenograft on mouse and use cells freshly isolated from the tumor tissue, since we hypothesize that these cells better represent tumor microenvironment than cultured cells and thus are closer to primary human tumor cells.

  2. 2.

    Primary normal bladder cells and NRK cells are used to eliminate phages that bind to normal epithelial cells. HUVEC cells are included to eliminate phages that nonspecifically bind to human cells. We used here equal number of tumor and normal cells during incubation with phage library. The number of normal cells relative to that of tumor cells can be much more increased to select phages with higher affinity to tumor cells.

  3. 3.

    Generally, three to four rounds of screening are carried out to enrich phages. You may add additional two or three screening rounds for more enrichment. However, different phages can be amplified at different rates in growth cycle that follows each round of selection (13). Thus, increasing the number of selection cycle may lead to a selection of phages that grow fast rather than phages that obtain affinity.

  4. 4.

    Plaques (phage clones) from screening rounds that show a significant enrichment are randomly picked for sequencing. We usually choose plaques from the last two rounds.

  5. 5.

    The most frequently occurring peptide sequences are considered as promising ones. In most cases, you can find predominant sequences. In some cases, however, you may find several shared motifs of 3–4 amino acid length among different peptide sequences. An example of bladder tumor-specific peptide we selected using phage display is the CSNRDARRC peptide (7). Fluorescein (or biotin) is usually attached at N-terminus of a peptide during synthesis.

  6. 6.

    BBN-induced tumors are very similar to human bladder transitional cell carcinomas and considered to be an excellent model of clinical bladder cancer (12). Moreover, orthotopic tumor model may mimic clinical cancer better than subcutaneous tumor xenografts.

  7. 7.

    For in vivo homing of a peptide targeting the vascular endothe-lium of tumors, intravenously administered peptide is allowed to circulate for a short period of time (e.g., 5–10 min). We reasoned that homing of a peptide to tumor epithelial cells would require longer circulation time (e.g., 1–3 h) than that to vascular endothelium, since it should diffuse and penetrate into tumor tissues.

  8. 8.

    Longer incubation of cells than overnight in ThinPrep™ Pre-servCyt™ solution is not recommended, since this may reduce or be detrimental to the fluorescence intensity of a fluores-cein-labeled peptide.

  9. 9.

    ThinPrep™ processor generates a thin and even layer of cellular material on glass slides, which is an important factor for observation under a microscope. Conventional cytospin method can also collect cells on glass slides and costs less compared with expensive ThinPrep™ vials. However, this method usually generates a multilayered and edge-oriented cell layer.