Cell lines and animal models
The 143B, G292, MG-63, U-2 OS, and Saos-2 human osteosarcoma cell lines, UM-SCC1 human head and neck squamous carcinoma and 293T human embryonic kidney cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA). The 143B cells were grown in RPMI1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS, Invitrogen) and 0.015 mg/ml 5-bromo-2′-deoxyuridine at 37°C in an atmosphere containing 5% CO2
. The U-2 OS, MG-63, G292 and Saos-2 cells were grown in Dulbecco’s modified Eagle medium (Invitrogen) supplemented with 15% (v/v) FBS at 37°C in an atmosphere containing 5% CO2
. UM-SCC1 and 293T cells were grown in Dulbecco’s modified Eagle medium (Invitrogen) supplemented with 10% (v/v) FBS at 37°C in an atmosphere containing 5% CO2
. The 143B tumor model was generated by subcutaneous injection of 5×106
cells into the left front flank of female athymic nude mice (Harlan Laboratories). The UM-SCC1 tumor model was established by injection of 5×106
cells into the right front flank of the same mice 2 weeks prior to 143B cell inoculation. The mice were used for microPET studies when the tumor volume reached about 300 mm3
(about 1–2 weeks for 143B, and about 3–4 weeks for UM-SCC1). All animal studies were conducted in accordance with the principles and procedures outlined in the Guide for the Care and Use of Laboratory Animals (11
) and were approved by the Institutional Animal Care and Use Committee of Clinical Center, the National Institutes of Health.
Selection of tumor cell binding peptides
For biopanning, a linear 12-amino-acid peptide library (Ph.D.™ −12 phage display peptide library, New England Biolabs Inc.) was used. Each selection round was conducted as follows: 1×1011 plaque-forming units were added to 293T cells for negative selection. The supernatant was then transferred to 143B cells for positive selection. After 1 h, the cells were washed 5 times with PBS plus 1% bovine serum albumin (BSA) to remove unbound phage particles. The cells and the bound phages were then incubated with E. coli host strain ER2738 to be amplified according to the manufacturer’s protocol. After four rounds of screening, 20 random phage clones were selected for DNA sequencing. The amino acid sequences of displayed peptides were deduced from the DNA sequence. The dominant peptide sequence, ASGALSPSRLDT, was identified and named as OSP-1 for further experiments ().
Scheme of the synthesis of 18F-FP-OSP-1 (A) and 18F-FP-OSP-S (B).
Dye-labeling of peptides and phage particles
The peptide OSP-1 and its scrambled peptide OSP-S (DLPSRTSALASG) were synthesized using a peptide synthesizer and purified with HPLC. For peptide labeling, OSP-1 (or OSP-S, 0.5 mg, 0.4 μmol) and DIPEA (7.5 μl) was added to a solution of Cy5.5 NHS ester (0.5 mg, 0.4 μmol) in DMF (150 μl), the reaction mixture was stirred at room temperature for 2 h and quenched with 10 μl of acetic acid. The crude product was purified by reserve phase HPLC on a semipreparative C-18 column. The desired fractions containing Cy5.5-OSP-1 (or Cy5.5-OSP-S) were collected and lyophilized to give a green fluffy powder. Yield: 57% (> 99% purity). The identity of the products were confirmed by TOF-MS ES+: Cy5.5-OSP-1, m/z 1035.79 for [M+H/2] (C89H123N17O32S4, calculated [MW] 2068.73), and Cy5.5-OSP-S, m/z 1035.75 for [M+H/2] (C89H123N17O32S4, calculated [MW] 2068.73), respectively.
OSP-1 phages (1×1012
pfu) were resuspended in 100 μl of a 0.3-M NaHCO3
(pH 8.6) solution containing 0.25 mg/mL FITC. The phage/fluorochrome reaction was allowed to continue for 1 hour at room temperature in the dark. Subsequent to incubation, the volume of the labeled OSP-1-phage was brought up to 1 ml with DPBS, and the OSP-1-phage was then PEG-precipitated twice and dialyzed extensively against 50 mM Tris–HCl and 150 mM NaCl (pH 7.5; TBS) to remove excess FITC (12
Live 143B, G292, MG-63, U-2 OS, Saos-2, UM-SCC1 and 293T cells were blocked with 10% bovine serum albumin for 60 min at 37°C, and stained with FITC-OSP-1-phage, Cy5.5-OSP-1 or Cy5.5-OSP-S (100 nM) for 60 min at 37°C or at room temperature in dark. After 5 washing steps, the fixed cells were mounted with 4’,6-diamidino-2-phenylindole (DAPI)-containing mounting medium and all cells were observed by an epifluorescence microscope (Olympus, X81).
Frozen 143B and UM-SCC1 tumor tissue slices (8–10 μm) from the tumor-bearing nude mice were fixed with cold acetone for 20 min and dried in the air for 30 min at RT. After blocking with 1% BSA for 30 min, the sections were incubated with Cy5.5-OSP-1 or Cy5.5-OSP-S (100 nM) for 60 min RT in dark. After 5 washing steps, the slices were mounted with DAPI-containing mounting medium under an epifluorescence microscope (Olympus, X81). Each experiment was performed in duplicate and repeated twice.
In order to determine cell surface expression pattern of heparan sulphate proteoglycan (HSPG) receptor, 143B, U-2 OS, MG-63, G292, Saos-2, UM-SCC1 and 293T cells were fixed with cold alcohol for 20 min. After blocking with 10% BSA for 30 min, the fixed cells were incubated with mouse anti-heparin/heparan sulfate monoclonal antibody (1:300, Millipore, Temecula, CA) for 1 h at room temperature and then visualized with Cy3-conjugated donkey anti-mouse secondary antibody (1:300; Jackson ImmunoResearch Laboratories, West Grove, PA). To confirm that OSP-1 peptide binds to HSPG receptor, fixed 143B cells were blocked with 10% BSA for 30 min and then incubated with OSP-1 (10 μM) for 1 h at room temperature, followed by mouse anti-heparin/hepararan sulfate monoclonal antibody (1:300, Millipore, Temecula, CA) for 1 h at room temperature and then visualized with Cy3-conjugated donkey anti-mouse secondary antibody (1:300; Jackson ImmunoResearch Laboratories, West Grove, PA). After 5 washing steps, the fixed cells were mounted with DAPI-containing mounting medium and all cells were observed by an epifluorescence microscope (Olympus, X81).
To a solution of 2-fluoropropionic acid (92 μg, 1 μmol) in DMF (9.2 μl) was added a solution of O-(N-succinimidyl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU, 0.3 mg, 1 μmol) in DMF (30 μl) and diisopropylethyalamine (DIPEA, 10 μl). The reaction mixture was heated at 60 °C for 20 min and was added to a solution of OSP-1 (or OSP-S, 1.2 mg, 1 μmol) in DMF (120 μl). The reaction mixture was heated for another 20 min at 60 °C and quenched with 20 μl of acetic acid. The crude product was purified by reserve phase HPLC on a semipreparative C-18 column. The desired fractions containing FP-OSP-1 (or FP-OSP-S) conjugate were collected and lyophilized to give a white fluffy powder. Yield: >95% (purity >99%). The identity of the products were confirmed by TOF-MS ES+ : FP-OSP-1, m/z 1248.65 for [MH]+ (C51H87FN15O20, calculated [MW] 1248.32), and FP-OSP-S, m/z 1248.67 for [MH]+ (C51H87FN15O20, calculated [MW] 1248.32), respectively.
F labeling precursor, 4-nitrophenyl 2-18
F-NFP), was synthesized as previously reported (13
). The OSP-1 and OSP-S labeling were as follows: OSP-1 (or OSP-S, 500 μg) dissolved in 150 μl anhydrous DMSO was added to dried 18
F NFP in a 1ml reaction vial, followed by addition of 20 μl of DIPEA. The reaction mixture was allowed to stand at RT for 30 min and quenched with 800 μl of 5% aqueous acetic acid solution. The labeled peptide was purified by reserved phase HPLC on a semipreparative C-18 column. The desired fractions containing 18
F-FP-OSP-1 (or 18
F-FP-OSP-S) were collected and diluted with 20 ml of water. After trapping with a C-18 cartridge preactivated with 5 ml of ethanol and 10 ml of water, the product was washed with 2 ml of water and eluted with 2 ml of ethanol. The ethanol solution was blow dried with a slow stream of N2
at 60 °C. The 18
F labeled peptide was redissolved in PBS solution and passed through a 0.22 μm Millipore filter into a sterile multidose vial for in vitro
and in vivo
experiments. The labeling yield is 20% after the unlabeled peptide was efficiently separated from the product. The specific activity was estimated to be ~37 TBq/mmol on the basis of the labeling agent 18
Cell uptake and efflux
The cell uptake studies were performed as we have previously described with some modifications (14
). Briefly, 143B, UM-SCC1 or 293T cells were seeded into 12-well plates at a density of 5×105
cells per well and incubated (about 37 kBq/well) with 18
F-labeled tracers at 37°C for 15, 30, 60, and 120 min. Tumor cells were then washed three times with chilled PBS and harvested by trypsinization with 0.25% trypsin/0.02% EDTA (Invitrogen). The cell suspensions were collected and measured in a γ counter (Packard, Meriden, CT). The cell uptake was expressed as percentage of decay corrected total input radioactivity. Experiments were performed twice with triplicate wells. For efflux studies, 18
F -labeled tracers (about 37 kBq/well) were first incubated with 143B, UM-SCC1 or 293T cells in 12-well plates for 2 h at 37°C to allow internalization. Then cells were washed twice with PBS, and incubated with cell culture medium for 15, 30, 60 and 120 min. After washing three times with PBS, cells were harvested by trypsinization with 0.25% trypsin/0.02% EDTA. The cell suspensions were collected and measured in a γ counter. Experiments were performed twice with triplicate wells. Data are expressed as percent added dose after decay correction.
PET scans and image analysis were performed using an Inveon microPET scanner (Siemens Medical Solutions). Each 143B and UM-SCC1 tumor-bearing mouse was injected in a tail vein with 3.7 MBq (100 μCi, 0.1 nmol in 100 μl) of 18F-OSP-1 or 18F-OSP-S under isoflurane anesthesia (n = 6 per group). For static PET, 5-min scans were acquired at 30 min, 1 h, and 2 h after injection. The images were reconstructed using a two-dimensional ordered subsets expectation maximum (OSEM) algorithm and no correction was applied for attenuation or scatter. For each microPET scan, regions of interest (ROIs) were drawn over the tumor, normal tissue and major organs using vendor software ASI Pro 188.8.131.52 on decay-corrected whole-body coronal images. The maximum radioactivity concentrations (accumulation) within a tumor or an organ were obtained from mean pixel values within the multiple ROI volume and then were converted to megabecquerels per milliliter using a conversion factor. These values were then divided by the administered activity to obtain (assuming a tissue density of 1 g/ml) an image ROI-derived percent injected dose per gram (%ID/g), according to the following formulas: Cf = MBq/ml/MV; % ID/g = 100*MV/Cf/Total Activity in MBq (Cf: conversion factor; MBq: megabecquerel; MV: mean value of pixels in ROI).
Ex vivo biodistribution
Female athymic nude mice bearing 143B and UM-SCC1 xenografts were injected with 0.925 MBq (25 μCi, 25 pmol in 100 μl) of 18F-OSP-1 or 18F-OSP-S to evaluate the distribution of the tracers in the tumor tissues and major organs. At 2 h and 4 h after injection of the tracer, the tumor-bearing mice were sacrificed and dissected. Blood, tumor, major organs, and tissues were collected and wet-weighed. The radioactivity in the wet whole tissue was measured by γ counter (Packard, Meriden, CT). The results are presented as percentage injected dose per gram of tissue (%ID/g). For each mouse, the radioactivity of the tissue samples was calibrated against a known aliquot of the injectate and normalized to a body mass of 20 g. Values were expressed as mean ± SD for groups of four animals (n=4 per group).
Quantitative data were expressed as means ± SD. Means were compared using one-way analysis of variance (ANOVA) and a Student’s t test. P values <0.05 were considered statistically significant.