General
Insulin, transferrin, HEPES, and sodium pyruvate were purchased from Biological Industries (BI) (Kibbutz Beit Haemek, Israel). Sodium selenite, hydrocortisone, ethanolamine, O-phosphorylethanolamine, 3,3′,5-triiodo-l-thyronine (T3), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (Rehovot, Israel). Recombinant human EGF was purchased from PeproTech Asia (Rehovot, Israel).
Hsd:Athymic Nude-Fox1nu mice (male, 4 to 5 weeks) were obtained from Harlan (Rehovot, Israel). All animal studies were conducted under a protocol approved by the Animal Research Ethics Committee of the Hebrew University of Jerusalem and in accordance with its guidelines. Animals were allowed to acclimate for at least 3 days, prior to their inoculation with tumor cells, and were routinely kept in 12-h light/dark cycles and provided with food and water ad libitum.
Cell culture
The following human NSCLC cell lines were employed: QG56, HCC827, NCI-H1975, and NCI-H3255. The latter were purchased from the National Cancer Institute - Division of Cancer Treatment and Diagnosis (NCI-DCTD) tumor repository (Frederick, MD, USA) and were regularly maintained in ACL-4 medium, containing insulin (0.02 mg/mL), transferrin (0.01 mg/mL), sodium selenite (25 nM), hydrocortisone (50 nM), EGF (1 ng/mL), ethanolamine (0.01 mM), O-phosphorylethanolamine (0.01 mM), triiodothyronine (100 pM), BSA (0.2% (w/v)), HEPES (10 mM), sodium pyruvate (0.5 mM), and l-glutamine (2 mM) in RMPI-1640 medium (Invitrogen™, Life Technologies, Carlsbad, MA, USA). HCC827 and NCI-H1975 cells were maintained in RMPI-1640 (#30-2001, ATCC), and QG56 were grown in RPMI-1640 (Invitrogen™) at 37°C in a humidified atmosphere of 95% air and 5% CO2. All media were supplemented with fetal bovine serum (FBS, 10%) and antibiotics (penicillin 104 units/L, streptomycin 10 mg/L) (BI, Israel).
Inhibition of cell growth
QG56 (4,000 cells), HCC827, NCI-H3255 (5,000 cells), and NCI-H1975 (7,000 cells) were cultured in 96-well plates. After 24 to 48 h, cells were treated with increasing concentrations (0 to 100 μM) of erlotinib (Cayman Chemical Company, Ann Arbor, MI, USA). The media containing erlotinib (0.05% DMSO, 0.1% ethanol) were freshly prepared and replaced every 24 h. Following 72 h of treatment, cell growth was determined by methylene blue assay [30]. The median inhibitory concentration (IC50) of erlotinib for cell growth of each cell line was calculated using GraphPad Prism 5.0 software. Experiments were repeated thrice for each cell line, in three to six replicates per tested concentration.
NSCLC xenografts
Mice were anesthetized with isoflurane (1% to 2% in oxygen) and injected s.c. in each front flank with a suspension of five million cells in a medium containing Matrigel (BD Biosciences, Beit Haemek, Israel, 20% (v/v)).
Western blot
Cell lysates and tumor tissue extracts were prepared in cold (0°C to 4°C) modified RIPA buffer [31], supplemented with protease inhibitor cocktail for mammalian tissues (P8340, Sigma, 1%). Equal amounts of each sample (30 μg of total protein) were loaded and separated by SDS-PAGE (10%). Proteins were electrophoretically transferred to a nitrocellulose membrane, and the latter was blocked in 3% BSA in TBST buffer (50 mM Tris-HCl, pH 7.5, 0.1% Tween 20, and 150 mM NaCl) for 30 min. Corresponding parts of the membrane were incubated overnight (4°C with gentle shake) with either of the following primary antibodies, diluted in 3% BSA/TBST: (1) rabbit polyclonal EGFR 1005 antibody (sc-03, Santa Cruz Biotechnology Inc., Dallas, TX, USA), (2) mouse monoclonal phosphotyrosine PY20 antibody (sc-508, Santa Cruz Biotechnology Inc.), or (3) mouse monoclonal anti-β-actin antibody (mAbcam 8224, Abcam, Cambridge, UK). The membrane parts were washed thoroughly with TBST buffer and incubated for 1 h with the corresponding horseradish peroxidase-conjugated IgGs (Santa Cruz Biotechnology Inc.) in 3% BSA/TBST. Finally, the membranes were washed in TBST, and immunoreactive proteins were visualized using the EZ-ECL kit (BI, Israel). Densitometry was performed using TINA 2.10 g software, and the intensity of each EGFR band was normalized to that of the corresponding β-actin band to correct for possible differences in the content of total loaded protein.
[11C]Erlotinib synthesis
The radiosynthesis of [11C]erlotinib was based on a previously published procedure [19,20], with slight modifications. [11C]CO2 (50.5 ± 2 GBq (n = 25)) was trapped at −160°C. Subsequently, the temperature of the cooling trap was increased to −5°C, and the activity was transferred by a stream of argon (40 mL/min) into the first reactor at −50°C, containing 300 μL of 0.25 N lithium aluminum hydride (ABX, Radeberg, Germany) in dry tetrahydrofuran. After 2.5 min, the solvent was removed under reduced pressure, and the reactor temperature was increased to 160°C. Next, hydroiodic acid (0.5 mL, Merck, White House Station, NJ, USA) was added, and [11C]CH3I was distilled through a NaOH column (Merck) under argon flow (25 mL/min) and transferred into a second reactor at −15°C, containing 10 mg of 6-O-desmethyl erlotinib (OSI-774, Selleck Chemicals, Houston, TX, USA) dissolved in 0.3 mL N,N-dimethylformamide, containing 3.5 mg of sodium hydride. At the end of the 1-min distillation step, 20.6 ± 1 GBq (n = 25) was trapped in the second reactor. The reactor was sealed and heated to 90°C for 1.5 min. Then, the reactor temperature was increased to 120°C for an additional 5 min. Following a 6.5-min reaction, volatiles were removed under argon flow (at reduced pressure) at a temperature of 90°C. The mixture was cooled to 40°C, 0.6 mL of acetonitrile/water was added, and the crude product was injected into a semi-preparative HPLC column, equipped with a variable wavelength UV detector (254 nm) and a radioactivity detector with NaI crystals. A Phenomenex C18 column (5 μm, 10 mm × 250 mm; Torrance, CA, USA) was used, with a mobile phase system of acetonitrile:acetate buffer 0.1 M, pH 3.8 (4:6), at a constant flow rate of 4 mL/min. The retention time of [11C]erlotinib was 10.5 min, and the product was collected into a flask containing 24 mL HPLC water and 350 μL of 1 M NaOH. Subsequently, the solution was passed through a Sep-Pak Plus C18 cartridge (Waters Corporation, Milford, MA, USA), which was pre-activated with 5 mL EtOH, and washed with 10 mL HPLC water prior to the synthesis. The cartridge was washed with 4 mL of water, and [11C]erlotinib was eluted using 1.3 mL of EtOH followed by 11 mL of sterile isotonic saline (B. Braun, Melsungen, Germany). Quality control analysis was performed on an analytical HPLC, equipped with a variable wavelength UV detector (254 nm) and a radioactivity detector with NaI crystals. A Phenomenex C18 column (5 μm, 4.6 mm × 250 mm) was used, with a mobile phase system of acetonitrile:acetate buffer 0.1 M, pH 3.8 (37:63) for 30 min, at a constant flow rate of 1 mL/min.
MicroPET/CT studies
Tumor-bearing mice (30 ± 1 g (n = 27)) were anesthetized with isoflurane (1% to 2.5% in O2) and maintained normothermic using a heating pad. Following a CT attenuation-correction scan, PET acquisitions were carried out in list mode using an Inveon™ MM PET-CT small-animal dedicated scanner (Siemens Medical Solutions, Malvern, PA, USA). PET scans were started at the time of [11C]erlotinib injection via the lateral tail vein (16.36 ± 0.6 MBq (n = 27)) and lasted for 1 h. Subsequently, mice were maintained at the same position and injected i.v. with [18F]FDG (5.67 ± 0.2 MBq (n = 24)). After a 40-min uptake period, a second 20-min PET acquisition was performed. Carrier-added studies were carried out in HCC827 tumor-bearing mice (n = 5), wherein erlotinib (6.7 mg/kg, dissolved in Cremophor EL/ethanol/saline (1:1:8)) was co-injected with the radiolabeled compound.
Emission sinograms were normalized and corrected for attenuation, scatter, randoms, dead time, and decay. Image reconstruction was performed using Fourier rebinning and two-dimensional ordered-subsets expectation maximization (2D-OSEM), with a voxel size of 0.776 × 0.776 × 0.796 mm3. Image analysis and quantification were performed using Inveon Research Workplace 4.2 (Siemens Medical Solutions). Delineation of tumors' volumes of interest (VOIs) was performed by manual segmentation, based on the fused [18F]FDG and CT images, and the corresponding [11C]erlotinib time-activity curves (TACs) were calculated. Distribution of activity was calculated as the percentage of injected dose per milliliter of tissue (%ID/mL). Standardized uptake values (SUVs) were calculated as the product of %ID/mL and the total body weight of the animal.
Statistics
Statistical analysis was made using GraphPad Prism 5 software. Unless otherwise stated, data is expressed as mean ± SEM. Comparisons of [11C]erlotinib uptake in tumors in imaging studies were made using one-way ANOVA, followed by the Bonferroni post hoc test. The level of significance was regularly set at p < 0.05.