Animal tumor models and treatment
All animal experimental procedures were conducted in accordance with Italian law for animal protection and were approved by the Italian Ministry of Health-Animal Welfare Direction (Protocol No. DGSAF21940-A-16/11/2013). Female BALB/c (nu/nu) mice, 6 weeks old, weighing 15–20 g were purchased from Charles River Laboratories (Milan, Italy). Two NSCLC cell lines were obtained from the American Type Culture Collection. In particular, H1975 cells bear an activating point mutation in exon 21 (L858R) and also harbor the T790M mutation in the kinase domain of EGFR [17]. Despite the T790M-mediated resistance, H1975 cells remain EGFR-driven. H1993 cells are reported to have a high level of MET gene amplification (15 copy numbers) [18, 19] and wild-type EGFR, thus showing resistance to erlotinib [8]. All cells were grown in Roswell Park Memorial Institute (RPMI) medium supplemented with 10 % fetal bovine serum, 100 IU/mL penicillin, and 50 μg/mL streptomycin in a humidified incubator with 5 % CO2 at 37 °C, and then, 5–10 × 106 cells were resuspended in 200 μl RPMI medium and injected s.c. into the flank of nude mice. When tumors reached a mean volume of approximately 100 mm3, animals were randomized into treatment groups (four animals for each cell line and for each treatment) and subjected to imaging studies. Tumor-bearing animals were treated daily for 3 days by oral gavage with 50 mg/kg of erlotinib, WZ4002 [20, 21] (an irreversible EGFR TKI with a higher affinity for T790M mutant EGFR than for wild-type EGFR), crizotinib [22, 23] (a MET inhibitor), or vehicle as described in Fig. 1.
Two additional subgroups of H1975- and H1993-tumor-bearing animals underwent longitudinal studies and were treated with 100 mg/kg erlotinib, WZ4002, crizotinib, or vehicle for 9 days. Tumor size was measured daily by caliper, and volume was determined using the following formula: volume = 0.5 × greatest diameter × (shortest diameter)2.
Imaging studies with 18F-FDG and small-animal PET/CT
Each animal underwent a baseline and a post-treatment scan using a small-animal PET/CT scanner (eXplore Vista Pre-Clinical PET Scanner GE Healthcare). After fasting for 8 h, animals received 7.4 MBq of 18F-FDG by i.v. injection through the tail vein. Animals were anesthetized using 2 % isoflurane and then subjected to PET/CT scan at 60 min post-injection.
Body temperature of the animals was held constant during tracer biodistribution and imaging studies by heating pad or heat lamp. One bed position including the tumor was scanned, and CT images were acquired with the x-ray source set at 35 kVp and 200 μA for 10 min followed by PET image acquisition for 20 min. After acquisition, the images were reconstructed by a combined algorithm based on Fourier rebinning (FORE) followed by 2D iterative image reconstruction using ordered-subset expectation maximization (OSEM). The reconstructed images had a matrix size of 175 × 175 and a voxel size of 0.3875 × 0.3875 × 0.7750 mm3. PET images were corrected for decay and converted to SUV. No statistically significant change of animal weight was observed after treatment. PET/CT data were transferred in DICOM format to an OsiriX workstation (Pixmeo, Switzerland). Three-dimensional regions of interest were drawn around the tumor on transaxial PET images of the baseline and post-treatment scans, and a volume of interest was determined using an automated isocontouring program [23, 24]. The maximum SUV (SUVmax) within the tumor volume of interest was then registered for each study. Finally, the percentage change of the 18F-FDG uptake in the post-treatment scan relative to baseline scan was determined for each animal. All quantitative data from animal imaging studies were expressed as mean ± SE.
Analysis of excised tumors
After treatment, tumors were surgically removed, immediately frozen in liquid nitrogen, and stored at −80 °C until used. Tumor samples (at least three for each animal model and each treatment) were homogenized on ice in RIPA lysis buffer with protease and phosphatase inhibitors (Sigma-Aldrich) using a dounce homogenizer followed by passages through a 26-gauge needle. The suspension was clarified by centrifugation at 13,000×g for 30 min at 4 °C and subjected to western blot analysis using a standard procedure.
Since in a previous study [16] we showed that inhibition of EGFR signaling in NSCLC cells reduces aerobic glycolysis and restores oxidative phosphorylation through the concerted downregulation of HKII and p-PKM2 Tyr105 and upregulation of mitochondrial complexes (OXPHOS), we tested the levels of these selected markers of energy metabolism in tumors from untreated and treated animals. Antibodies used for western blotting included mouse monoclonal antibodies against actin (Sigma; 1 μg/mL) and α-tubulin (Sigma; 1 μg/mL), and the OXPHOS cocktail (Mitoscience, Eugene, OR; 1:1000) that targets the following proteins: 20-kD subunit of Complex I (20 kD), COX II of Complex IV (22 kD), 30-kD Ip subunit of Complex II (30 kD), core 2 of Complex III (~50 kD), and F1α (ATP synthase) of Complex V (~60 kD); rabbit monoclonal antibody against PKM2 (Cell Signaling; 0.1 mg/mL); and rabbit polyclonal antibodies against hexokinase II (Cell Signaling; 1:1000), phospho-PKM2 Tyr105 (Cell Signaling; 1:1000), cyclin D1 (Cell Signaling; 1:1000), and PGC-1α (Santa Cruz Biotechnology, 1:1000). A commercially available ECL kit (GE Healthcare, UK) was used to reveal the reaction.
The western blotting signal was then quantified by morphodensitometric analysis using ImageJ software (NIH, Bethesda, MD, USA). Briefly, the product of the area and the optical density of each band were determined and normalized to the same parameter derived from the actin control. Data were expressed as relative protein levels of each treated sample compared to the corresponding vehicle-treated internal control.
Statistical analysis
Statistical analysis was done using the software MedCalc for Windows, version 12.7.0.0 (MedCalc Software, Mariakerke, Belgium). Unpaired or paired Student’s t test was used when appropriate to compare means. In particular, a paired t test was used to compare 18F-FDG uptake in the same tumors before and after treatment, whereas an unpaired t test was used to examine differences between untreated controls and treated groups. Differences between means were considered statistically significant for p < 0.05 (*) and highly statistically significant for p < 0.01 (**).