The human NSCLC cell lines H322, H292, Calu-3, H1299, A549, H1703 and Calu-1 were obtained from American Type Culture Collection (Manassas, VA, USA) and were cultured as recommended. The PC9, HCC827 and HCC827GR5 cell lines were kindly provided by Dr P. Jänne (Dana-Farber Cancer Institute, Boston MA, USA). All cells were maintained under standard cell culture conditions at 37°C in a water-saturated atmosphere of 5% CO2 in air. As previously reported  cells showing by proliferation assays IC50 for erlotinib < 1 μM were considered sensitive (H322, H292, Calu-3, PC9, HCC827) while cell lines with IC50 > 5 μM (H1299, A549, H1703, Calu-1, HCC827GR5) were considered resistant.
Erlotinib, gefitinib, cetuximab, trastuzumab and rituximab were from inpatient pharmacy. RAD001, NVP-BKM-120 and NVP-BYL-719 were from Novartis.
Stock solutions of 20 mM drugs were prepared in dimethylsulfoxide (DMSO) (with the exception of mAbs), stored at −20°C and diluted in fresh medium for use. The final concentration of DMSO never exceeded 0.1% v/v.
Western blot analysis
Procedures for protein extraction, solubilization, and protein analysis by 1-D PAGE are described elsewhere [32, 33]. Fifty μg of proteins from lysates were resolved by 7.5% SDS-PAGE and transferred to PVDF membranes. Membranes were incubated with: 1:1000 rabbit polyclonal anti-EGFR; 1:1000 rabbit mAb anti-HER2/ErbB2; 1:1000 rabbit mAb anti-Phospho-p70S6K (Thr421/Ser424); 1:1000 mouse mAb anti-Phospho-p44/42 MAPK (ERK1/2); 1:1000 rabbit mAb anti-p44/42 MAPK (ERK1/2) (Cell Signaling Technology, Beverly, MA, USA); 1:1000 mouse mAb anti- Transferrin Receptor (Invitrogen Corporation, Camarillo, CA, USA); 1:3000 mouse mAb anti-Actin (Sigma–Aldrich, St Louis, MO, USA). Blots were then washed and incubated with HRP-anti-mouse or HRP-anti-rabbit antibodies at 1:20000 dilution (Pierce, Rockford, IL, USA). Immunoreactive bands were visualized using an enhanced chemiluminescence system (Immobilion™ Western Cemiluminescent HRP Substrate, Millipore USA).
Cell surface protein isolation
Calu-3 cells were grown in T75 flasks and treated with 0.5 μM erlotinib for 24 h. Cells were incubated with EZ-LINK Sulfo-Biotin (Pierce) for 2 h at 4°C with gentle rotation. The reaction was stopped by washing twice with 25 mM Tris–HCl (pH 7.5) in PBS (phosphate-buffered saline) and cells were scraped into ice-cold lysis buffer (50 mmol/l HEPES, pH 7.0, 10% glycerol, 1% TritonX-100, 5 mmol/l EDTA (ethylenediaminetetraacetic acid), 1 mmol/l MgCl2, 25 mmol/l NaF, 50 μg/ml leupeptin, 50 μg/ml aprotinin, 0.5 mmol/l orthovanadate, and 1 mmol/l phenylmethylsulfonyl fluoride). Lysates were centrifuged at 15000 g for 20 min at 4°C, and supernatants were removed and assayed for protein concentration using the DC Protein assay (Bio-Rad, CA, USA). A volume of 500 μl of lysis buffer containing equal amount of proteins was incubated with UltraLink Immobilized NeutrAvidin protein (Pierce) for 2 h at 4°C with gentle rotation, washed three times with lysis buffer before suspension in SDS (sodium dodecyl sulfate)-loading buffer and then resolved by SDS-PAGE.
For the determination of EGFR and HER2 protein membrane levels, NSCLC cell lines H322, Calu-3 and H292 were treated with 1 μM erlotinib for 24 h. One million cells per condition were then incubated with Isotype control Monoclonal Mouse IgG1/R-PE (Ancell IRP, Bayport, MN, USA), PE mouse anti-Human EGFR (Calu-3 and H322 cells) (BD Biosciences, San Josè, CA, USA) or PE mouse anti-Human HER2 (H322 and H292) (BD Biosciences). After the incubation the analysis was performed with an EPICS-XL flow cytometer.
For the relative quantization of EGFR or HER2 binding sites, NSCLC cell lines H322, Calu-3, H292 were treated with 1 μM erlotinib for 24 h. One million cells were then dispensed for each condition and treated with either 20 μg/ml rituximab (Isotype control), cetuximab (Calu-3 and H322) or trastuzumab (H292) for 1 h. After the incubation with PE-anti-human-IgG (BD Biosciences), the analysis was performed with an EPICS-XL flow cytometer.
The values of mean fluorescence intensity (MFI) were converted in units of equivalent fluorochrome (MEF) using the FluoroSpheres 6-Peak Kit (Dako, CA, USA).
Quantitative real-time PCR
Total RNA was isolated by the TRIzol® reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed as previously described .
The transcript levels of EGFR gene were assessed by Real-Time qRT-PCR on an iCycler iQ Multicolor RealTime PCR Detection System (Bio-Rad, Hercules, CA, USA).
Primers and probes included: EGFR-F (5′-GCCTTGACTGAGGACAGCA-3′), EGFR-R (5-TTTGGGAACGGACTGGTTTA-3), EGFR-probe (5′-FAM CTTCCTCC3′DQ); PGK1-F (5′-GGAGAACCTCCGCTTTCAT-3′), PGK1-R (5′-CTGGCTCGGCTTTAACCTT-3′), PGK1-probe (5′-FAM GGAGGAAG 3′DQ); RPL13-F (5′-ACAGCTGCTCAGCTTCACCT-3′), RPL13-R (5′-TGGCAGCATGCCATAAATAG-3′), RPL13-probe (5′-FAMCAGTGGCA3′DQ); HPRT-F (5′-TGACCTTGATTTATTTTGCATACC-3′), HPRT-R (5′CGAGCAAGACGTTCAGTCCT-3′), HPRT-probe (5′-FAM GCTGAGGA 3′DQ).
The amplification protocol consisted of 15 min at 95°C followed by 40 cycles at 94°C for 20s and at 60°C for 1 min.
The relative transcript quantification was calculated using the geNorm algorithm for Microsoft Excel™ after normalization by expression of the control genes [phosphoglycerate kinase1 (PGK1), ribosomal protein L13 (RPL13) and hypoxanthine-guanine-phosphoribosyltransferase (HPRT)] and expressed in arbitrary units (a.u.).
The cells were seeded into 96-well plate in quadruplicate and were exposed to various treatments. After 96 h, 100 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) solution (1 mg/ml, Sigma-Aldrich) was added to each well and incubated. After 4 h, crystalline formation was dissolved with DMSO and the absorbance at 570 nm was measured using the microplate-reader 550 (BioRad).
Isolation and culture of NK cells
Human PBMC were isolated from buffy coat of healthy donors by using a Lympholyte-H density gradient centrifugation (Cederlane Burlington, Ontario, Canada). Highly purified CD56+ natural killer (NK) cells were obtained by magnetic separation using the NK Cell Isolation Kit and the autoMACS Separator (Miltenyi Biotec, Cologne, Germany) according to the user manual.
Purified NK cells were resuspended in culture medium (RPMI 1640 without phenol red and supplemented with heat inactivated 10% FCS, 50 U/ml penicillin, 50 U/ml streptomycin, 2 mmol/l glutamine) plated and preincubated at 37°C for up to 18 h in the presence of human Interleukin-2 (IL-2, 100 U/ml, Miltenyi Biotec).
Antibody-dependent cell-mediated cytotoxicity (ADCC) was measured with the CytoTox 96 non-radioactive cytotoxicity assay (Promega, Madison, WI, USA) according to manufacturer’s instructions. 2x103
Calu-3, H322, H292 or H1299 cells were treated for 24 h with 1 μM erlotinib, and then seeded with purified NK cells (ratio of 1:25 and 1:50) in a 96-well plate and incubated with 10 μg/ml cetuximab or trastuzumab. After 4 hours the lactate dehydrogenase (LDH) release was determined and the percentage of cytotoxicity was calculated after correcting for background absorbance values according to the following formula:
All experiments involving animals and their care were performed with the approval of the Local Ethical Committee of University of Parma, in accordance with the institutional guidelines that are in compliance with national (DL116/92) and international (86/609/CEE) laws and policies. Twenty-four Balb/c-Nude female mice (Charles River Laboratories, Calco, Italy) were housed in a protected unit for immunodeficient animals with 12-hour light/dark cycles and provided with sterilized food and water ad libitum. At the time of xenograft establishment, mice were 8 weeks old and weighted ~20g. 200 μl of matrigel (BD Biosciences) and sterile PBS (1:1) containing 1x107 Calu-3 cells, were subcutaneously injected on the right flank of each mouse (using a 1 ml syringe, needle G25). When tumour volume reached an average size of 300 mm3, 14 days after injection, animals were randomized into four groups and the treatment started. After 4 weeks, mice were euthanized by cervical dislocation and tumours collected for immunohistochemistry and histological analysis.
Erlotinib (25 mg/Kg) was administered orally in 1% methylcellulose, 0.2% Tween 80 in sterilized water 5 days/week. Cetuximab (2 mg/Kg) was intraperitoneally injected in sterile saline solution 2 days/week. Control group received both oral gavage of 1% methylcellulose, 0.2% Tween 80 in sterilized water 5 days/week and i.p. injection of sterile saline solution (0.9%) 2 days/week.
Dosages of drugs were chosen halving the one used in a previous study in NSCLC-xenograft models, in order to avoid the complete inhibition of tumour growth by the single agent treatment and to better highlight the effect of erlotinib-cetuximab combination [19, 34].
Tumour xenografts were measured twice a week, tumour volume was determined using the formula: (length x width2)/2. Final data are expressed as percent of volume increase: (tumour volume/pre-treatment tumour volume) x 100.
Morphometric and immunohistochemical analysis of tumour xenografts
Formalin fixed samples were embedded in paraffin. From each tumour serial sections of 5 μm thickness were obtained and stained with Haematoxylin and Eosin (H&E), Masson’s Trichrome and for immunohistochemistry. Morphometric analysis was performed in order to evaluate: (a) the numerical density of neoplastic cells, (b) the volume fraction of interstitial inflammatory cells, (c) the volume fraction of fibrosis and (d) the fraction of proliferating and apoptotic cells.
In particular, for each section stained with H&E, a quantitative evaluation of tissue composition was performed. To better define the fraction occupied by neoplastic and non neoplastic cells, sections were stained with pancytokeratin antibodies (monoclonal mouse, 1:500, o.n. 4°C, Dako) revealed through biotin-streptavidin-DAB system, as repeatedly described. The numerical density (n/mm2) of pancytokeratin positive neoplastic cells was computed.
In addition, cell proliferation and apoptotic death were investigated by fluorescence microscopy. Thus, Ki67 labeling (rabbit polyclonal antibody, Vector) and the Terminal deoxynucleotidyltransferase (TdT)–mediated dUTP nick end labeling (TUNEL) assay (Roche Diagnostics, Italy) on cytokeratinpos neoplastic cells were revealed by specific fuorescent probes.
The area occupied by interstitial cells was expressed as percentage of the total area explored. By the same approach, the volume fraction of fibrosis was calculated on Masson’s Trichrome stained sections. To define the volume fractions, the number of points overlying each tissue components was counted and expressed as percentage of the total number of points explored.
All these morphometric measurements were obtained with the aid of a grid defining a tissue area of 0.23 mm2 and containing 42 sampling points each covering an area of 0.0052 mm2.
All these evaluations were performed on the entire section of each tumour sample of each experimental group of animals using an optical microscope (250X final magnification).
Statistical analyses were carried out using GraphPad Prism version 5.0 software (GraphPad Software Inc., San Diego, CA, USA). Results are expressed as mean values ± standard deviations (SD) for the indicated number of independent measurements. Differences between the mean values recorded for different experimental conditions were evaluated by Student’s t-test, and P values are indicated where appropriate in the figures and in their legends. A P value <0.05 was considered as significant.
For in vivo studies comparison among groups was made using analysis of variance (two-way ANOVA repeated measures) followed by Bonferroni’s post-test. Analysis was performed using Prism 5.0 (GraphPad Software) and differences were considered significant when P value was below 0.05. The nature of the interaction between erlotinib and cetuximab was calculated using the Bliss interaction model .