Cell lines
A panel of non-small cell lung cancer cells, H460 (large cell carcinoma), H647 (adenosquamous carcinoma), A549 (adenocarcinoma) and SKMES1 (squamous cell carcinoma) were used. H460 and H647 cells were purchased from the American Tissue Culture Collection (ATCC), while A549 and SKMES1 cells were purchased from the European Cell and Culture Collection (ECACC). H460 and H647 cells were maintained in Roswell Park Memorial Institute (RPMI-1640) medium in a humidified atmosphere of 5% CO2 in air at 37°C. A549 cells were maintained in Ham’s F12 supplemented with 4 mM L-glutamine, while SKMES1 cells were cultured in EMEM media supplemented with 2 mM L-glutamine and 1% non-essential amino acids (NEAA). All media were supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 μg/ml) (Lonza, UK). All cells were maintained as monolayer cultures and exponentially growing cultures were used in all experiments. All cell lines were tested and authenticated six months prior to this study using the PowerPlex® 16 HS System (Source BioScience, UK), a multiplex STR system.
Analysis of mRNA expression by RT-PCR
Total RNA was isolated using Tri-reagent (MRC Inc, OH, USA). First-strand cDNA was prepared from 1 μg of total RNA using Superscript III reverse transcriptase (Invitrogen, UK) according to manufacturer’s instructions. PCR reactions were carried out for the VEGF receptors, VEGFR-1, VEGFR-2, NP1 and NP2. The endothelial cell line, EAhy926, was used as a positive control. Primer sequences used were as follows:
VEGFR-1 Forward: 5′CAAGTGGCCAGAGGCATGGAGTT3′
Reverse: 5′GATGTAGTCTTTACCATCCTGTTG3′
VEGFR-2 Forward: 5′GAGGGCCTCTCATGGTGATTGT3′
Reverse: 5′TGCCAGCAGTCCAGCATGGTCTG3′
NP1 Forward: 5′ATGGAGAGGGGGCTGCCG3′
Reverse: 5′CTATCGCGCTGTCGGTGTA3′
NP2 Forward: 5′CCCCGAACCCAACCAGAAGA3′
Reverse: 5′GAATGCCATCCCAGATGTCCA3′
VEGF Forward: 5′CGCAAGCTTAGGAGTACCCTGATGAG3′
Reverse: 5′CCGTCTAGAACATTTGTTGTGCTGT′
β-actin amplification was carried out in parallel to account for loading differences between samples:
β-actin Forward: 5′TGTTTGAGACCTTCAACACCC3′
Reverse: 5′AGCACTGTGTTGGCGTACAG3′
Specificity of all primers was confirmed by comparing the primer sequence for each gene against the Genbank database. PCR products were analyzed on a 1% agarose gel and images acquired using the BioSpectrum® Imaging System (UVP, CA, USA).
Western blot analysis
Total protein was extracted from cells using ice-cold RIPA buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton-X 100, 0.1% (w/v) SDS) supplemented with phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktail (2 mM AEBSF, 1 mM EDTA, 130 μM Bestatin, 14 μM E-64, 1 μM Leupeptin, 0.3 μM Aprotinin). Protein concentrations were determined using the bicinchoninic acid assay (BCA) as per manufacturer’s instructions. Protein (40 μg) from whole cell lysates was fractionated on 8% or 12% SDS-PAGE gels and transferred to a PVDF membrane (PALL Corporation, FL, USA). Transfer efficiency and loading was confirmed by reversible staining of the membrane with Ponceau S solution (Sigma-Aldrich, UK) following protein transfer. Membranes were blocked at room temperature with 5% non-fat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T), followed by incubation with the appropriate primary antibodies at room temperature or otherwise stated: rabbit anti-VEGF (Millipore, CA, USA), 1:2000 at 4°C in 5% BSA TBS-T (0.05%); mouse anti-Flt-1 (Millipore, CA, USA), 1:500 at 4°C in 3% Marvel TBS-T (0.05%); rabbit anti-KDR (Upstate, USA), 1:5000 at room temperature in 5% Marvel TBST-T (0.05%); goat anti-NP1 and rabbit anti-NP2 (Santa Cruz Biotech, CA, USA), 1:400 at room temperature in 5% Marvel TBS-T (0.1%); anti β-actin (Merck Biosciences, UK), 1:20000 at room temperature in 5% Marvel TBS-T (0.1%). Membranes were washed in TBST and incubated with a secondary horseradish peroxidase (HRP)-labeled antibody for 1 h at room temperature (1:2000 at room temperature in 0.1% TBS-T). Membranes were washed in TBST following incubation with secondary antibody. Bound antibody complexes were detected and visualized using SuperSignal™ West Pico enhanced chemiluminescence substrate (Pierce, IL, USA).
Cell proliferation
Cell survival/proliferation was measured using the bromodeoxyuridine (BrdU) cell proliferation ELISA according to manufacturer’s instructions (Roche Diagnostics, Germany). BrdU labeling solution was added at a final concentration of 10 μM. Cells were fixed for 60 min followed by incubation for 90 min anti-BrdU antibody (1:100). Wells were washed and incubated in substrate solution. Absorbance was measured at 450 nm using a reference wavelength at 690 nm.
High content imaging & confocal microscopy
NSCLC cells were seeded (1 × 104) in MatriPlate™ 96-well glass bottomed micro-well plates (Matrical Bioscience, WA, USA) and allowed to adhere overnight. Following serum depletion (0.5% FBS), cells were treated with recombinant human VEGF (100 ng/ml), VEGF neutralizing antibodies (1 μg/ml) or in combination, for 6 h. Cells were fixed in 3% paraformaldehyde and washed in PBS. After washing, cells were blocked in 5% normal goat serum for 1 h followed by incubation with primary rabbit phospho-Akt (1:400) (Millipore) and phospho-p44/p42 MAPK (1:400) (Cell Signaling Technology) primary antibodies in 4% BSA overnight at 4°C. Cells were washed in blocking buffer and incubated with a secondary Alexa Fluor® 488 (Invitrogen) goat anti-rabbit antibody (1:1000), red phalloidin (1:1,000) and Hoechst 33342 (1:500) at room temperature for 1 h. After washing in PBS, localization and expression levels of phospho-Akt and phospho-Erk1/2 were examined on the In Cell 1000 analyzer (GE Healthcare, UK) using IN Cell Investigator high-content image analysis software (version 1.5). For confocal microscopy analysis, NSCLC cells were seeded in glass chamber slides and allowed to adhere overnight. Following serum depletion (0.5% FBS), cells were treated with recombinant human VEGF (100 ng/ml) or VEGF neutralizing antibodies (1 μg/ml) for 6 hrs. Cells were fixed in 3% paraformaldehyde and washed in PBS. After washing, cells were incubated in blocking buffer containing 5% bovine serum albumin (BSA) for 1 h and incubated with rabbit phospho-Akt (1:200) and p44/p42 MAPK (Erk1/2) (1:50) primary antibodies (Cell Signaling Technology) at 4°C overnight. Cells were then washed in PBS and incubated with a secondary Alexa Fluor® 488 (Invitrogen) goat anti-rabbit antibody (1:1000) and Hoechst 33342 for nuclear staining at room temperature for 1 h. After washing in PBS, localization and expression levels of phospho-Akt and phospho-Erk1/2 were examined using a Zeiss LSM 510 laser scanning confocal microscope (Carl Zeiss International, Germany).
Cell cycle analysis
Cells were detached and pelleted by centrifugation at 1300 rpm for 3 min. Supernatants were discarded and cells were suspended in 1 ml phosphate-buffered saline (PBS) and fixed in 90% ice-cold ethanol. Following incubation at room temperature for 30 min, cells were resuspended in 1 ml PBS containing propidium iodide (25 μg/ml) and DNase-free RNase A (100 μg/ml) and left at 37°C for 30 min. DNA synthesis and cell cycle distribution was measured by FACS (Becton Dickinson, UK).
siRNA transient transfections
siRNA ON-TARGETplus SMART pool siRNA for NP1, NP2, VEGF and VEGFR-2 (KDR) were designed and synthesized (Dharmacon Inc, USA). Each siRNA pool contains four individual sequences to silence target gene expression at the mRNA level by at least 75%. A non-targeting scrambled control was also included for each target gene of interest. Cells at 60% confluence were transfected in penicillin/streptomycin-free media with each siRNA (100 nM) using DharmaFect1 transfection reagent (Dharmacon Inc, USA) according to manufacturer’s instructions. After 6 h, siRNAs were removed and cells were maintained in complete media for 24, 48 and 72 h. At each time point, total protein was extracted from A549 and SKMES1 cells for Western blot analysis to determine knockdown of each gene at the protein level. As an alternative to siRNA, due to low levels of knockdown of VEGFR-2, a blocking antibody to VEGFR-2 (sc-19530) (Santa Cruz Biotech, Germany) was also used.
Generation of NP1 stable transfected NSCLC cells
NP1 plasmid DNA was inserted into the site of the mammalian vector pcDNA3.1(-) (Invitrogen Corporation, CA, USA) to generate pcDNA3.1(-)-NP1 plasmid constructs. The NP1 plasmid constructs, including a pcDNA3.1(-) empty vector control, were individually transfected into the NP1 negative cell line, H460. Stable transfections were carried out using FuGENE HD™ transfection reagent (Roche Diagnostics Ltd., UK). Cells (3 × 105) were cultured in their respective supplement-free medium and transfected with either 1 μg pcDNA3.1(-)-NP1 or pcDNA-3.1(-) (control vector) in antibiotic-free media containing 3 μL/mL FuGENE HD™ according to manufacturers’ instructions. Following transfection, cells were further incubated for 24 h at 37°C. Antibiotic selection was then carried out by treating the cells with Geneticin G418 (800 μg/mL). Following several rounds of antibiotic selection, clones were selected and characterized at the mRNA and protein levels in order to examine relative NP1 expression levels.
In vivo tumor growth studies
Nude mice on a BALB/c background (CBy.CG-Foxn1
nu) were purchased from Jackson Laboratories (Bar Harbor, MD, USA). Female mice, 10 weeks of age were utilized. Animals were housed under specific pathogen-free conditions in individually ventilated and filtered cages under positive pressure. All animal experiments were performed in compliance with Irish Department of Health and Children regulations (Licence B100/3250) and approved by the Trinity College Dublin BioResource Ethical Review Board. Mice were anaesthetized with isofluorane and injected subcutaneously on the left-hand side dorsal flank with 3 × 106 H460 empty vector control cells (n = 8) or 3 × 106 NP1 stable transfectant cells (n = 8). Mice were monitored and weighed weekly. Final tumor volume was recorded using digital callipers and calculated based on the equation (D1)2 × D2 × 0.524, where D1 is the smaller of the two diameters of the tumor measured in both directions. Experiments were terminated when the tumor volume reached 2 cm3. Tumors were excised and retained for further analyses. H460 cells (NP1-negative) were transfected with a NP1 plasmid to over-express this receptor for the in vivo component of this study.
Statistical analysis
Statistical analysis was carried out using analysis of variance (ANOVA) with post-hoc analysis using Bonferroni multiple comparisons test, unless otherwise stated. Where the means of two data sets were compared, an unpaired Students t-test was used. Data is graphically represented as mean ± standard error of the mean (SEM) following three independent experiments, where p < 0.05 was considered statistically significant. All data were analyzed using GraphPad InStat™ (version 3.0) statistical software.