Tumour and normal tissue were collected at the time of resection from 60 patients (35 male, 25 female), median age 69 years (range 43–91 years) with colorectal cancer at The Queen Elizabeth Hospital, Adelaide, Australia, and snap frozen in liquid nitrogen. All tumour tissue was histologically confirmed as adenocarcinoma, and staged using the ACPS classification system. Liver metastases consistent with colorectal primaries were also collected from five of these patients after hemi-hepatectomy. Informed consent was obtained in all cases and ethics approval was obtained from The Queen Elizabeth Hospital Ethics Committee.
Colon cancer cell lines SW480, SW620, SW48, LIM1215, LIM2405, HT29, LIM1863, LIM2412 and LIM2099 were cultured in HEPES-buffered RPMI 1640 medium (Life Technologies, Grand Island, N.Y.), pH 7.4. The medium was supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 160 μg/ml L-glutamine and 10% heat-inactivated foetal bovine serum (JRH Biosciences, Lenexa, KS) in 75 cm2 vented tissue culture flasks at 37°C in a 5% CO2 environment. Cells were collected at >90% confluency by trypsin digestion and centrifugation for 5 min at 1000 rpm, resuspended in phosphate buffered saline (PBS) and counted using a haemocytometer. The LIM cell lines were kindly provided by Dr R. Whitehead (Ludwig Institute for Cancer Research, Melbourne, Australia); the remainder were purchased from the American Type Culture Collection (ATCC, Rockville, MD).
RNA and protein extraction from tissues and cell lines
RNA and protein were isolated sequentially from tissue samples and cell lines using Trizol™ solution (Sigma, St. Louis, MO). Growth medium was aspirated from the flask of growing cells (>90% confluency) and the cells washed with phosphate-buffered saline (PBS) before being directly lysed using 1 ml of the Trizol™ solution. After a five min incubation with gentle rocking, the cell lysate was removed to a 2 ml eppendorf tube. Tissue samples (0.5 – 1.0 g) were ground in liquid nitrogen using a mortar and pestle, then transferred to an eppendorf tube containing 1 ml Trizol™ solution. The RNA and protein fractions were extracted from each of these samples using the manufacturer's recommendations. Protein concentrations were determined using the DC Protein Assay Kit from Biorad (Sydney, NSW, Australia) following the manufacturer's protocol and using bovine serum albumin diluted from 0.2 mg/ml to 1.5 mg/ml to determine the standard curve.
cDNA array analysis
Two duplicate (from the same manufacturing batch) Atlas Cancer cDNA arrays (Clontech, Palo Alto, CA) were differentially screened with cDNA probes made using mRNA extracted from tumour and matched normal tissue from two different patients and the supplied control mRNA. Total RNA (25 μg) was DNase treated (Life Technologies, Bethesda, MD) then re-extracted using Trizol™ solution. mRNA was prepared from these samples using the Oligotex mRNA purification kit (Qiagen, Germany) following the manufacturer's protocol. cDNA probes were synthesised using the method supplied with the cDNA array filters. These 32P-labelled probes were hybridised to different arrays in 15 ml ExpressHyb™ solution (Clontech) overnight at 68°C, then washed at high stringency in the recommended wash solutions. Blots were exposed to phosphorimager plates overnight, then visualised using a Fuji-BAS phosphorimager. The blots were re-hybridised with a new probe after removal of the previous probe by incubation in boiling 0.1% SDS for 10 min and confirmation by phosphorimaging that there was no residual signal retained.
Semi-quantitative and Relative RT-PCR
Total RNA (2 μg) was reverse transcribed at 37°C using 3 μl pD(N)6 primers (Life Technologies), 200 μM each deoxyribonucleoside triphosphate (dNTP) (Pharmacia, Uppsala, Sweden) and 200 U MMLV reverse transcriptase (Life Technologies) in a reaction volume of 30 μl. Primers specific to either EphB4 (EphB4F1 5'- GAG AGG TAC CTC CTG CAG TGT C and EphB4R1 5'- CCA TGT CCG ATG AGA TAC TGT CCG-3' or EphB4 F2 5'-CGG ACA GTA TCT CAT CGG ACA TG-3' and EphB4 R2 5'-GCT TGG CCT GGG ACT TCA TGT G-3') or CK19 (CK19 F 5'-GAC TAC AGC CAC TAC TAC ACG ACC and CK19 R 5'-AGC CGC GAC TTG ATG TCC ATG AGC C), were used in a PCR reaction carried out in a Perkin Elmer thermocycler with the following conditions: 1.5 mM MgCl2, 200 μM each dNTP, 50 ng of each primer, and 0.5 units of Tth polymerase in 1 × PCR buffer (10 × PCR buffer is 670 mM Tris-HCl pH 8.8, 166 mM [NH4]2SO4, 4.5% Triton X-100, 2 mg/mL Gelatin) (Biotech International, Perth, Australia). Cycling conditions included an initial denaturation at 94°C for 5 min, followed by 30 cycles of 94°C for 30 sec, 60°C (EphB4 set 1) or 63°C (EphB4 set 2) or 68°C (CK19) for 30 sec, and 72°C for 30 sec, with a final extension of 72°C for 7 min. The QuantumRNA™ 18S Internal Standards kit from Ambion (Austin, TX) was used to calculate the relative abundance of transcripts corresponding to EphB4. One microlitre of a 3:7 ratio of 18S primer to 18S competimer (product of 489 bp) was added to each reaction tube and the annealing temperature increased to 62°C as recommended in the Ambion protocol. PCR products were separated by electrophoresis through 1.5% agarose gels and the results analysed using the Kodak Digital Science ID 2.0.2 program (Eastman Kodak Company, New Haven, CT, USA). The level of EphB4 expression in each sample was calculated relative to the amount of 18S product within that sample (expressed as a ratio). The relative expression of tumour compared to normal was compared using the GraphPad Instat™ program (http://www.graphpad.com/instat3/instat.htm).
Protein samples (20 μg) extracted from tumour and matched normal tissue were analyzed in duplicate using 6% SDS-PAGE gels [24
]. One of these gels was stained with coomassie blue to visualise the proteins and compare loading of samples and the second was used for transfer to Immobilon-P PVDF membrane (Millipore Corporation, Bedford, MA). EphB4 antigens were detected using a 1:1000 dilution of the rabbit polyclonal antibody EphB4 (H-200) (Santa Cruz Biotechnology, Santa Cruz, CA) raised to human EphB4. After an overnight incubation at 4°C, the primary antibody was detected using the Lumi-LightPLUS
Western Blotting kit (Roche Biochemicals, Mannhein, Germany) following the manufacturer's recommendation, and between 10s and 30s exposure to Hyperfilm™ ECL™ (Amersham Pharmacia Biotech, UK).
Sections (8 μm) of fresh frozen tissue were mounted on poly-L-lysine coated slides, fixed in acetone, air dried, then frozen until required at -80°C. Endogenous peroxidase activity was blocked with 0.5% hydrogen peroxide in methanol for 30 min at room temperature. Nonspecific binding sites were blocked with 3% normal goat serum in PBS for 20 min at room temperature and the Vector Laboratories (Burlingame, CA) Avidin/Biotin blocking kit following the manufacturer's instructions. The sections were then incubated overnight at 4°C with a 1:200 dilution of the EphB4-specific antibody used for the western analysis. After rinsing with PBS, the sections were incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories) for 30 min at room temperature followed by washing with PBS. Immunoreactivity was detected with the avidin-biotin system (Vector Laboratories) using 18.5 mM 3,3'-diaminobenzidine tetrahydrochloride (Sigma) as a chromogen for 2 min. The sections were then counterstained using Harris haematoxylin, dehydrated, cleared using SUB-X clearing solution (Surgipath Medical Industries, Inc. Richmond, IL) and mounted using SUB-X mounting medium (Surgipath).