Patients and tissues
Pancreatic cancer tissue samples were collected from 55 patients who underwent pancreatic cancer resection in the Department of General Surgery at the University of Heidelberg, Germany, and in the Department of Visceral and Transplantation Surgery at the University of Bern, Switzerland. Twelve cases were stage I, 13 cases stage II, 23 cases stage III, and 7 cases stage IV pancreatic adenocarcinomas, according to the Union International Contre Le Cancer (UICC) system. According to routine pathological grading, 16 cases were well-differentiated, 22 moderately differentiated, and 17 poorly differentiated. Normal pancreatic tissues were collected from 34 healthy organ donors. Pancreatic tissues were either frozen in liquid nitrogen and stored at -80°C (for RNA and protein extraction) or immediately fixed in 4% paraformaldehyde solution and subsequently embedded in paraffin. In order to determine MIA serum concentrations, sera from 50 pancreatic cancer patients (35 male, 15 female; median age 59 years; range 29–80 years) and healthy volunteers (14 male; median age 27 years; range 25–35 years) were collected at the Department of General Surgery, University of Heidelberg, Germany. Written informed consent was obtained from all patients. The study was approved by the Ethics Committees of the Universities of Bern, Switzerland, and Heidelberg, Germany.
Cell lines and culture conditions
Mia PaCa-2, T3M4, Aspc-1, Bxpc-3, Capan-1, Colo-357, SU8686 and Panc-1 pancreatic cancer cells and B16 (cloneB78/H1) mouse melanoma cells were grown in RPMI 1640 medium containing 10% FBS (fetal bovine serum), 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen, Karlsruhe, Germany). Cells were maintained in a 37°C humidified atmosphere saturated with 5% CO2. For TGF-β1 induction experiments, pancreatic cancer cells were seeded in 10 cm dishes in 10% FBS growth medium and allowed to attach for 12 hrs. Growth medium was replaced by serum-reduced medium (0.5% FBS), supplemented with 200 pM TGF-β1 for the indicated time periods. For experimental hypoxia, cells were subjected to a hypoxic microenvironment by one hour-long flushing in a special incubator chamber with an anoxic gas mixture (89.25% N2, 10% CO2, 0.75%O2) and sealing of the unit.
Real-time quantitative polymerase chain reaction (QRT-PCR)
All reagents and equipment for mRNA/cDNA preparation were purchased from Roche Applied Science (Mannheim, Germany). mRNA was prepared by automated isolation using MagNA Pure LC instrument and isolation kits I (for cells) and II (for tissue). cDNA was prepared using a 1st strand cDNA synthesis kit for RT-PCR according to the manufacturer's instructions. Real-time PCR was performed with the Light Cycler Fast Start DNA SYBR Green kit . The number of specific transcripts was normalized to housekeeping genes (cyclophilin B and hypoxanthine guanine phosphoribosyltransferase, HPRT). All primers were obtained from Search-LC (Heidelberg, Germany).
Briefly, consecutive paraffin-embedded tissue sections (5 μm thick) were deparaffinized and rehydrated. Antigen retrieval was performed by pretreatment of the slides in citrate buffer (pH 6.0) in a microwave oven for 10 min. Thereafter, slides were cooled to room temperature in deionized water for 5 min. After blocking of endogenous peroxidase activity with 0.3% hydrogen peroxide and washing in deionized water 3 times for 10 min, the sections were blocked for 1 h at room temperature with normal rabbit serum (DAKO, Hamburg, Germany), then incubated with primary goat polyclonal anti-MIA antibody (A-20, Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:35 in normal rabbit serum) overnight at 4°C. The slides were rinsed with washing buffer (Tris-buffered saline with 0.1% BSA) and incubated with secondary rabbit anti-goat HRPO-labeled IgG (Sigma-Aldrich, Taufkirchen, Germany), diluted 1:200 for 45 min at room temperature. After color reaction, tissues were counterstained with Mayer's hematoxylin. For negative control, appropriately diluted goat IgG was used instead of the primary antibody.
Enzyme-linked immunosorbent assay (ELISA)
The amount of secreted MIA protein in cell culture supernatants and serum samples was determined using a one-step MIA ELISA (Roche Diagnostic GmbH, Mannheim, Germany) according to the manufacturer's instructions.
Cells were washed with ice-cold PBS and collected in lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 2 mM EDTA, 1% SDS) containing the Complete mini-EDTA-free protease inhibitor cocktail tablets from Roche (Roche Applied Science, Mannheim, Germany). Lysates were centrifuged at 13,000 rpm at 4°C for 30 min, the supernatants were collected, and protein concentrations were measured with the BCA protein assay (Pierce Chemical Co., Rockford, IL, USA) using BSA as protein standard. 20 μg of protein were mixed with loading buffer, heated at 95°C for 5 min, separated on 12% SDS polyacrylamide gels, and transferred onto nitrocellulose membrane at 100 V for 90 min. Membranes were blocked in 5% non-fat milk in TBS-T (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) for 1 h, incubated overnight at 4°C with anti-MIA antibody (A-20, Santa Cruz) and exposed to secondary HRPO-labeled donkey anti-goat antibody (Santa Cruz) for 1 h at room temperature. The signal detection was performed using the ECL system (Amersham Life Science, Amersham, UK).
For immunoprecipitation, pancreatic cell lines (Mia PaCa-2 and SU8686) were suspended in lysis buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 25 mM NaF, 10% glycerol, 1 mM PMSF) supplemented with the Complete-TM mixture of proteinase inhibitors (Roche Diagnostic, Mannheim, Germany) and incubated for 30 min on ice. After centrifugation, the supernatant was transferred into a fresh vial, pre-cleared with protein A-Sepharose beads (Santa Cruz) and incubated with 50 μl anti-MIA antibody (A-20, Santa Cruz) overnight at 4°C. Following addition of 30 μl of protein A-Sepharose for 1 h at 4°C, the mixture was pelleted, washed three times with lysis buffer, and resuspended in Laemmli sample buffer.
MTT cell growth assays
Cell growth experiments were performed using the 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Pancreatic cancer cells were seeded at a density of 5000 cells/well in 96-well plates, grown overnight, and then exposed to different concentrations of recombinant MIA protein as indicated. After 24 h, MTT was added (50 μg/well) for 4 hours. Formazan products were solubilized with acidic isopropanol, and the optical density was measured at 570 nm.
Invasion assays were performed in a BD Biocoat Matrigel Invasion Chamber with 8-μm pore size (BD Biosciences, Heidelberg, Germany) according to the manufacturer's instructions. The Matrigel was rehydrated with 500 μl DMEM (serum-free) and incubated in a 37°C, 5% CO2 atmosphere for 2 h. 5 × 104 cells were incubated for 24 h and subsequently treated with MIA (100 ng/ml) , which was added to the top chamber and incubated for 24 h. The non-invading cells were removed from the upper surface of the membrane with cotton-tipped swabs. Cells adhering to the lower surface were fixed with 75% methanol mixed with 25% acetone and stained with 1% toluidine blue (Sigma-Aldrich, Taufkirchen, Germany). The whole membrane was scanned using the software of the Zeiss KS300 and Zeiss AxioCam HR system (Jena, Germany). To calculate the total number of all invading cells, the cells were counted in every cut-out of the mosaic image of the whole membrane using the same software. The assays were performed in duplicate and repeated three times.