Cell lines and cell culture
The cell lines, 6606PDA, 6606l and 7265PDA were a kind gift from Prof. Tuveson, Cambridge, UK. The 6606PDA and 6606l cell lines were originally isolated from a pancreatic adenocarcinoma or the respective liver metastasis of a mouse with C57BL/6J background, which expressed the KRASG12D oncogene in the pancreas (p48-cre induced expression of the oncogene) . The 7265PDA cell line was isolated from a pancreatic adenocarcinoma of a mouse, which expressed the KRASG12D oncogene and in addition the p53R172H allele in the pancreas (Pdx1-creER induced expression of the two alleles). All cell lines were maintained in DMEM high glucose medium with 10% fetal calf serum. For the injection of 6606PDA cells, subconfluent cultures of cells were trypsinized and the trypsinization was stopped by medium. After centrifugation the cells were resuspended in PBS, the suspension was mixed with an equal volume of Matrigel (BD Bioscience, San José, Calif., USA, Nr: 354248) and kept on ice (at a concentration of 1.25x107 cells/ml) until injection . For re-isolation of cells from carcinomas, tumors were isolated and cut up into small pieces. The pieces and outgrowing cells were cultivated in DMEM high glucose medium with 10% fetal calf serum.
Evaluation of cells
Western blots were performed by separating cell lysate on SDS polyacryl gels and transferring the proteins to a polyvinyldifluoride membrane (Immobilon-P; Millipore, Eschborn, Germany). The membranes were blocked with 2.5% (wt/vol.) BSA or 5% (wt/vol.) milk powder (for the analysis of CD133) and incubated overnight at 4°C with a rabbit anti-ALDH1a1 (Cell Signaling, Boston, USA, code 12035, 1:1000), rat anti-CD133 (eBioscience Inc., San Diego, USA, code 14-1331, 1:500) or goat anti-GFAP (Abcam, Cambridge, UK, code ab53554,1:2000) antibody followed by incubation with a secondary peroxidase-linked anti-rabbit antibody (Cell Signaling, code 7074, 1:1000), anti-rat antibody (Santa Cruz Biotechnology, Santa Cruz, USA, code sc3823, dilution 1:10,000), or anti-goat (Santa Cruz Biotechnology, sc-2020, 1:20.000). For analysis of β-actin production, membranes were stripped, blocked by 2.5% (wt/vol.) BSA and incubated with mouse anti-β-actin antibody (Sigma-Aldrich, St Louis, MO, code A5441, dilution 1:20000) followed by peroxidase-linked anti-mouse antibody (Sigma-Aldrich, USA; code A9044, dilution 1:60,000). Protein production was visualized by luminol-enhanced chemiluminescence (ECL plus; GE Healthcare, Munich, Germany) and digitalised with Chemi- Doc XRS System (Bio-Rad Laboratories, Munich, Germany). Signals were densitometrically assessed and corrected with the signal intensity of β-actin (Quantity One; Bio-Rad Laboratories).
For the analysis of CD133 mRNA by PCR total RNA from cells or kidney was isolated using a RNeasy Mini Kit (Qiagen, Germany) according to the manufacturer‘s instructions. After a quality control of the isolated RNA by agarose gel electrophoresis first strand cDNA was synthesized by reverse transcription of 2 μg of total RNA using oligo(dT)18 primer (Biolabs, Frankfurt am Main, Germany) and Superscript II RNaseH-Reverse Transcriptase (Invitrogen, Karlsruhe, Germany). After heat inactivation of the reverse transcriptase 1/20 of the cDNA was amplified (27 cycles: 94°C for 30, 68°C for 40, 72°C for 60 seconds) using CD133 specific primers (forward primer: CCCTCCAGCAAACAAGCAAC, reverse primer: ACAGCCGGAAGTAAGAGCAC) and the PCR product of 325 bp was visualized by agarose gel electrophoresis.
For the quantification of cell proliferation rates, cells were plated on 96 well plates, so that the cells were 20% confluent, when BrdU was added to the medium. The BrdU incorporation was measured after 24 hours of incubation by the colorimetric cell proliferation assay as specified by the manufacturer (Roche Applied Science, Penzberg, Germany).
For this study male B6.V-Lepob/ob mice (obese mice) were compared with male B6.V-Lep+/? littermates (lean mice). The therapy with metformin was performed on male C57BL/6J mice. The mouse strains were originally purchased from The Jackson Laboratory (Bar Harbor, ME) and bred in our local animal facility. For defining the border between carcinoma and the desmoplastic reaction, carcinoma cells were injected in the pancreas of C57BL6-TgACTB-eGFP1Osb/J mice (with a corresponding phenotype to lean B6.V-Lep+/? mice) . Animals were kept on water and standard laboratory chow ad libitum. All experiments were executed in accordance with the EU-directive 2010/63/EU and approved by the Landesamt für Landwirtschaft, Lebensmittelsicherheit und Fischerei Mecklenburg-Vorpommern (7221.3-1.1-069/12).
Syngeneic orthotopic carcinoma model
For injection of carcinoma cells general anesthesia was induced in 93 ± 32 day old mice (average ± standard deviation) by 1.2-2.5% isoflurane. Perioperative analgesia was ensured by sc injection of 5mg/kg carprofen (Rimadyl, Pfizer GmbH, Berlin, Germany) and eyes were protected by eye ointment. After shaving and disinfection of the skin, the abdominal cavity was opened by transverse laparotomy and the head of the pancreas was identified. Duodenum and pancreas was gently lifted by tweezers and 20 μl cell suspension containing 2.5x105 carcinoma cells were injected slowly into the head of the pancreas using a precooled ga22s 710 RN 100 ul syringe (Hamilton Syringe, Reno, Nev., USA). The pancreas was placed back into the abdominal cavity and the cavity was closed by a coated 5-0 vicryl suture (Johnson & Johnson MEDICAL GmbH, Norderstedt, Germany). The skin was then closed by a 5-0 prolene suture (Johnson & Johnson MEDICAL GmbH). On day 8 after the injection of carcinoma cells, chronic pancreatitis was induced over 2 weeks by administration of three ip injections of 50 μg/kg cerulein (Sigma-Aldrich Chemie GmbH), 3 days a week, at a rate of one every hour per day. Control mice were sham treated appropriately with 0.9% saline solution instead of cerulein and tissues were analyzed on day 20. For the evaluation of the impact of metformin on cancer pathophysiology 250 mg/kg 1,1-dimethylbiguanide hydrochloride (Sigma-Aldrich, code 150959) was ip injected daily from day 8 to day 15 followed by daily injection of half of this dose from day 16 to day 29 and analysis of the tumor on day 29 (3-6 hours after the last metformin administration). Control mice were sham treated appropriately with PBS instead of metformin and tumors were analyzed on day 29. For pain relief, 800 mg/L metamizol (Ratiopharm GmbH, Ulm, Germany) was added to the drinking water during the entire timespan of all in vivo experiments. In order to assess cell proliferation 50 mg/kg 5-bromo-2-deoxyuridine (BrdU) was injected ip 2.5 hours before tissue asservation. For blood samples and organ harvest, animals were anesthetized with 90 mg/kg ketamine (bela-pharm, Vechta, Germany) and 7 mg/kg xylazine (Bayer Health Care, Leverkusen, Germany).
Analysis of the blood
Blood glucose concentrations were measured with the blood glucose meter Contour (Bayer Vital, Leverkusen, Germany) on day 0 before injection of carcinoma cells and on day 20 before the first cerulein injection of this day. Blood samples for assessing lipase activity were taken two hours after the third cerulein injection on day 8. The activity of lipase in blood plasma was analysed using the Cobas c111 spectrophotometer (Roche Diagnostics, Mannheim, Germany).
Evaluation of tissue
The pancreas and tumor weight was measured after careful separation of the carcinoma from the pancreas. Evaluation of CD133 expression was performed on 7 μm cryo-sections. These sections were fixed with 4% paraformaldehyde in PBS for 15 min, reactive groups were then quenched in 50 mM NH4Cl for 10 min and the cell membranes were permeabilised with 0.3% saponin in PBS for 15 min, before CD133 immunohistochemistry was performed. All other data were obtained on 4μm paraffin sections after fixing the tissue in 4% (wt/vol.) phosphate-buffered formalin for 2–3 days. Histology was evaluated after staining paraffin sections with haematoxylin and eosin (H/E). Planimetric analysis of necrotic areas was performed on 10 randomly chosen pictures (taken with a 20x objective) of each carcinoma by using Adobe Photoshop CS5 (Adobe, San Jose, CA, USA). Apoptosis was analysed using the ApopTag Plus Peroxidase in situ detection kit (Millipore, Eschborn, Germany). To evaluate the cellular inflammatory response to cerulein injection, naphthol AS-D chloroacetate esterase (CAE) staining was performed on sections. Cell proliferation, chronic pancreatitis, and desmoplastic reaction were evaluated by immunohistochemistry using mouse anti-BrdU (Dako, Hamburg, Germany, clone Bu20a, dilution 1:50), rabbit anti-collagen-I (Abcam, code ab 34710, dilution 1:200), or rabbit anti-α-smooth muscle actin (Abcam, code ab5694, dilution 1:800) antibody. To verify desmoplastic reaction by the host, carcinoma cells were assessed in GFP expressing mice with goat anti-GFP antibody (Gene Tex, San Antonio, Texas, USA, GTX26673, 1:500). Cancer cells were further characterized by immunohistochemistry using rabbit anti-ALDH1a1 (Cell Signaling, code 12035, 1:800), goat anti-GFAP (Abcam, code ab7260,1:2000) or rat–anti CD133 (a generous gift by Denis Corbeil, Dresden, Germany, 1:200). Additional immunohistochemistry was performed using rat-anti-cytokeratin 19 (The Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City, USA, clone TROMA-III, dilution 1:50), rat anti-F4/80 (AbD Serotec, Oxford, UK, MCA497, 1:10) or goat anti-vimentin (Santa Cruz Biotechnology, Santa Cruz, USA, sc7557, dilution 1:50) antibody. The following secondary antibodies were used: the Universal LSAB+ Kit/HRP (Dako) for primary goat, rabbit or mouse antibodies or alkaline phosphatase conjugated anti-rat (Santa Cruz Biotechnology, sc2021, 1:200) antibody for primary rat antibodies. All quantifications of cells or of necrotic areas were performed 120 to 270 μm from the tumor margin.
Data presentation and statistics were performed as described previously . The significance of differences was evaluated using a Mann-Whitney rank-sum test, followed by the correction for the accumulation of the α error by considering the number of meaningful comparisons. Differences with P ≤ 0.05, divided by the number of meaningful comparisons, were considered to be significant.