Establishment of primary pancreatic cancer xenografts
Animal experiments were carried out using protocols approved by University Health Network Animal Welfare Committee. The establishment of the primary pancreatic cancer xenografts was done as previously described [8, 9, 11]. Fresh pancreatectomy samples that were superfluous to diagnostic needs were obtained from the University Health Network Tumour Tissue Bank according to institutional human ethical guidelines. Primary xenografts were established at the orthotopic site of 4-to 5-week-old mice by attaching tumour fragments to the surface of the exposed pancreas by a small incision in the upper left abdomen under general anaesthesia. Three orthotopic primary pancreatic cancer xenografts, designated as OCIP 19, 21, and 23, were used for these experiments.
Drug preparation and treatment protocols
The MEK inhibitor RDEA119/BAY869766 was provided by Ardea Biosciences, Inc. (San Diego, CA). Rapamycin was purchased from Calbiochem (San Diego, CA). Rapamycin was dissolved in DMSO at 1 mg/ml, aliquoted, and stored at -20°C. RDEA119, which has good oral bioavailability, was prepared freshly at 3.125 mg/ml in 10% Cremophor EL in saline, for oral gavage in vivo.
The 48 h combination therapy experiment included a total of 12 OCIP23 tumour-bearing mice with 3 animals randomly assigned to one of four groups: drug-vehicle control (10% Cremophor EL in saline, oral gavage; DMSO, i.p.), RDEA119 (6.25 mg/kg, oral gavage, b.i.d.), rapamycin (2 mg/kg, i.p.), and RDEA119 plus rapamycin groups (6.25 mg/kg, oral gavage, b.i.d. and 2 mg/kg, i.p., respectively). All mice were sacrificed 48 h after beginning the experiment (4 h after the final dose of RDEA119).
The chronic dosing combination therapy experiment included 36 tumour-bearing mice for each model, with 9 animals randomly assigned to one of four groups: drug-vehicle control, RDEA119, rapamycin, and RDEA119 plus rapamycin groups. In this experiment RDEA119 was administered 6.25 mg/kg, oral gavage, b.i.d. 5-day on and 2-day off, rapamycin was administered 2 mg/kg i.p. once weekly. Due to the different growth rates of these 3 models, the drug administration was initiated on Day 52, 24, and 12 after implantation in OCIP19, OCIP21, and OCIP23, respectively. Animal body weight, and tumour condition were recorded thrice weekly for the duration of the study. After 2-4 weeks of treatment, the animals were sacrificed and the tumours were harvested immediately (4 h after the final dose of RDEA119 or/and 48 h after the final dose of rapamycin). Depending on the subsequent analyses, all harvested tumours were cut into pieces, either snap frozen in liquid nitrogen, homogenized in lysis buffer as for the cell lysates, fixed in 10% formalin for 24 h and paraffin embedded.
Immunoblotting and quantification
Minced tumour pieces were homogenized in 1 ml lysis buffer. Equivalent amounts of protein were separated on 12% SDS-PAGE gels. Proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) and probed with the appropriate antibodies. Rabbit monoclonal antibodies against phospho-4E-BP1 (Thr37/46) (236B4), rabbit polyclonal antibodies against phospho-p44/42 MAPK (Thr202/Tyr204), phospho-S6 Ribosomal Protein (Ser235/236), phospho-S6 Ribosomal Protein (Ser240/244) and antibodies directed against their nonphosphorylated counterparts, were purchased from Cell Signalling Technology, Inc. (Danvers, MA). The secondary antibodies for western blot (anti-mouse and anti-rabbit IgG antibodies) were from Amersham Biosciences (Buckinghamshire, United Kingdom). Membranes were developed with enhanced chemiluminescence (ECL) Plus detection reagents and imaged using a Typhoon™9410 system (GE Healthcare, Piscataway, NJ). Quantification of immunoblots was performed using ImageQuant 5.2 software by calculating an index based on signal intensity multiplied by signal area.
Flow cytometric analysis
To study the effect of RDEA119 or/and rapamycin on the cell cycle, snap frozen tumours were minced and permeabilized with 0.1% Triton X-100 and stained with 50 μg/ml propidium iodide (PI)/RNase A (1 mg/ml). DNA histograms were analyzed using ModFit LT™(Verity, Topsham, ME). To determine the effect of RDEA119 or/and rapamycin administration on DNA synthesis in tumour cells in vivo and on tumour kinetics, tumour-bearing mice were injected i.p. with 100 mg/kg 5-bromo-2'-deoxyuridine (BrdU) (Sigma Chemical Co., St. Louis, MO) dissolved in PBS 30 min before the mice were sacrificed and the tumour removed. Single-cell suspensions from tumours were prepared by an enzymatic technique for dual label flow cytometry as described previously [12]. Briefly, single-cell suspensions were fixed in 80% ethanol, denatured, neutralized and then stained with anti-BrdU (PRBQD-Alexa 488, Phoenix Flow Systems, Inc., San Diego, CA) and 1 μg/ml 4', 6-diamidino-2-phenylindole (DAPI). BrdU labelling index was analyzed using WinList™6.0 (Verity, Topsham, ME).
Immunofluorescent, immunohistochemical staining and image analysis
Serial sections were cut from paraffin-embedded tumour tissue: one of these was stained with H&E for transmitted light microscopy and used for selecting the tumoural areas, on which the further image analysis was to be done. The 48h-treatment sections were labelled with primary antibodies against phosphorylated ERK (Cell Signalling Technology, Inc., Danvers, MA) and BrdU (Clone IU-4, MD5000; Caltag Laboratories, Inc., Burlingame, CA). Secondary antibodies used alone were control for nonspecific background. All these sections were counterstained with 1 μg/ml DAPI to outline the nuclear area. p53 (DO-7, Vector Laboratories, Inc., Burlingame, CA) and phosphorylated ERK (Cell Signalling Technology, Inc., Danvers, MA) were stained for transmitted light microscopy for all the chronic dosing sections.
The analysis included the entire viable tumoural area identified on the slide, using a scanning autostage to create composite images of individual fields at 20 × magnification (2.24 mm2) as described previously [13]. A tumoural map that included only viable tumour was made using the H&E sections and then carefully adjusted on the composite DAPI images. For each marker, we measured at least two variables: the intensity of the signal (integrated absorbance) and the percentage of positive stained area/nuclei, based on the binary images.
Analysis of plasma RDEA119 concentrations
Plasma samples were harvested 4 h after the final dose of RDEA119 or/and 48 h after the final dose of rapamycin. Plasma samples were analyzed for RDEA119 using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method [6]. The method involved the addition of the internal standard ([13C6]RDEA119), protein precipitation with acetonitrile, and final analysis by high-performance LC-MS/MS. An API 5000 triple quadruple mass spectrometer was used to monitor the precursor!product ion transitions of m/z 573!394 and m/z 579!400 for RDEA119 and [13C6]RDEA119 in positive electrospray ion mode. The calibration curves covered the concentration range from 10 to 10,000 ng/ml.
Statistical analysis
Data from quantitative experiments are presented as mean ± SE. Data obtained from the xenograft models (tumour weight and animal body weight) and comparisons between four treatment groups were analyzed using a repeated measure one-way ANOVA test with Newman-Keuls multiple comparison post test. P < 0.05 was considered statistically significant.