The CCA cell lines HuCCT1 and KKU-100, as well as MMNK-1 (a normal bile duct cell line), were obtained from the Japanese Collection of Research Bioresources Cell Bank (JCRB; Osaka, Japan). All of the established cell lines used in this study were validated by short tandem repeat analysis performed by the Division of Transfusion Medicine, Taipei Veterans General Hospital. The HuCCT1 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The KKU-100 and MMNK-1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.). All the cells were supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 µg/ml streptomycin, 100 µg/ml penicillin, and 2 mM L-glutamine in a humidified atmosphere containing 5% CO₂ at 37°C.

Cell growth was measured using a clonogenic assay. HUCCT1 or KKU-100 cells that were either treated or not treated with BMS-777607 (Bristol-Myers Squibb, Taiwan) were cultured for 10 days to allow clonogenic growth, as previously described (18).

Cell viability was determined using the TACS tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assay kit (Trevigen, Gaithersburg, MD, USA), according to the manufacturer's instructions. MTT is used to determine cell viability in cell proliferation and cytotoxicity assays. Briefly, HUCCT1 or KKU-100 cells were seeded at a concentration of 1,000 cells/well in 100 µl culture medium into 96-well microplates. At 24 h post-seeding, the cells were treated with dimethyl sulphoxide (DMSO) or BMS-777607 for 6 days; the cells were then incubated in medium containing MTT for 2 h. The optical density at 570 nm was measured using a microplate reader (Spectral Max250; Molecular Devices, Sunnyvale, CA, USA).

Whole cell lysates from the CCA cell lines were obtained using Pierce radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc., Rockford, IL, USA). Protein samples were separated on 6–8% gradient dodecyl sulfate-polyacrylamide gels and transferred to Immobilion-PVDF membranes (Millipore, Billerica, MA, USA). Antigen-antibody complexes were detected using an electrochemiluminescence blotting analysis system (Millipore). The following primary antibodies were used: RON (Abcam, Cambridge, UK), MET (Cell Signaling Technology Inc., Danvers, MA, USA), phospho-RON (Abcam) and β-actin (Abcam).

Eighteen adult male Sprague-Dawley (SD) rats (310±14 g) were used in our animal experiments. They were equally divided (n=6) into the following 3 groups: i) A control group; ii) a gemcitabine/oxaliplatin treatment group; and iii) a BMS-777607 treatment group. The rats were housed in an animal room with lighting set to a 12:12 h light-dark cycle (lights on from 08:00 a.m. to 08:00 p.m.) and an ambient temperature set at 22°C. Food and water were provided ad libitum. The rats were administered 300 mg/l thioacetamide (TAA) via drinking water daily for up to 20 weeks (19). TAA-induced rat CCA was induced as previously reported and followed the Animal Experimental Guidelines of Chang Gung Memorial Hospital, Chang Gung University (Taoyuan, Taiwan) with approval code: IACUC: 2010121409. The gemcitabine/oxaliplatin treatment group received gemcitabine [50 mg/kg, intraperitoneal injection (i.p.)] and oxaliplatin (2 mg/kg, i.p.) once every 2 weeks over a 4-week period starting at the 21st week. The MET-RON dual inhibitor treatment group received BMS-777607 [30 mg/kg, per os (p.o.)] 5 days/week (Monday through Friday) starting at the 21st week. The control group rats received i.p. injections of PBS following the same schedule.

To evaluate the changes in glycolysis in live animals with liver tumors, we conducted 2-deoxy-2-[F-18] fluoro-d-glucose (FDG)-positron emission tomography (PET) studies of the rats at the Molecular Imaging Center of Chang Gung Memorial Hospital. In total, 18 rats were treated with TAA and received serial PET scanning at weeks 21, 23 and 25 using the Inveon™ system (Siemens Medical Solutions USA Inc., Knoxville, TN, USA). Equal numbers of animals were assigned to the control and treatment groups based on their baseline PET results, ensuring that the control and treatment groups possessed similar PET-positive rates. The details of the radioligand preparation, scanning protocols, and determination of optimal scanning time used in the present study have been previously described (20). Briefly, animals were fasted overnight prior to scanning. At 90 min post-¹⁸F-FDG injection [intravenous injection (i.v.)], 30 min static scans were obtained for all of the animals. All imaging studies were performed using a temperature-(set to 37°C) and anesthesia-(2% isoflurane vaporized in 100% oxygen) controlled imaging bed (Minerve System, Esternay, France). PET images were reconstructed using the 2D ordered subset expectation-maximization method (4 iterations and 16 subsets) without attenuation and scatter corrections. All imaging data were processed using the PMOD image analysis workstation (PMOD Technologies Ltd., Zurich, Switzerland). The largest liver tumor for each animal was identified by careful study of all 3 image sets for each rat. ¹⁸F-FDG uptake into the largest liver tumor, as well as apparent normal liver tissue, was quantified by calculating the standardized uptake value (SUV). These values were calculated according to the recommendations made by the European Organization for Research and Treatment of Cancer (21). The tumor regions of interest (ROIs) were determined using transverse images of the selected tumors and measuring the largest diameter. The normal liver ROIs were also determined using the same transverse images. The mean SUV (SUV_mean) of the normal liver and tumor tissue was calculated, and the tumor-to-liver radioactivity ratio was calculated for comparison.

We analyzed the impact of expression of MET-RON on the characteristics and prognosis of 96 patients with mass-forming CCA (MF-CCA) who had received hepatectomies between 1989 and 2006 at the Department of Surgery, Chang Gung Memorial Hospital. The study was approved by the local Institutional Review Board of Chang Gung Memorial Hospital (clinical study nos. 99–2886B, 99–3810B and 102–5813B). Informed written consent for immunohistochemical tumor analysis was obtained from each patient.

MET and RON expression levels in the 96 MF-CCA patients were examined by immunohistochemical staining. Tissue sections (4 µm) were prepared from formalin-fixed, paraffin-embedded hepatectomy specimens, and incubated with anti-RON primary antibody (EP1132Y; 1:100 dilution; Abcam) and anti-MET primary antibody (8F11; 1:100 dilution; Abcam) overnight at 4°C. After three 5-min washes with TBST, bound antibody signal was visualized using Dako Labelled Streptavidin-Biotin2 (LSAB2) System-HRP (Dako A/S, No. K0675; Dako, Glostrup, Denmark). Control slides were incubated with the secondary antibody only. For the assessment of immunohistochemical staining of MET-RON, the percentage of stained target cells was determined based on 10 random microscopic fields of view per tissue section (magnification, ×400) using microscope Olympus BX51 and CCD capture for Olympus DP21 (Olympus Corp.). Their average scores were then calculated. Staining intensities were assigned scores of 1 (mild), 2 (moderate) or 3 (strong). H-scores were calculated as the percentage of positive staining (0–100) × the corresponding staining intensity (0–3). Specimens with H-scores of <160 or ≥160 were classified into those having low or high expression, respectively (range, 5–300; median, 160).

The follow-up evaluation included physical examinations and blood chemistry tests during each visit. Additionally, serum levels of CEA and CA 19-9 were measured, and the remnant liver was examined by ultrasound (US) every 3 months. When a new lesion was detected by US or elevated levels of CEA/CA 19-9 were noted, the patients received abdominal CT or magnetic resonance cholangiopancreatography (MRCP) for confirmation. If the patients complained of bone pain, bone scans were performed to detect metastasis. Whether any of these procedures suggested recurrence, the patient in question was readmitted for a more comprehensive assessment, including angiographic evaluation or magnetic resonance imaging (MRI). The methods for treating recurrence included surgery, systemic chemotherapy, external beam radiotherapy, intraluminal radiotherapy, interventional radiological therapy and conservative treatment.

All data are presented as the mean ± SD. Differences between the experimental and control groups were calculated using the Student's t-test. Progression-free survival (PFS) and overall survival (OS) rates were evaluated with the Kaplan-Meier method. Several clinicopathological variables were considered for the initial univariate analysis, which was performed using the log-rank test. The Cox proportional hazards model was applied for multivariate regression. All statistical operations were performed using SPSS for Windows (version 17.0; SPSS, Inc., Chicago, IL, USA). A value of P≤0.05 derived from 2-tailed tests was considered significant.

HuCCT1 and KKU-100 cells were selected as model cell lines since we found them to have higher levels of MET and RON than the normal bile duct cell line, MMNK-1, as determined by western blot analysis. We aimed to investigate the possible antiproliferative effects that BMS-777607 may have on CCA cells. To determine the potential effects, we used clonogenic assays to measure the growth of the two human intrahepatic CCA cell lines, HuCCT1 and KKU-100, in the presence of varying concentrations of BMS-777607. We found that BMS-777607 had a concentration-dependent antiproliferative effect on both the HuCCT1 and KKU-100 cell lines (Fig. 1A and B). In the HuCCT1 and KKU-100 cell lines, the IC₅₀ values of MET-RON dual inhibitor (BMS-777607) 6 days after treatment were 11.4 and 5.9 µM, respectively (Fig. 1C). We also demonstrated that the expression of MET and RON protein in HuCCT1 and KKU-100 cells was higher than that observed in the MMNK-1 cells. In addition, we also demonstrated that the expression of phospho-RON was decreased in both HuCCT1 and KKU-100 cell lines after treatment with BMS-777607 (Fig. 1D).

We were also interested in assessing the possibility of using BMS-777607 to treat this cancer in vivo, as the rats with TAA-induced CCA displayed overexpression of MET and RON (Fig. 2A and B). To conduct this assessment, we compared the therapeutic efficacy of BMS-777607 to that of a combination of gemcitabine and oxaliplatin (e.g., a standard therapy for CCA) in treating the rats with TAA-induced CCA. As can be seen in Fig. 3A, animal PET-CT showed that the rats in each group had at least one FDG-avid tumor in the liver after 20 weeks of TAA treatment. These CCA rats were then treated with the vehicle alone, the MET-RON dual inhibitor alone (30 mg/kg, p.o., 5 days/week) (Monday through Friday), or gemcitabine plus oxaliplatin (gemcitabine 50 mg/kg + oxaliplatin 2 mg/kg, i.p., two times in 1 week) for 4 weeks. As of 2 to 4 weeks from the beginning of treatment, the group receiving the vehicle alone had steady increases in the mean tumor-to-liver (T/L) ratio of the SUV (that is, elevation from 33.0 to 50.0%). However, as of 2 weeks after beginning treatment, both the CCA rats receiving BMS-777607 and those receiving gemcitabine/oxaliplatin were found to have significant decreases in the T/L ratio of the SUV (P=0.041 for BMS-777607 and 0.006 for gemcitabine/oxaliplatin, respectively) (Fig. 3B). These findings indicate that MET-RON treatment significantly suppressed the in vivo growth of CCA tumors in our animal model.

Forty-five of the 96 MF-CCA patient specimens (46.9%) were found to have high cytoplasmic immunostaining for MET-RON (H score ≥160, Fig. 4). The overexpression of MET-RON was associated with elevated alkaline phosphatase, elevated CEA, tumor size larger than 5 cm, and positive resection margin, but only elevated alkaline phosphatase and elevated CEA were independently associated with it (Table I). With regard to survival, univariate log-rank analysis of the 96-post hepatectomy patients with MF-CCA identified the following factors as having adverse influences on OS: presence of symptoms, elevated alkaline phosphatase, elevated CEA, decreased albumin, tumor size >5 cm, positive surgical margin and lymph node status, and higher MET-RON immunostaining (Table II). Multivariate Cox proportional hazard analysis, however, revealed that both positive symptoms and higher MET-RON immunostaining scores independently predicted worse OS (Table II and Fig. 4).