Animals and tumor cells
All of the experiments on animals were approved by the Regional Administrative Authority in Darmstadt, Germany.
Twenty inbred male ACI-rats (Harlan Winkelmann; Borchen, Germany) weighing 220–240 g were used. The animals were kept under conventional conditions with a temperature of 22 ± 2 °C, a relative humidity of 55 ± 10 %, a dark-light rhythm of 12 h, and they were fed with standard laboratory chow and tap water ad libitum. The hepatoma cell line used in the current study was obtained from the German Cancer Research Center (DKFZ; Heidelberg, Germany). The injected cell line (Morris hepatoma 3924A) represents a rapidly growing, poorly differentiated hepatocellular carcinoma.
Chemotherapeutics and agents
For TACE a dose of 0.1 mg Mitomycin (Roche, Grenzach-Wyhlen, Germany) was dissolved in 0.1 ml 0.9 % NaCl solution 10 min before application. The embolization was performed using a dose of 0.1 ml lipiodol (Guerbet GmbH, Sulzbach, Germany) and 5.0 mg degradable starch Microspheres (Spherex©, Pharmacia, Erlangen, Germany).
Survivin siRNA was provided from Ruibo Gentech Co (Wuhan, PR. China). The target sequence of Survivin siRNA is as follows: CCGAGAATGAGCCTGATTT. A dose of 2.5 nmol Survivin siRNA was stable at 2–8 °C for 10 min before administration.
A combination of intra-peritoneal injection of Ketamine Hydrochloride (Ketanest Parke-Davis, Germany; 100 mg/kg), Xylazine Hydrochloride (Rompun, Bayer Germany; 15 mg/kg) and Atropine Sulfate (Atropin Sulfat Braun, Braun, Germany; 0.1 mg/kg) was used for anesthesia in all Orthotopic, interventional and imaging procedures.
Tumor implantation (day 0)
The technique for tumor implantation was basically similar to that described by Yang et al.  with minor modifications . The Morris Hepatoma 3924A tumor tissue, recovered from the passaged animals 12 days after subcutaneous implantation (corresponding to 5 × 106 tumor cells), was cut into small cubes about 2 mm3. A small sub-capsular incision on the left lateral lobe of the liver was made in the recipient ACI-rats under anesthesia. The tumor fragment was gently placed into created pocket with a small cotton swab on the liver surface and the abdominal wall was then closed.
Interventional therapy (day 13)
For interventional studies a second laparotomy was performed. By using a binocular operative microscope (M651, Leica; Wetzler, Germany), a PE-10 polyethylene Micro-catheter (inner diameter 0.28 mm, outer diameter 0.61 mm; Wenzel; Heidelberg, Germany) was retrogradely inserted into the Gastro-duodenal artery and pushed forward to the hepatic artery. Different agents were then injected into the hepatic artery using the sandwich technique (subsequent injection of Mitomycin +/− Survivin siRNA + Lipiodol + degradable starch Microspheres) within 20 min. Each group of animals received treatment according to the following protocols:
Group A (TACE + Survivin siRNA, test group, n = 10): Mitomycin (0.1 mg) + Lipiodol (0.1 ml) + degradable starch Microspheres (5.0 mg) + Survivin siRNA (2.5 nmol)
Group B (TACE alone, control group, n = 10): Mitomycin (0.1 mg) + Lipiodol (0.1 ml) + degradable starch Microspheres (5.0 mg)
MR imaging and analysis (day 12 and 25)
MRI imaging before (on day 12) and after (on day 25) treatment was performed using a 3.0 Tesla MRI unit (Magnetom, Siemens; Erlangen, Germany) by using a wrist coil (Small field of view). T1-weighted (SE: TR/TE, 500/12 ms) and T2-weighted (TSE: TR/TE, 3870/80 ms) transverse images with a section thickness of 2 mm and 184 × 256 matrix were acquired. There was no gap between sections and no contrast medium was administered. The tumor volume was determined and evaluated in the T2-weighted image according to the formula : V = 0.5 × d1 × d22, where d1 is the maximum diameter of the tumor and d2 is the minimum diameter perpendicular to d1. Image evaluations and size assessments were performed by a single radiologist with more than 15 years’ experience in abdominal MRI imaging and who was blinded to the group assignment of the animal in the study.
Western blot (day 25)
Western blot analysis was carried out to determine the expression level of the VEGF in the two groups. After the MRI examination, all the rats were sacrificed using an over-dose of intravenous Sodium Pentobarbital. To homogenize the tumors, Precellys Homogenizer IV (Peqlab Biotechnologie GmbH; Erlangen, Germany) at 4 °C was used in a lysis buffer which is composed of 50 mM HEPES, 200 mM NaCl, 0.2 mM MgSO4, 0.4 mM Phenylmethylsulfonyl fluoride, 2 % Triton- X-100, 10 μg/mL Leupeptine, 10 μg/mL Aprotinine, 0.02 % soybean trypsin inhibitor and 0.2 mM Orthovanadate (Sigma-Aldrich; Taufkirchen, Munich, Germany). The resulting Cell lysates were centrifuged for 10 min at 12,000 × g at 4 °C. Coomassie Plus protein assay kit (Pierce; Rockford, IL, USA) was used to measure the protein concentration in the supernatants. The protein concentration results were obtained Spectrophotometrically by Tecan Infinite® M 200 microplate reader (Tecan-Deutschland; Crailsheim, Germany) at 595 nm. Protein was then denatured in Laemmli sample buffer (Bio-Rad Laboratories; Munich, Germany) with β mercaptoethanol (Sigma; Taufkirchen, Germany), boiled for 5 min, and transferred on ice. Sodium Dodecyl Sulfate Polyacrylamide gel electrophoresis (SDS-PAGE) (50 μg per lane) was then conducted. The molecular weight standards used were PeqGold prestained protein markers IV (Peqlab Biotechnologie GmbH; Erlangen, Germany). After separation using gel electrophoresis, protein was blotted onto a Polyvinylidene Difluoride membrane (Hybond P; GE Healthcare; Munich, Germany). Blots were then blocked with 10 % low-fat milk for 1 h at room temperature followed by overnight incubation at 4 °C with primary antibody from Santa Cruz Biotechnology (Rabbit polyclonal VEGF 1:200; Rabbit polyclonal MMP-9 and mouse monoclonal β Actin, (clone AC-15, 1:1000; Sigma). Blots were then washed 3 times with Towbin buffer with 0.5 % Tween 20 followed by incubation for 30 min at room temperature with secondary antibody from Millipore GmbH; Schwalbach/Ts, Germany (Polyclonal goat Anti-rabbit IgG, 1:5000; goat Anti-mouse IgG, 1:5000, both HRP conjugated). All antibodies were diluted in Towbin Buffer with 0.5 % Tween 20 and 0.5 % bovine serum albumin. Blots were then washed and incubated withEnhanced Chemiluminescence detection kit (GE Healthcare; Munich, Germany). Signal intensity was finally detected and captured by Fusion FX-7 (Vilber Lourmat, Marnee la Vallee, France), documented and analyzed by Bio1D software (Vilber Lourmat). β Actin was used as the loading control.
Immunohistochemical examination (day 26)
The Liver samples were embedded and frozen in a Tissue-Tek (Sakura, Zoeterwoude, Netherlands) and 5 μM cryo-sections were prepared. These Sections were fixed in 100 % acetone and equilibrated in PBS followed by overnight incubation at 4 °C with anti-VEGF rabbit polyclonal antibody (Santa Cruz Biotechnology Inc., USA) which was diluted with Dako antibody diluents (DAKO, Hamburg, Germany). The cryosections were then incubated with Anti-rabbit Alkaline Phosphatase supervision polymer system (DCS Innovative Diagnostik-Systeme, Hamburg, Germany). Staining was visualized using the Neu Fuchsin substrate Chromogen (DCS Innovative Diagnostik-Systeme, Hamburg, Germany) and were counterstained with Hematoxylin and mounted in Kaisers Glycerol Gelatin (Merck, Darmstadt, Germany). To evaluate the expression of VEGF, all slides were examined and scored by two independent pathologists who were blinded to the animal data. The percentage staining was scored as follows: 0 (No staining, 1 (0-5 %), 2 (6-25 %), 3 (26-50 %), 4 (51-75 %), 5 (76-100 %).
The mean tumor growth ratio (V2/V1, V2 tumor volume after treatment and V1 tumor volume before treatment) by MRI and the mean expression ratio (VEGF/β-actin) level of VEGF by Western blot from each group were measured and the significance of differences between the two groups were analyzed using the paired-t-test, the statistical software used was GraphPad Prism (version 3.02, La Jolla, CA, USA). Immunohistochemical staining of VEGF was evaluated using descriptive and semi-quantitative methods. The differences between both groups in the Western-Blot analysis and Immunohistochemical analysis were tested for statistical significance using the unpaired-t-test and the Wilcoxon signed rank test respectively. Differences with a p value less than 0.05 were considered statistically significant.