Radiopharmaceutical Preparation
Synthesis of the labeling precursor, kit preparation, and subsequent 99mTc-labeling were performed as previously described[35]. Briefly, the kit for the preparation of 99mTc-3PRGD2 was formulated by combining 20mg of hydrazinonicotinamide-3PRGD2, 5 mg of trisodium triphenylphosphine-3,39,399-trisulfonate (TPPTS), 6.5 mg of tricine, 40mg of mannitol, 38.5 mg of disodium succinate hexa- hydrate, and 12.7 mg of succinic acid. For 99mTc radiolabeling, to the kit vial was added 1 mL of 1,110–1,850 MBq (30–50 mCi) of 99mTcO4- saline solution, and then the vial was water-bathed at 100℃for 15–20 min. The resulting solution was analyzed by instant thin-layer chromatography using Gelman Sciences silica-gel paper strips and a 1:1 mixture of acetone and saline as eluant. The radiochemical purity was always greater than 95%. The reaction mixture was then diluted to approximately 370 MBq/mL (10 mCi/mL) with saline and was filtered with a 0.20-mm Millex- LG filter (EMD Millipore). Each animal was injected with 7.4-11.1 MBq (0.2-0.3 mCi) of 99mTc-3PRGD2 per mice. The resulting solution was analyzed by instant thin-layer chromatography using paper strips and acetone as eluent. The radiochemical purity was >95%.
Animal Model Establishment.
Female BALB/c mice (5 weeks of age) were purchased from Vital River Lab Animal Technology Co., Ltd. PANC-1 mice model was established by subcutaneous injection of 2 × 106 PANC-1cells into the right rear legs of mice. Once the tumor diameter reached 5-7mm, the mice were initiated with treatment (2 weeks after inoculation of PANC-1cells).
Treatment Protocol.
The study flowchart is provided in Fig. 1. PANC-1 tumor-bearing BALB/c mice with a tumor size of 5-7mm were randomly assigned to four groups (n = 7 mice per group). The mice were randomly assigned to four major groups. The first group was injected 10 mg/kg of Endostar, the second one with 10 mg/kg of Gemcitabine, the third one with 10 mg/kg of Endostar and 10 mg/kg of Gemcitabine at the same time ,and the control group with 0.9% saline. The treatments were performed daily for 28 days continuously. Tumor dimensions were measured every day with digital calipers, and the tumor volume was calculated using the formula (volume = 1/2 length × width × width). To assess potential toxicity, body weight was measured daily. All mice were euthanized and the tumor tissues were harvested for further immunohistochemical staining when the treatment was complete.
Imaging Protocol
The scanner was dual-head g-cameras, using low-energy high-resolution collimators and a 20% energy window centered on 140 keV. After intravenous injection of 99mTc-3PRGD2, static planar scans of the mice were obtained at 1.5h(the highest time point of tumor uptake). The acquisition count was 3 × 105. The matrix is 256 × 256, and the magnification is 1.33. The region of interest (ROI) of the tumor (T) and contralateral corresponding site (NT) were delineated, and the ratio of radioactivity (T/NT) was calculated. The study flowchart is provided in Fig. 1.
Immunofluorescence staining
Vascular endothelium was labeled using immunohistochemical staining with isolectin B4. The fluorescence staining study was carried out as previously described [37]. Slides were incubated with the fluorescein-labeled Griffonia Simplicifolia Lectin I (GSLI) Isolectin B4 (FL-1201, 1: 50, Vector Laboratories, USA) overnight at 4°C and sealed with DAPI Fluoromount-G® mounting medium (Southern Biotech, USA). Microvessel density was counted on the isolectin B4-stained slides in three fields in a blinded way using a fluorescence microscope in three fields under 40×magnification and the results were averaged.
Statistical Analysis
Quantitative and semiquantitative data were expressed as the mean ± SD and analyzed using SPSS version 17.0 (IBM, Chicago, IL, USA). Mean values were compared using one-way analysis of variance (ANOVA) or Student’s t-test. Two-way repeated-measures analysis of variance (ANOVA) was used to evaluate the differences between different treatment groups.