In vivo experimental animal model and protocol

The orthotopic pancreatic cancer mice model was used as previously described [32, 46]. The mice were continuously anesthetized with 2% isoflurane (Pfizer Japan Inc., Tokyo, Japan), and a 5-mm incision was made in the left flank on the splenic silhouette. The distal pancreas was gently delivered from the peritoneal cavity with spleen thorough the incision. 5.0 × 10⁶ PANC-1 cells suspended in 40 µl of phosphate buffered saline (PBS) were injected into the tail of the pancreas. After injection, the pancreas was placed in the abdomen and the abdominal wound site was closed in two layers. At 4 weeks after the injection, the animals were randomly divided into four treatment groups, which were the same as the in vitro experimental groups (n = 4). Pomalidomide (0.5 mg/kg) was administered orally five times a week, and gemcitabine (50 mg/kg) and nab-paclitaxel (0.5 mg/kg) were injected intraperitoneally once a week. The doses of antitumor agents were determined according to the previous studies [32, 47]. In the C group, vehicle of pomalidomide (PBS) was administered orally five times a week, which vehicles of gemcitabine and nab-paclitaxel (distilled water and isotonic sodium, respectively) were injected intraperitoneally once a week. The tumor volume was evaluated sequentially once a week by magnetic resonance imaging (MRI). At five weeks after treatment, the animals were sacrificed and orthotopic pancreatic tumors were excised for the assessment.