All the treatments were performed twice a week and lasted for 2 wk. Tumor width
(W) and length (L) were measured every 4 d by calipers. The tumor volume (Tv) LY2606368 was calculated according to the following formula: Tv = 0.52 × L × W2. The treated mice were closely monitored and sacrificed if any signs of approaching death were shown. The mice in all groups were sacrificed 50 days after tumor establishment. All experiments involving mice were approved by the Institute’s Animal Care and Use Committee. Detection of microvessel density and apoptosis Frozen tissues were sectioned (5 μm) and fixed in acetone at 4°C. For detection of CD31 immunostaining, sections were probed with a monoclonal rat anti-mouse CD31 antibody (1:400, Santa Cruz Biotechnology, Santa Cruz, CA, US) at 4°C overnight, followed by incubation with biotinylated polyclonal goat anti-rat antibody (1:200, Vector Laboratories, Peterborough, UK) and Vectastain Elite ABC Kit (Vector Laboratories, Peterborough, UK). Positive reaction was visualized using 3,3-diaminobenzidine as chromagen (DAB substrate kit, Vector Laboratories, Peterborough, UK). Sections were counterstained with hematoxylin and mounted with glass coverslips. Apoptotic cells were identified using the TUNEL (terminal deoxynucleotidyl transferase-mediated nick end
labeling) assay (In Situ Cell Death Detection Kit, Roche, Basel, Switzerland) following the manufacturer’s guide. Images were captured by the Olympus fluorescence microscope at ×200 magnification. The quantification of microvessel density (MVD) (the maximum vascular area of the tumor) was assessed within hot spot. The apoptotic cells were Selleckchem VX770 counted in 5 high power fields in each slide in a blinded manner. The percentage of apoptotic cells among tumor cells were calculated as apoptotic index. Alginate encapsulation assay Alginate bead containing tumor cell assay was described in details previously. Briefly, cultured LLC cells were resuspended with 1.5% (m/v) sodium alginate (Sigma-Aldrich, St. Louis, MO, US), and then the tumor cell alginate solution was dropped into a swirling bath of 0.25 M CaCl2 in order
to form droplets containing about 1 × 105 tumor cells per bead. After anesthetized, the C57BL/6 mice were implanted Thymidine kinase s.c. with four beads into an incision on the back, the incisions were sutured with surgical clamps. Treatment of Ad-hEndo (1 × 109pfu/100 μl) or cisplatin (1 mg/kg) was performed on day 0, 4, 8, 12 after bead implantation, with Ad-null or saline as control. At 14 days, the mice were injected i.v. with 100 μl FITC-dextran solution (Sigma Chemical) (100 mg/kg) and were sacrificed 20 minutes later. Image of the alginate implants was taken by using SPOT FIEX camera. Alginate beads were transferred to tubes containing 2 ml of saline. The tubes were mixed by a vortex for 20 s and centrifuged (3 min; 1000 × g). Finally the fluorescence of the supernatant was measured to quantify blood vessel formation.