tumor xenograftsFor tumor angiogene

tumor xenograftsFor tumor angiogenesis study, mice aged between 8–12 weeks were injected with 80 mg per kg (weight) tamoxifen intraperitoneally, once daily for 5 days to induce Cds2 deletion before tumor cell inoculations. Cells were harvested in 100 μl RPMI-1640 or DMEM medium and gently mixed 2:1 (volume ratio) with matrigel (Corning, 356237). Then, mice were inoculated subcutaneously on the lateral flanks with 106 TC-1 cells or 0.5 million B16 cells (150 μl volume per injection). Vernier caliper was used to measure the approximate tumor volume calculated as length × width2/2, and final tumor weight measurements were taken at the termination of the experiments. Subsequently, tumors as well as other organs were harvested for histology analysis.For vessel regression determination, mice were injected with 3 × 106 TC-1 cells or 106 B16 cells (150 μl volume per injection) two days prior to a five-day consecutive injection of tamoxifen, allowing for a robust angiogenic response. Tumors were measured once two days for 25 days or until reaching an average diameter of 1.5 cm, and tumor necrosis was monitored daily during this process. For TC-1 tumors, ultrasound imaging and analysis were performed at day 7, 11 and 15 post-implantation to assess the blood perfusion. Meanwhile, a portion of tumors without necrosis were excised at day 9 or 13 for tumor weight measurements, histology and qRT-PCR analysis. To determine the Vegfa expression in tumor cells, GFP-labeled B16 cells were inoculated as described above. Then day 9 tumors were excised, cut into pieces and digested with collagenase/dispase (Roche, 1 mg ml−1) in DMEM at 37 °C for 30 min. GFP-positive and -negative cells were sorted by FACS to perform qPCR analysis. To pharmacologically inhibit PI3K signaling, BKM120 or BEZ235 dissolved in 10% N-methyl-2-pyrrolidone (NMP) with 90% PEG400 was orally administrated at 40 mg kg−1 daily from day 5 to day 9 after B16 cell inoculation. Then, tumors were excised at day 10 for weight measurement and immunohistochemistry analysis. For vivo morpholino (vMO) administration, Cds2 vMOs (Gene Tools) were administrated at 1 mg kg−1 daily through intravenous injection from day 1 post B16 cell implantation until the experimental termination. Tumor size was measured once two days for 15 days or until reaching size limitation guided by Animal Protocol.