2.10. Biodistribution in a Nude Mouse Xenograft Tumor Model

Human IgG (Jackson Immunoresearch Laboratories) and h4G3 were labeled with fluorescence dye CF750 using VivoBrite Rapid Antibody Labeling Kit (Biotium Inc., Hayward, CA, USA) in accordance with manufacturer’s instructions. To generate xenograft, 5 × 10⁶ OVCAR-3 cells in 100 μL PBS were injected subcutaneously into the right flank of 6 week old female athymic nude mice (Orient Bio, Seongnam, Gyeonggi, Korea). For the T47D xenograft model, 1 × 10⁷ T47D cells in 100 μL PBS were injected subcutaneously into athymic nude mice planted with 17β-estradiol pellets (Innovative Research of America, Sarasota, FL, USA). Tumor-bearing mice were treated intravenously with CF750-labeled antibodies as 100 μg/100 μL PBS. After 6, 24, 48, 72, and 96 h, the mice were anesthetized with Terrell^TM isoflurane (Piramal Critical Care Inc., Bethlehem, PA, USA) and placed in the IVIS Spectrum CT (Perkin Elmer, Waltham, MA, USA) to visualize CF750-labeled antibodies. The fluorescence was detected using an excitation filter (710 nm) and emission filter (780 nm). At the final time point, the mice were sacrificed and the liver, kidney, lung, spleen, intestine, and tumor were excised. The organs from each mouse were placed in 100 mm Petri dishes and imaged using IVIS Spectrum CT (Perkin Elmer). The intensity of fluorescence was analyzed using Live Imaging software (Perkin Elmer), and the average fluorescence intensity from organs was calculated by creating a region of interest over each organ. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU-190216-1).