Mouse Models and In Vivo Fluorescence Imaging

Athymic nude male mice (N = 16) were purchased from The Jackson Laboratory (Sacramento, CA, USA). Experimental protocols were approved by the Yale University Institutional Animal Care and Use Committee (IACUC; number 2013-11504) and adhered to the NIH Guide for the Care and Use of Laboratory Animals. A freshly prepared mixture (50 μL complete culture medium, 5 × 10⁵ Hep3B-Luc or SNU-449-Luc cells, 50 μL Corning Matrigel Matrix HC, Phenol Red-free, and LDEV-free) was directly injected into mouse liver under inhalation isoflurane anesthesia. The injection point was 2 mm below the angle, which formed by the xiphoid and the left costal margin of the mouse. All mice were then maintained for 15 days and examined by In Vivo Imaging System (IVIS) Lumina LT Imaging System (Xenogen/Caliper Life Sciences, USA) with Living Image 4.3.1 software (Caliper Life Sciences, USA). Intraperitoneal (i.p.) injection of D-luciferin was used to provide substrate bioluminescent imaging. All mice were then divided into an LV group (LNP-VA) (N = 4) and NC group (LNP-ddH₂O) (N = 4). 100 μL LV at a concentration of 100 mg/kg was administered to each mouse by tail injection once a week for 4 consecutive weeks, and images of tumor sizes were collected every week. Mice were monitored daily for 70 days to collect survivorship data. Mice were sacrificed in a CO₂ chamber when ethically necessary due to clinical symptoms or substantial loss in body weight. To compare survival differences between LV-treated and control groups, log rank (Mantel-Cox) test was used for survival analysis in GraphPad Prism (version 8.2.1).