Xenograft animal model and cancer cell imaging

Male Balb/c nude mice were obtained from the National Laboratory Animal Center of Taiwan (Taipei City, Taiwan) and maintained under the supervision of the Institutional Animal Care and Use Committee (IACUC) of Chung Shan Medical University. All the protocols for animal experiments have been approved by the IACUC (No. 1947), and all the experiments were performed in accordance with the approved guidelines. After a 1-week acclimation period, the mice were randomly divided into the following three groups (n = 6 per group): Normal (no cancer cell implantation), Sham (implantation of cancer cells treated with 0.1% DMSO/PBS for 24 h), and Delphinidin (implantation of cancer cells treated with 100 μM delphinidin for 24 h). Cancer cell implantation was performed by intraperitoneal injection of luciferase-transfected DLD-1 cells (2 × 10⁶ cells in 50 μL of PBS), and the animals were maintained for two weeks. After the maintenance period, the mice were anesthetized and intraperitoneally injected with luciferin (at 150 mg/kg in 100 μL). Images were captured at 20 min after injection using an IVIS-200 Imaging System (Xenogen, Alameda, CA, USA) and processed using Living Image software (Xenogen) by region-of-interest analysis of the total photons per second for each tumor, with background subtraction. After the image analysis was completed, the mice were sacrificed to examine the organs.