In vivo tumor xenograft study

Athymic (nu/nu) male nude mice were purchased from the NCI (Frederick, MD). The treatment protocol was approved by the Institutional Animal Care and Use Committee of the University of Colorado Denver. About 1 million 22Rv1 cells were suspended in 50 μL of serum free medium, mixed with 50 μL of matrigel, and injected s.c. in flanks of each mouse. Once the xenografts started growing, mice were randomly divided into 2 groups: Group I mice (vehicle control group) orally administered with 200 μL of 0.5% CMC (w/v) in sterile water; Groups II mice orally administered with 200 mg/kg body weight dose of silibinin in 200 μL of 0.5% CMC. Mice were imaged by FDG-PET to assess glucose uptake before the beginning of the silibinin treatment (day 0) as well as on day 4 and day 15. DCE-MRI and DW-MRI were performed for tumor vascularity and cellularity, respectively, on day 1, day 6 and day 16 of the experiment. At the end, tumor tissues were collected and analyzed for metabolites by quantitative ¹H-NMR metabolomics. A part of the tumor tissues was also fixed in formalin and analyzed by IHC for various biomarkers. Throughout the experiment, tumor sizes were measured twice weekly using digital caliper and tumor volume was calculated by the formula: 0.5236 L₁(L₂)², where L₁ is long diameter, and L₂ is short diameter.