Xenograft models and immunohistochemistry

Six 6-week-old immunodeficient male mice (BACL/CAnN.Cg-Foxnl^nu/CrlNarl) were subcutaneously injected with 5×10⁶ luciferase-expressing CWR22Rv1 cells. Mice were divided into two groups for control (Teklad, TD.01804) and arginine-free diets (Teklad, TD.09152). Mouse diets were started 2 weeks before tumor inoculation allow for habituation. Body weight and tumor size were measured regularly. Tumor size was calculated by the equation (L×W²)/2 (L representing tumor length and W tumor width). Mice were sacrificed until either group reached a tumor size 20 mm in diameter. Tumor growth was evaluated by the IVIS system (IVIS Lumina II, Caliper Life Sciences) before euthanasia. All studies followed the protocol (NHRI-IACUC-106077) approved by the NHRI IACUC. Tumors were fixed with formalin for 48 h and subjected to sectioning and staining. Briefly, tumor sections were deparaffined and rehydrated. Then sections were blocked with 3% hydrogen peroxide/methanol followed by antigen retrieval with a 10 mM sodium citrate buffer at 95 ℃ for 10 min. Antibodies used were Ki-67 (Thermo, MA5-14520), CD45 (Cell Signaling, 70257), F4/80 (Cell Signaling, 70076), CD11c (Cell Signaling, 45581), IFNα2 (abcam, 193055) and NKp46 (R&D, AF2225).