Orthotopic Mouse Renal Tumor Model

All experiments were approved by the Animal Ethics Committee of the University of Tokyo. The housing and handling conditions of the mice were consistent with the method above. Mouse renal orthotopic tumor models were conducted as previously described [22 (link), 29 (link)]. Briefly, BALB/c‐nu/nu male mice (5 weeks old) were purchased from Sankyo Labo Service Corporation (Tokyo, Japan). ccRCC cells (1.0 × 10⁵) expressing Luc2 and mCherry were inoculated into the subrenal capsule of mice. For in vivo bioluminescence imaging, D‐luciferin potassium salt (200 mg·kg⁻¹; Promega) was diluted in PBS and injected into mice intraperitoneally. For ex vivo bioluminescence imaging, the harvested kidneys and lungs were reacted with d‐luciferin potassium solution for 10 min, and images were captured using NightOWL LB981 (Berthold Technologies, Bad Wildbad, Germany). Quantitative analysis was conducted using the IndiGO software (Berthold Technologies). Everolimus was reconstituted in saline solution (Otsuka, Tokyo, Japan) containing 5% Tween20 and 30% propylene glycol (Sigma‐Aldrich) and administered to mice (2.5 mg·kg⁻¹) thrice weekly.