Orthotopic Grafting of OSCC13 Cells in the Tongue and Bioluminescence Detection

Nude mice (8 weeks of age) or WT and TNCKO (C57Bl/6J) mice (bred in house) were grafted in the first third part of the tongue. In particular, 3 × 10⁶ OSCC13 cells in 10 µl PBS were injected using a U-100 insulin syringe (BD Micro-Fine) in C57Bl/6J mice. Some 1 × 10⁶ cells OSCC13-LUC (luciferase expression vector expressing cells) were similarly grafted. Tumors were visible 2 weeks upon engraftment and mice were sacrificed for analysis at 3 weeks (bioluminescence experiment), 2 and 4 weeks (NIR) or 4 weeks (IR). For irradiation analysis, a 2 Gy unique dose of photons was delivered two weeks after tumor cell engraftment (3 × 10⁶ cells). Mice were sacrificed two weeks later and tissue was analyzed by staining. For bioluminescence detection, a RediJect D-Luciferin Ultra Bioluminescent Substrate (Perkinelmer 770505) solution at 30 mg/ml was injected intraperitoneally 7 min before imaging. Images were acquired for 5 min using a live imager (NightOwl, Berthold). All mice were housed and handled according to the guidelines of INSERM and the ethical committee of Alsace, France (CREMEAS) (Directive 2010/63/EU, 01386.02, C-67-482-033 on the protection of animals used for scientific purposes).