Orthotopic HCC model in double transgenic mice

DEN (N0756, Sigma-Aldrich) was diluted in 0.9% sterile NaCl to a working concentration of 2.5 mg/ml. Male CreER^T2+/− and CreER^T2−/− Tp53^flox/flox C57BL/6J mice received DEN (25 mg/kg, i.p.) at the age of 2 weeks. After 41 to 43 weeks, after developing HCC, the mice received tamoxifen (100 mg/kg) by oral gavage for five consecutive days, followed by a 1-week washout phase. Tamoxifen (Molekula) was prepared fresh as a working solution (30 mg/ml) in 90% peanut oil and 10% ethanol. Then, 4 cycles of treatment were conducted: Mice were either fed ad libitum or fasted two times per week for 24 hours in the periodically fasted group. Both groups received sorafenib (30 mg/kg) by oral gavage two times per week, after the 24-hour fasting in the fasted group, and on the same day in the fed group. After 4 cycles and an additional 2 to 3 days of ad libitum feeding, the mice were sacrificed. The whole liver was weighed, then all HCC nodules were dissected from the liver and weighed together to assess total tumor mass.