To investigate the mechanism of Treg fragility and the downregulated suppressive function of Tregs after cryo-thermal therapy, the level of serum TNF-α in tumor-bearing control mice, RFA-treated mice or cryo-thermal treated mice was measured at different time points (1 d, 3 d, 7 d, 14 d, and 21 d after treatment) by western blotting and further verified by ELISA on day 21 after treatment. Here, TNF is linked to neoplasm.