Cryoinjury of MCF-7 human breast cancer cells and its enhancement using tumor necrosis factor-alpha (TNF-α) as an adjuvant, were investigated. Through a series of experiments in a two level factorial design critical parameters affecting cryotherapy responses were identified. The cryoinjury was investigated by quantifying the effects of four freeze/thaw (F/T) parameters, selected to be within the expected range for a cryosurgical iceball. Thermal parameters considered were cooling rate (5 and 50 °C/min), end temperature (-20 and -80 °C), hold time (0 and 10 min), and thawing rate (20 and 100 °C/min). After exposing the cells to the selected F/T conditions, survival was assessed and statistically analyzed to determine the effect of each parameter and their interactions. A statistical analysis shows that the end temperature and hold time were the two most significant parameters in the range studied. This suggests that proper control of these two parameters is important to achieve desired cryodestruction of MCF-7 cells. Enhancement of cryoinjury by TNF-α was also investigated in a tissue equivalent cryoinjury model in which a cryosurgical iceball is formed. MCF-7 cells cultured in a collagen matrix underwent a controlled F/T with or without TNF-α pre-treatment at 100 ng/ml for 24 hours. Post-thaw viability of MCF-7 cells was assessed at three hours, and at one and three days after freezing. Although the TNF-α treatment alone induced neither apoptotic nor necrotic cell death, the combination of TNF-α pre-treatment and freezing enhanced the immediate cryoinjury of MCF-7 cells, and significantly impaired the post-thaw recovery. Without TNF-α treatment, MCF-7 cell cultures were repopulated, reaching approximately 80% survival at day 3 even after severe cryoinjury (≤ 20% survival) at three hours. In contrast, this repopulation was significantly inhibited by TNF-α pre-treatment, in which case the viability of the frozen region remained below 40% at day 3. The effects of TNF-α on the cryoinjury of MCF-7 cells suggest that TNF-α may serve as a potent adjuvant to cryosurgery of breast cancer.

Key words: Cryoinjury; Breast cancer; Post-thaw recovery; TNF-alpha; MCF-7; and Tissue equivalent.