Investigation of the Impact of Cell Cycle Stage on Freeze Response Sensitivity of Androgen-Insensitive Prostate Cancer

Background: Cryoablation, an effective means of ablating cancer, is often used in conjunction with adjuvants that target cancer cells in a specific cell cycle stage to increase treatment efficacy. The objective of this study was to investigate the impact of cell cycle stage on cancer freeze response as well as investigate the potential cellular kinetic effect of calcitriol, the active metabolic of vitamin D 3 , when used as a cryosensitizing adjuvant in order to maximize prostate cancer cell death.Methods: Cell cycle distribution of PC-3 cells was analyzed via flow cytometry to compare gap 1, synthesis, and gap 2/mitosis phase subpopulations pre- and postfreeze as well as changes elicited by calcitriol pretreatment. Distinct gap 1, synthesis, and gap 2/mitosis phase populations were obtained through fluorescence-activated cell sorting and synthesis phase thymidine synchronization. Posttreatment viability was assessed using alamarBlue and fluorescence microscopy to assess live, apoptotic, and necrotic subpopulations.Results: A small but statistically significant increase in synthesis phase and decrease in gap 2/mitosis phase populations was noted at 6 hours postfreeze in asynchronous samples. Synchronization in synthesis phase yielded an increase in cell death when combined with freezing to both −15°C and −20°C. Calcitriol pretreatment increased the gap 1 phase population by 20% and a synergistic decrease in viability following freezing. However, gap 1–sorted populations combined with calcitriol treatment did not exhibit this synergistic effect. Fluorescence microscopy of fluorescence-activated cell sorting-sorted cells revealed necrosis as the predominant form of cell death in all phases, though apoptosis did play a role.Conclusion: Although initial results suggested a potential sensitivity, PC-3 cells exposed to freezing as sorted populations did not reveal significant differences in cell death. As such, the data from this study suggest that there is no difference in cell cycle stage sensitivity to freezing injury.