Background: Critical to the continual improvement of cryoablation efficacy is deciphering the biochemical responses of cells to low-temperature exposure. The identification of delayed-onset cell death has allowed for the manipulation of cellular responses through the regulation of apoptosis. We hypothesized that in addition to delayed apoptotic events associated with mild subfreezing temperatures (10 to -25 °C), cells exposed to ultra-low temperatures (<-30 °C) may undergo rapid, early-onset apoptosis.
Methods: Human prostate cancer model and cells (PC-3) were exposed to temperatures of -60, -30 and -15 °C to simulate a cryoablative procedure. Using a combination of flow-cytometry, fluorescent microscopy and western blot analyses, samples were assessed at various times post thaw to identify the presence, levels and the pathways involved in cell death.
Results: Exposure to temperatures <-30 °C yielded a significant apoptotic population within 30 min of thawing, peaking at 90 min (~40%), and by 6 h, only necrosis was observed. In samples only reaching temperatures >-30 °C, apoptosis was not noted until 6-24 h post thaw, with the levels of apoptosis reaching ~10% (-15 °C) and ~25% (-30 °C) at 6 h post thaw. Further, it was found that early-onset apoptosis progressed through a membrane-mediated mechanism, whereas delayed apoptosis progressed through a mitochondrial path.
Conclusions: These data demonstrate the impact of apoptotic continuum, whereby the more severe cryogenic stress activated the extrinsic, membrane-regulated pathway, whereas less severe freezing activated the intrinsic, mitochondrial-mediated path. The rapid induction and progression of apoptosis at ultra-low temperatures provides an explanation as to why such results have not previously been identified following freezing. Ultimately, an understanding of the events and signaling pathways involved in triggering apoptosis following freezing may provide a path for selective induction of the rapid-onset and delayed programmed cell death pathways in an effort to improve the overall cryoablation efficacy.