9. Multi-dimensional assessment of pancreatic cancer cell responses to cryoablation in vitro

Abstract

As the incidence of pancreatic cancer (PaCa) continues to increase worldwide with little improvement in the overall prognosis, there is a drive to identify alternative strategies for the treatment of PaCa to prolong patient survival. To this end, several reports have described the potential benefits of the use of cryoablation for the treatment of PaCa. While promising, few studies have been reported examining the physical and molecular response of PaCa to cryoablation. An understanding of these responses is critical to serve as supportive and guidance information if the use of cryo for the treatment of PaCa is to continue to grow. In an effort to detail the effects of freezing in pancreatic cancer (PaCa), a human PaCa cell line (Bx-PC3) was evaluated in vitro and using an in vivo -like tissue engineered model ( PaCaTEM ). Bx-PC3 were exposed to a range of freezing temperatures from −10 °C to −20 °C and compared to non-frozen controls. PaCaTEM models were frozen in situ following a single 5 min freeze protocol using the supercritical nitrogen (SCN) cryoablation system with a 1.5 mm × 3 cm cryoprobe. Following freezing, cell survival was assessed in the in vitro studies using the metabolic activity indicator, alamarBlue and microfludic flow cytometry (MFC). Assessment of PaCaTEM samples included monitoring of iceball size and isothermal distribution during the freezing process as well as assessment of cell ablation at 1 and 24 h post-freeze via fluorescence microscopy (Vybrant apoptosis assay). Results from in vitro experiments showed that freezing to −10 °C did not affect Bx-PC3 viability, while −15 °C and −20 °C resulted in a significant loss of viability (90% and 98%, respectively). A complete loss of cell viability was evident at temperatures of −25 °C and colder in vitro . Temporal analysis of the cell response to freezing via MFC revealed that following exposure, there was an initiation of both apoptotic and necrotic responses. Specifically, following freezing to −15 °C, apoptotic cell death was found to peak at 4hrs and necrosis at 8hrs post-thaw. Analysis of the PaCaTEM samples revealed the creation of a 3.0 cm (±0.2 cm) diameter iceball with extension of the −25 °C isotherm to a diameter of 2.0 cm (±0.1 cm). Fluorescence imaging of the PaCaTEM revealed complete PaCa cell ablation to a diameter of 1.9 cm (±0.2 cm) with a transitional zone of cell death extending to the margin of the iceball. This zone of ablation correlated to complete cell destruction below 25 °C in in vivo -like tissue. The results of this study show that cryoablation is an effective tool for the targeted ablation of pancreatic cancer. Further, the data suggest that a temperature of −25 °C may serve as a target temperature to be reached at the margin of a tumor to assure complete PaCa destruction.