106 Responses of pancreatic cancer cells under cryoablative conditions

Abstract

Pancreatic cancer (PaCa) incidence and mortality rates continue to increase, driving the need for improved diagnostics and more effective therapies.Combinatorial approaches in PaCa have focused on the use of multiple chemotherapeutic drugs, while cryotherapeutic options have been underutilized.Given past success in prostate cancer, in this study we investigated the potential for multi-modal treatment utilizing cryoablation in conjunction with low dose chemotherapy for the treatment of PaCa.Both standard tissue culture models and the 3-D tissue engineered (PaCaTEM) approach were studied, providing a more robust assessment of the cellular responses to cryotherapy and the associated adjuncts.The human PaCa cell line (Bx-PC3) was evaluated in vitro and in a 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 with and without pretreatment with 0.025 μg/ml 5-FU. 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.Cell survival following the treatment regimes were 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 followed by fluorescence microscopic examination of cell death at 1 and 24 h (Vybrant apoptosis assay).Freezing exposure to −10 °C did not impact overall Bx-PC3 viability, however exposure to −15 °C and −20 °C resulted in a significant loss of viability (90% and 98%, respectively), which was found to be more effective with concomitant 5-FU . At temperatures of −25 °C and colder in vitro, no metabolic activity remained throughout the observational period.Temporal analysis of the cell death response to freezing by MFC revealed that an initiation and sustained levels of both apoptosis and necrosis.Focusing on the −15 °C isotherm, apoptotic cell death was found to peak at 4 h and necrosis at 8 h post-thaw.Within the PaCaTEM samples, a 3.0 cm (±0.2 cm) diameter iceball was observed.Fluorescence imaging of the PaCaTEM revealed complete PaCa cell ablation to a diameter of 1.9 cm (±0.2 cm)’ correlating with the −25C isotherm.A transitional zone of cell death extending to the margin of the iceball was also observed.The results of this study indicate that cryoablation, alone or as a combinatorial therapy, may serve as an effective tool for the targeted ablation of pancreatic cancer.At minimum, −25 °C should be achieved as a target tissue temperature at the margin of a tumor to assure complete PaCa destruction. Source of funding: CPSI Biotech. Conflict of interest: None declared. ksnyder@cpsibiotech.com