Characterization of Pancreatic Cancer Cell Thermal Response to Heat Ablation or Cryoablation

Abstract

One of the most lethal carcinomas is pancreatic cancer. As standard treatment using chemotherapy and radiation has shown limited success, thermal regimens (cryotherapy or heat ablation) are emerging as viable alternatives. Although promising, our understanding of pancreatic cancer response to thermal ablation remains limited. In this study, we investigated the thermal responses of 2 pancreatic cancer cell lines in an effort to identify the minimum lethal temperature needed for complete cell death to provide guidance for in vivo applications. PANC-1 and BxPC-3 were frozen (-10°C to -25°C) or heated (45°C-50°C) in single and repeated exposure regimes. Posttreatment survival and recovery were analyzed using alamarBlue assay over a 7-day interval. Modes of cell death were assessed using fluorescence microscopy (calcein acetoxymethyl ester/propidium iodide) and flow cytometry (YO-PRO-1/propidium iodide). Freezing to -10°C resulted in minimal cell death. Exposure to -15°C had a mild impact on PANC-1 survival (93%), whereas BxPC-3 was more severely damaged (33%). Exposure to -20°C caused a significant reduction in viability (PANC-1 = 23%; BxPC-3 = 2%) whereas -25°C yielded complete death. Double freezing exposure was more effective than single exposure. Repeat exposure to -15°C resulted in complete death of BxPC-3, whereas -20°C severely impacted PANC-1 (7%). Heating to 45°C resulted in minimum cell death. Exposure to 48°C yielded a slight increase in cell loss (PANC-1 = 85%; BxPC-3 = 98%). Exposure to 50°C caused a significant decline (PANC-1 = 70%; BxPC-3 = 9%) with continued deterioration to 0%. Double heating to 45°C resulted in similar effects observed in single exposures, whereas repeated 48°C resulted in significant increases in cell death (PANC-1 = 68%; BxPC-3 = 29%). In conclusion, we observed that pancreatic cancer cells were completely destroyed at temperatures <-25°C or >50°C using single thermal exposures. Repeated exposures resulted in increased cell death at less extreme temperatures. Our data suggest that thermal ablation strategies (heat or cryoablation) may represent a viable technique for the treatment of pancreatic cancer.

Keywords: apoptosis; cryoablation; hyperthermia; lethal temperature; pancreatic cancer; thermal therapy.

Figure 1.

Assessment of posttreatment viability and recovery of PaCa cells following exposure to a mild freezing insult. PANC-1 (A) and BxPC-3 (B) cells were subjected to freezing, and survival was assessed over 7 days posttreatment. Viability assessment indicated complete cell death was achieved following exposure to temperatures below −25°C for both cell types. (*P < .05). PaCa indicates pancreatic cancer.

Figure 2.

Assessment of PaCa cell viability following exposure to mild hyperthermic treatment. Impact on PANC-1 (A) or BxPC-3 (B) cell proliferation was assessed over a 7-day interval following mild hyperthermia. The results suggest that following exposure to 50°C or warmer complete cell death was attained in both cell models. (*P < .05). PaCa indicates pancreatic cancer.

Figure 3.

Fluorescence micrographs of PaCa cells following thermal exposures. Following thermal exposures, PANC-1 (A) or BxPC-3 (B) cells were stained with propidium iodide (red) and Calcein-AM (green) at 1, 4, 8, and 24 hours and visualized under ×20 magnification. Green fluorescence indicates live, whereas red fluorescence indicates dead cells. Fluorescence images illustrate an increase in cell death at temperatures below −15°C and above 50°C at 24 hours posttreatment. These findings appear to correlate with postthaw viability results obtained from flow cytometry and alamarBlue analysis. AM indicates acetoxymethyl ester; PaCa, pancreatic cancer.

Figure 4.

Assessment of the modes of cell death following thermal treatment. PANC-1 (A and C) and BxPC-3 (B and D) cells were subjected to selected temperatures and assessed using microfluidic flow cytometry. Samples were stained with propidium iodide and YO-PRO-1 to detect levels of necrosis and apoptosis at 1, 4, 8, and 24 hours postthermal exposure. Data illustrate necrosis is the predominant mode of cell death following both mild freezing and heat treatment of PaCa cells, whereas apoptosis contributes to a lesser degree. PaCa indicates pancreatic cancer.

Figure 4.

Assessment of the modes of cell death following thermal treatment. PANC-1 (A and C) and BxPC-3 (B and D) cells were subjected to selected temperatures and assessed using microfluidic flow cytometry. Samples were stained with propidium iodide and YO-PRO-1 to detect levels of necrosis and apoptosis at 1, 4, 8, and 24 hours postthermal exposure. Data illustrate necrosis is the predominant mode of cell death following both mild freezing and heat treatment of PaCa cells, whereas apoptosis contributes to a lesser degree. PaCa indicates pancreatic cancer.

Figure 5.

Comparison of single versus repeat thermal exposure in PaCa cells. PANC-1 (A) or BxPC-3 (B) cells were subjected to select freezing or heating temperatures either as a single exposure or a double exposure. Viability was assessed over a 7-day recovery period to compare initial death response and recovery in each condition. Data suggest that repeat exposure results in increased cell death at milder temperatures compared to a single exposure. (*,^,#,+P < .05). PaCa indicates pancreatic cancer.