Yan Li1, Russell E Brown, Robert C G Martin. 1. Division of Surgical Oncology, Department of Surgery, University of Louisville, Louisville, Kentucky 40202, USA.
Abstract
INTRODUCTION: Local recurrence after thermal ablation of hepatocellular carcinoma (HCC) is common, problematic, and poorly understood. The continued effects of the heat sink around major vessels have been hypothesized to increase local ablation failure and possibly enhance the growth of the incompletely ablated malignancy. The aim of this study was to evaluate the critical temperature to which incomplete ablation would occur. METHODS: Established murine (Hep-6L), rodent (Hep-4IIE), and human (Hep-G2) HCC cells were cultivated and then exposed to variable 2° temperatures from 37 °C-54 °C and incubated at 37 °C for 72 h. After incubation, 3-(4, 5-dimthyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays were used to measure proliferation, metabolic activity, and apoptosis. RESULTS: Reliable cell death occurred in all three cell lines by microscopy and MTT assay after treatment at temperatures above 48 °C. However, in the 72 h after treatment, we observed successive recovery or maintenance of cellular proliferation at each time endpoint at ≤47 °C. This effect was present across all cell lines at 45 °C, and was most pronounced in the Hep-6L and Hep-4IIE cell lines (P < 0.05). Hep-G2 cells demonstrated maintenance of proliferation by MTT assay over the 72 h following treatment at 45 °C. TUNEL assays were strongly positive at temperatures ≥48 °C. TUNEL positivity suggests death via apoptotic mechanisms as opposed to coagulative processes seen at higher temperatures. Cells treated at these higher temperatures had a statistically significant (P < 0.05) higher apoptotic index as measured by TUNEL positivity, despite being morphologically similar by light microscopy. CONCLUSIONS: Cellular proliferation and resistance to apoptosis are significantly increased after incomplete thermal ablation, with a 2° difference in complete versus incomplete ablation.
INTRODUCTION: Local recurrence after thermal ablation of hepatocellular carcinoma (HCC) is common, problematic, and poorly understood. The continued effects of the heat sink around major vessels have been hypothesized to increase local ablation failure and possibly enhance the growth of the incompletely ablated malignancy. The aim of this study was to evaluate the critical temperature to which incomplete ablation would occur. METHODS: Established murine (Hep-6L), rodent (Hep-4IIE), and human (Hep-G2) HCC cells were cultivated and then exposed to variable 2° temperatures from 37 °C-54 °C and incubated at 37 °C for 72 h. After incubation, 3-(4, 5-dimthyl-thiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays were used to measure proliferation, metabolic activity, and apoptosis. RESULTS: Reliable cell death occurred in all three cell lines by microscopy and MTT assay after treatment at temperatures above 48 °C. However, in the 72 h after treatment, we observed successive recovery or maintenance of cellular proliferation at each time endpoint at ≤47 °C. This effect was present across all cell lines at 45 °C, and was most pronounced in the Hep-6L and Hep-4IIE cell lines (P < 0.05). Hep-G2 cells demonstrated maintenance of proliferation by MTT assay over the 72 h following treatment at 45 °C. TUNEL assays were strongly positive at temperatures ≥48 °C. TUNEL positivity suggests death via apoptotic mechanisms as opposed to coagulative processes seen at higher temperatures. Cells treated at these higher temperatures had a statistically significant (P < 0.05) higher apoptotic index as measured by TUNEL positivity, despite being morphologically similar by light microscopy. CONCLUSIONS: Cellular proliferation and resistance to apoptosis are significantly increased after incomplete thermal ablation, with a 2° difference in complete versus incomplete ablation.
Authors: Erica Locatelli; Yan Li; Ilaria Monaco; Wei Guo; Mirko Maturi; Luca Menichetti; Paolo Armanetti; Robert C Martin; Mauro Comes Franchini Journal: Int J Nanomedicine Date: 2019-03-13