Tumor treating fields (TTFields) in combination with sorafenib inhibit hepatocellular carcinoma in vitro and in vivo.

Abstract

464 Background: Hepatocellular carcinoma (HCC) is a highly malignant liver cancer and a leading cause of cancer related mortality. Sorafenib was the first approved systemic treatment for HCC, and remains one of few front-line treatments for this malignancy. Tumor treating fields (TTFields) are low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields that exert antimitotic effects on cancerous cells. Results of the phase 2 HEPANOVA study of TTFields (150 kHz) plus sorafenib for advanced HCC support investigation of TTFields in a randomized controlled phase 3 study. The current research aimed to describe the in vitro and in vivo efficacy of this combination and to elucidate details regarding the underlying mechanism of action. Methods: In vitro examinations were performed in HepG2 and Huh-7D12 human HCC cell lines, to which TTFields at a frequency of 150 kHz were applied using the inovitro system. Autophagy was examined by western blot and fluorescence detection of microtubule-associated protein light chain 3 (LC3) levels, an accepted autophagy marker. The effect of TTFields in combination with sorafenib was evaluated using cytotoxic, clonogenic, and apoptotic assays. In vivo, SD rats were inoculated orthotopically into the left hepatic lobe with N1S1 HCC cells. 7 days later, TTFields or sham (heat) were applied to the abdominal region of the rats, continuously for 6 days. Daily intraperitoneal injections of sorafenib (10 mg/kg/day) or vehicle were performed during this time. To determine tumor volume growth, MRI images were acquired before and after treatment. Levels of autophagy and apoptosis were examined in tumor sections by immunohistochemistry for LC3 and cleaved PARP, respectively. Results: Application of TTFields induced autophagy in HCC cells. TTFields delivery was cytotoxic to the cells, reduced their colony forming ability, and induced apoptosis while combination with sorafenib elevated these effects. In vivo, tumor volume increased 6-fold in control animals vs 1.6-fold in animals treated with TTFields plus sorafenib. This effect was accompanied by significantly elevated levels of cleaved PARP and LC3 within the tumors of treated relative to control rats. Conclusions: The results demonstrate induction of autophagy and apoptosis in HCC following treatment with TTFields. Concomitant application of TTFields with sorafenib enhanced efficacy via a mechanism that may involve overwhelming autophagy, in vitro and in vivo.