Mechanism of Action of Tumor Treating Fields (TTFields) in Combination with Sorafenib for Treatment of Hepatocellular Carcinoma

Abstract

Purpose/Objective(s)Hepatocellular carcinoma (HCC) is a highly malignant liver cancer and is one of the leading causes of cancer-related mortality worldwide. Sorafenib, a multikinase inhibitor, is the main first-line treatment for advanced HCC, yet its survival benefits are limited. Tumor Treating Fields (TTFields) therapy is an antimitotic treatment, in which low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields are delivered to tumors non-invasively and loco-regionally. This research aimed to evaluate the efficacy of TTFields for the treatment of HCC in vitro and in vivo, alone and in combination with sorafenib, and to elucidate the underlying mechanism of action.Materials/MethodsHCC cell lines (HepG2 and Huh-7D12) were treated with various frequencies of TTFields to identify which is the most cytotoxic frequency. The effect of TTFields at optimal frequency in combination with sorafenib was tested. Cytotoxic and apoptotic effects were examined, as well as ER stress levels and responsive pathways. In vivo, N1S1 HCC cells (50,000) were orthotopically injected into the left hepatic lobe of SD rats, and tumors allowed to develop for 7 days. TTFields (at optimal frequency) or sham were then continuously applied for 6 days to the abdominal region of the rats, with concomitant sorafenib (10 mg/kg/day) or vehicle injected daily. Tumor volume growth was determined by MRI, and levels of cellular death were examined by immunohistochemistry.ResultsTTFields were found to be most cytotoxic to HCC cells at 150 kHz. Cells displayed dose-dependent sensitivity to sorafenib, and concomitant delivery of 150 kHz TTFields augmented this effect. Each modality alone led to increased levels of autophagy induced by elevated ER stress. The combination of TTFields and sorafenib triggered stress responsive pathways resulting in increased cellular death. In the animal model, tumor growth was significantly reduced and levels of cleaved PARP were elevated, in the combination group compared to other treatment groups, indicative of increased apoptosis.ConclusionThe results demonstrate that TTFields and sorafenib each induce ER stress leading to autophagy in HCC, and when combined, the elevated stress increases cellular death, both in vitro and in vivo. The ongoing phase 2 HEPANOVA (NCT03606590) clinical trial will investigate the safety and efficacy of TTFields plus sorafenib in patients with unresectable, locally advanced HCC. Hepatocellular carcinoma (HCC) is a highly malignant liver cancer and is one of the leading causes of cancer-related mortality worldwide. Sorafenib, a multikinase inhibitor, is the main first-line treatment for advanced HCC, yet its survival benefits are limited. Tumor Treating Fields (TTFields) therapy is an antimitotic treatment, in which low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields are delivered to tumors non-invasively and loco-regionally. This research aimed to evaluate the efficacy of TTFields for the treatment of HCC in vitro and in vivo, alone and in combination with sorafenib, and to elucidate the underlying mechanism of action. HCC cell lines (HepG2 and Huh-7D12) were treated with various frequencies of TTFields to identify which is the most cytotoxic frequency. The effect of TTFields at optimal frequency in combination with sorafenib was tested. Cytotoxic and apoptotic effects were examined, as well as ER stress levels and responsive pathways. In vivo, N1S1 HCC cells (50,000) were orthotopically injected into the left hepatic lobe of SD rats, and tumors allowed to develop for 7 days. TTFields (at optimal frequency) or sham were then continuously applied for 6 days to the abdominal region of the rats, with concomitant sorafenib (10 mg/kg/day) or vehicle injected daily. Tumor volume growth was determined by MRI, and levels of cellular death were examined by immunohistochemistry. TTFields were found to be most cytotoxic to HCC cells at 150 kHz. Cells displayed dose-dependent sensitivity to sorafenib, and concomitant delivery of 150 kHz TTFields augmented this effect. Each modality alone led to increased levels of autophagy induced by elevated ER stress. The combination of TTFields and sorafenib triggered stress responsive pathways resulting in increased cellular death. In the animal model, tumor growth was significantly reduced and levels of cleaved PARP were elevated, in the combination group compared to other treatment groups, indicative of increased apoptosis. The results demonstrate that TTFields and sorafenib each induce ER stress leading to autophagy in HCC, and when combined, the elevated stress increases cellular death, both in vitro and in vivo. The ongoing phase 2 HEPANOVA (NCT03606590) clinical trial will investigate the safety and efficacy of TTFields plus sorafenib in patients with unresectable, locally advanced HCC.