Correction: Davidi et al. Tumor Treating Fields (TTFields) Concomitant with Sorafenib Inhibit Hepatocellular Carcinoma In Vitro and In Vivo. Cancers 2022, 14, 2959.

The authors wish to make minor corrections to Figure 1 and Figure 2 of the following paper [...].

Tumor Treating Fields (TTFields) Concomitant with Sorafenib Inhibit Hepatocellular Carcinoma In Vitro and In Vivo.

Hepatocellular carcinoma (HCC), a highly aggressive liver cancer, is a leading cause of cancer-related death. Tumor Treating Fields (TTFields) are electric fields that exert antimitotic effects on cancerous cells. The aims of the current research were to test the efficacy of TTFields in HCC, explore the underlying mechanisms, and investigate the possible combination of TTFields with sorafenib, one of the few front-line treatments for patients with advanced HCC. HepG2 and Huh-7D12 human HCC cell lines were treated with TTFields at various frequencies to determine the optimal frequency eliciting maximal cell count reduction. Clonogenic, apoptotic effects, and autophagy induction were measured. The efficacy of TTFields alone and with concomitant sorafenib was tested in cell cultures and in an orthotopic N1S1 rat model. Tumor volume was examined at the beginning and following 5 days of treatment. At study cessation, tumors were weighed and examined by immunohistochemistry to assess autophagy and apoptosis. TTFields were found in vitro to exert maximal effect at 150 kHz, reducing cell count and colony formation, increasing apoptosis and autophagy, and augmenting the effects of sorafenib. In animals, TTFields concomitant with sorafenib reduced tumor weight and volume fold change, and increased cases of stable disease following treatment versus TTFields or sorafenib alone. While each treatment alone elevated levels of autophagy relative to control, TTFields concomitant with sorafenib induced a significant increase versus control in tumor ER stress and apoptosis levels, demonstrating increased stress under the multimodal treatment. Overall, TTFields treatment demonstrated efficacy and enhanced the effects of sorafenib for the treatment of HCC in vitro and in vivo, via a mechanism involving induction of autophagy.

Tumor Treating Fields (TTFields) downregulate the Fanconi Anemia-BRCA pathway and increase the efficacy of chemotherapy in malignant pleural mesothelioma preclinical models.

OBJECTIVES: Tumor Treating Fields (TTFields) are low intensity, intermediate frequency, alternating electric fields with antimitotic effects on cancerous cells. TTFields concomitant with pemetrexed and a platinum agent are approved in the US and EU as first line therapy for unresectable, locally advanced or metastatic malignant pleural mesothelioma (MPM). The goal of the current study was to characterize the mechanism of action of TTFields in MPM cell lines and animal models. METHODS: Human MPM cell lines MSTO-211H and NCI-H2052 were treated with TTFields to determine the frequency that elicits maximal cytotoxicity. The effect of TTFields on DNA damage and repair, and the cytotoxic effect of TTFields in combination with cisplatin and/or pemetrexed were examined. Efficacy of TTFields concomitant with cisplatin and pemetrexed was evaluated in orthotopic IL-45 and subcutaneous RN5 murine models. RESULTS: TTFields at a frequency of 150 kHz demonstrated the highest cytotoxicity to MPM cells. Application of 150 kHz TTFields resulted in increased formation of DNA double strand breaks, elevated expression of DNA damage induced cell cycle arrest proteins, and reduced expression of Fanconi Anemia (FA)-BRCA DNA repair pathway proteins. Co-treatment of TTFields with cisplatin or pemetrexed significantly increased treatment efficacy versus each modality alone, with additivity and synergy exhibited by the TTFields-pemetrexed and TTFields-cisplatin combinations, respectively. In animal models, tumor volume was significantly lower for the TTFields-cisplatin-pemetrexed combination compared to control, accompanied by increased DNA damage within the tumor. CONCLUSION: This research demonstrated that the efficacy of TTFields for the treatment of MPM is associated with reduced expression of FA-BRCA pathway proteins and increased DNA damage. This mechanism of action is consistent with the observed synergism for TTFields-cisplatin vs additivity for TTFields-pemetrexed, as cisplatin-induced DNA damage is repaired via the FA-BRCA pathway.

In Vivo Safety of Tumor Treating Fields (TTFields) Applied to the Torso.

BACKGROUND: Tumor Treating Fields (TTFields) therapy is a non-invasive, loco-regional, anti-mitotic treatment modality that targets rapidly dividing cancerous cells, utilizing low intensity, alternating electric fields at cancer-cell-type specific frequencies. TTFields therapy is approved for the treatment of newly diagnosed and recurrent glioblastoma (GBM) in the US, Europe, Israel, Japan, and China. The favorable safety profile of TTFields in patients with GBM is partially attributed to the low rate of mitotic events in normal, quiescent brain cells. However, specific safety evaluations are warranted at locations with known high rates of cellular proliferation, such as the torso, which is a primary site of several of the most aggressive malignant tumors. METHODS: The safety of delivering TTFields to the torso of healthy rats at 150 or 200 kHz, which were previously identified as optimal frequencies for treating multiple torso cancers, was investigated. Throughout 2 weeks of TTFields application, animals underwent daily clinical examinations, and at treatment cessation blood samples and internal organs were examined. Computer simulations were performed to verify that the targeted internal organs of the torso were receiving TTFields at therapeutic intensities (≥ 1 V/cm root mean square, RMS). RESULTS: No treatment-related mortality was observed. Furthermore, no significant differences were observed between the TTFields-treated and control animals for all examined safety parameters: activity level, food and water intake, stools, motor neurological status, respiration, weight, complete blood count, blood biochemistry, and pathological findings of internal organs. TTFields intensities of 1 to 2.5 V/cm RMS were confirmed for internal organs within the target region. CONCLUSIONS: This research demonstrates the safety of therapeutic level TTFields at frequencies of 150 and 200 kHz when applied as monotherapy to the torso of healthy rats.

Tumor-treating fields (TTFields) induce immunogenic cell death resulting in enhanced antitumor efficacy when combined with anti-PD-1 therapy.

Tumor-treating fields (TTFields) are alternating electric fields in a specific frequency range (100-300 kHz) delivered to the human body through transducer arrays. In this study, we evaluated whether TTFields-mediated cell death can elicit antitumoral immunity and hence would be effectively combined with anti-PD-1 therapy. We demonstrate that in TTFields-treated cancer cells, damage-associated molecular patterns including high-mobility group B1 and adenosine triphosphate are released and calreticulin is exposed on the cell surface. Moreover, we show that TTFields treatment promotes the engulfment of cancer cells by dendritic cells (DCs) and DCs maturation in vitro, as well as recruitment of immune cells in vivo. Additionally, our study demonstrates that the combination of TTFields with anti-PD-1 therapy results in a significant decline of tumor volume and increase in the percentage of tumor-infiltrating leukocytes in two tumor models. In orthotopic lung tumors, these infiltrating leukocytes, specifically macrophages and DCs, showed elevated expression of PD-L1. Compatibly, cytotoxic T-cells isolated from these tumors demonstrated increased production of IFN-γ. In colon cancer tumors, T-cells infiltration was significantly increased following long treatment duration with TTFields plus anti-PD-1. Collectively, our results suggest that TTFields therapy can induce anticancer immune response. Furthermore, we demonstrate robust efficacy of concomitant application of TTFields and anti-PD-1 therapy. These data suggest that integrating TTFields with anti-PD-1 therapy may further enhance antitumor immunity, hence achieve better tumor control.

The antiangiogenic role of the pro-inflammatory cytokine interleukin-31.

Pro-inflammatory cytokines in the tumor microenvironment are known for their ability to either inhibit or promote cancer progression. Here we evaluated the role of Interleukin-31 (IL31), a protein belonging to the pro-inflammatory IL-6 cytokine family which has been characterized in autoimmune disease, in tumorigenesis. We show that IL31 and its receptor, IL31RA, are highly expressed in various human and mouse cancer cell lines, as well as in tumor specimens from cancer patients. MC38 murine colon carcinoma cells depleted of IL31 exhibit an increase in invasive and migratory properties in vitro, effects that are reversed by supplementing the cells with exogenous IL31. In vivo, IL31-depleted MC38 tumor cells implanted to mice grow faster than control tumors. In contrast, MC38 tumor-bearing mice infused with recombinant IL31, exhibit a significant reduction in tumor growth than control mice. Furthermore, IL31 infusion reduces the number of metastatic lesions in the lungs of mice bearing 4T1 murine metastatic breast carcinoma. Lastly, injecting tumor-bearing, chemotherapy-treated mice with a long-lived IL31-IgG fusion protein reduces tumor growth, angiogenesis and pulmonary metastasis to a greater extent than when chemotherapy is used alone. The IL31 anti-tumor activity is explained, in part, by the anti-angiogenic effects demonstrated both in vitro and in vivo highlighting the potential use of IL31 as an anti-cancer drug.

Anti-VEGF-A affects the angiogenic properties of tumor-derived microparticles.

Tumor derived microparticles (TMPs) have recently been shown to contribute to tumor re-growth partially by inducing the mobilization and tumor homing of specific bone marrow derived pro-angiogenic cells (BMDCs). Since antiangiogenic drugs block proangiogenic BMDC mobilization and tumor homing, we asked whether TMPs from cells exposed to an antiangiogenic drug may affect BMDC activity and trafficking. Here we show that the level of VEGF-A is reduced in TMPs from EMT/6 breast carcinoma cells exposed to the anti-VEGF-A antibody, B20. Consequently, these TMPs exhibit reduced angiogenic potential as evaluated by a Matrigel plug and Boyden chamber assays. Consistently, BMDC mobilization, tumor angiogenesis, microvessel density and BMDC-colonization in growing tumors are reduced in mice inoculated with TMPs from B20-exposed cells as compared to mice inoculated with control TMPs. Collectively, our results suggest that the neutralization of VEGF-A in cultured tumor cells can block TMP-induced BMDC mobilization and colonization of tumors and hence provide another mechanism of action by which antiangiogenic drugs act to inhibit tumor growth and angiogenesis.