Tumor Treating Fields (TTFields) are an anti-neoplastic treatment modality delivered via application of alternating electric fields using insulated transducer arrays placed directly on the skin in the region surrounding the tumor. A Phase 3 clinical trial has demonstrated the effectiveness of continuous TTFields application in patients with glioblastoma during maintenance treatment with Temozolomide. The goal of this study was to evaluate the efficacy of combining TTFields with radiation treatment (RT) in glioma cells. We also examined the effect of TTFields transducer arrays on RT distribution in a phantom model and the impact on rat skin toxicity. The efficacy of TTFields application after induction of DNA damage by RT or bleomycin was tested in U-118 MG and LN-18 glioma cells. The alkaline comet assay was used to measure repair of DNA lesions. Repair of DNA double strand breaks (DSBs) were assessed by analyzing γH2AX or Rad51 foci. DNA damage and repair signaled by the activation pattern of phospho-ATM (pS1981) and phospho-DNA-PKcs (pS2056) was evaluated by immunoblotting. The absorption of the RT energy by transducer arrays was measured by applying RT through arrays placed on a solid-state phantom. Skin toxicities were tested in rats irradiated daily through the arrays with 2Gy (total dose of 20Gy). TTFields synergistically enhanced the efficacy of RT in glioma cells. Application of TTFields to irradiated cells impaired repair of irradiation- or chemically-induced DNA damage, possibly by blocking homologous recombination repair. Transducer arrays presence caused a minor reduction in RT intensity at 20 mm and 60 mm below the arrays, but led to a significant increase in RT dosage at the phantom surface jeopardizing the “skin sparing effect”. Nevertheless, transducer arrays placed on the rat skin during RT did not lead to additional skin reactions. Administration of TTFields after RT increases glioma cells treatment efficacy possibly by inhibition of DNA damage repair. These preclinical results support the application of TTFields therapy immediately after RT as a viable regimen to enhance RT outcome. Phantom measurements and animal models imply that it may be possible to leave the transducer arrays in place during RT without increasing skin toxicities.

Tumor Treating Fields (TTFields) are noninvasive, alternating electric fields within the intermediate frequency range (100–300 kHz) that are utilized as an antimitotic cancer treatment. TTFields are loco-regionally delivered to the tumor region through 2 pairs of transducer arrays placed on the skin. This novel treatment modality has been FDA-approved for use in patients with glioblastoma and malignant pleural mesothelioma based on clinical trial data demonstrating efficacy and safety; and is currently under investigation in other types of solid tumors. TTFields were shown to induce an anti-mitotic effect by exerting bi-directional forces on highly polar intracellular elements, such as tubulin and septin molecules, eliciting abnormal microtubule polymerization during spindle formation as well as aberrant cleavage furrow formation. Previous studies have demonstrated that TTFields inhibit metastatic properties in cancer cells. However, the consequences of TTFields application on cytoskeleton dynamics remain undetermined. In this study, methods utilized in combination to study the effects of TTFields on cancer cell motility through regulation of microtubule and actin dynamics included confocal microscopy, computational tools, and biochemical analyses. Mechanisms by which TTFields treatment disrupted cellular polarity were (1) interference with microtubule assembly and directionality; (2) altered regulation of Guanine nucleotide exchange factor-H1 (GEF-H1), Ras homolog family member A (RhoA), and Rho-associated coiled-coil kinase (ROCK) activity; and (3) induced formation of radial protrusions of peripheral actin filaments and focal adhesions. Overall, these data identified discrete effects of TTFields that disrupt processes crucial for cancer cell motility.

Tumor Treating Fields (TTFields) are electric fields that exert physical forces to disrupt cellular processes critical for cancer cell viability and tumor progression. TTFields induce anti-mitotic effects through the disruption of the mitotic spindle and abnormal chromosome segregation, which trigger several forms of cell death, including immunogenic cell death (ICD). The efficacy of TTFields concomitant with anti-programmed death-1 (anti-PD-1) treatment was previously shown in vivo and is currently under clinical investigation. Here, the potential of TTFields concomitant with anti- PD-1/anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) or anti-programmed death-ligand 1 (anti-PD-L1) immune checkpoint inhibitors (ICI) to improve therapeutic efficacy was examined in lung tumor-bearing mice. Increased circulating levels of high mobility group box 1 protein (HMGB1) and elevated intratumoral levels of phosphorylated eukaryotic translation initiation factor 2α (p-eIF2α) were found in the TTFields-treated mice, indicative of ICD induction. The concomitant application of TTFields and ICI led to a significant decrease in tumor volume as compared to all other groups. In addition, significant increases in the number of tumor-infiltrating immune cells, specifically cytotoxic T-cells, were observed in the TTFields plus anti-PD-1/anti-CTLA-4 or anti-PD-L1 groups. Correspondingly, cytotoxic T-cells isolated from these tumors showed higher levels of IFN-γ production. Collectively, these results suggest that TTFields have an immunoactivating role that may be leveraged for concomitant treatment with ICI to achieve better tumor control by enhancing antitumor immunity.

Despite the availability of numerous therapeutic substances that could potentially target CNS disorders, an inability of these agents to cross the restrictive blood–brain barrier (BBB) limits their clinical utility. Novel strategies to overcome the BBB are therefore needed to improve drug delivery. We report, for the first time, how Tumor Treating Fields (TTFields), approved for glioblastoma (GBM), affect the BBB’s integrity and permeability. Here, we treated murine microvascular cerebellar endothelial cells (cerebEND) with 100–300 kHz TTFields for up to 72 h and analyzed the expression of barrier proteins by immunofluorescence staining and Western blot. In vivo, compounds normally unable to cross the BBB were traced in healthy rat brain following TTFields administration at 100 kHz. The effects were analyzed via MRI and immunohistochemical staining of tight-junction proteins. Furthermore, GBM tumor-bearing rats were treated with paclitaxel (PTX), a chemotherapeutic normally restricted by the BBB combined with TTFields at 100 kHz. The tumor volume was reduced with TTFields plus PTX, relative to either treatment alone. In vitro, we demonstrate that TTFields transiently disrupted BBB function at 100 kHz through a Rho kinase-mediated tight junction claudin-5 phosphorylation pathway. Altogether, if translated into clinical use, TTFields could represent a novel CNS drug delivery strategy.

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) are electric fields that induce cancer cell death. Genomic analysis of glioblastoma tumors resected from TTFields-treated patients suggested a potential link between a reduced or absent response to TTFields and activating mutations in the phosphatidylinositol 3-kinase (PI3K) p110α subunit (PIK3CA). Our study aimed to investigate the role of the PI3K/AKT pathway in the response to TTFields. We tested changes in signaling pathways in control versus TTFields-treated U-87 MG glioblastoma, A2780 ovarian carcinoma, and H1299 non-small cell lung cancer (NSCLC) cells using the Luminex multiplex assay, validated by western blot analysis and inhibition assays. We also performed in vivo validation using immunohistochemistry on tumor sections from animals bearing orthotopic N1-S1 hepatocellular, MOSE-L ovarian, or LL/2 lung tumors that were treated with TTFields or sham. Finally, we examined the efficacy of concomitant treatment with TTFields and PI3K inhibitors in cell lines and mouse models. Our findings elucidate the mechanisms driving PI3K/AKT activation following TTFields treatment, revealing that the AKT signaling amplitude increases over time and is influenced by cell-surface and cell-cell interactions. Specifically, focal adhesion kinase (FAK) and N-cadherin were found to promote AKT phosphorylation, activating cell survival pathways. Furthermore, our investigation revealed that pharmacological inhibition of PI3K sensitized cancer cells to TTFields, both in vitro and in vivo. Our research suggests that the PI3K/AKT pathway is involved in cancer cell response to TTFields, and that inhibition of this pathway may serve as a potential therapeutic target for sensitizing cancer cells to TTFields.