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.
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.
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).
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.
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.
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
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
and
, via a mechanism involving induction of autophagy.
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.
BackgroundTumor Treating Fields (TTFields) are a noninvasive, antineoplastic treatment delivered locoregionally to tumor bed via low intensity (1–3 V/cm), intermediate frequency (100–500 kHz), ...alternating electric fields. This treatment modality has been shown to be cytotoxic to rapidly dividing cells, with highest efficacy demonstrated at different optimal frequencies depending on tumor cell-type. TTFields therapy is FDA-approved for the treatment of newly diagnosed and recurrent glioblastoma (GBM), with the overall tolerable safety profile (EF-11 and EF-14 clinical trials) attributed to the low rate of mitotic events in normal, quiescent brain cells. Further evaluation of the safety profile of TTFields is needed for treating cancer in different body regions where there are high rates of cellular proliferation, i.e. torso. Many solid malignant tumors may reside in the torso region – mesothelioma and non-small cell lung carcinoma (NSCLC) in the thoracic segment; pancreatic cancer, hepatocellular carcinoma, and gastric cancer in the abdomen; and ovarian cancer in the pelvis. Hence, we investigated the safety of delivering TTFields to the torso of healthy rats at conditions previously deemed effective for treating the aforementioned cancer cell types.MethodsTTFields were applied using the Novo-TTF100L system at frequencies of 150 or 200 kHz and intensities of 1–2 V/cm RMS to torsos of Sprague Dawley (SD) female rats for a duration of 2 weeks. Throughout treatment, animals underwent daily clinical examinations. Blood samples and comparative histological evaluation of major internal organs were performed at treatment cessation.ResultsNo significant differences were observed for the TTFields treated groups in comparison to control groups for the following parameters: activity level, food and water intake, stools, motor neurological status, respiration, weight, complete blood count, blood biochemistry, and pathological findings.ConclusionsThese results demonstrate the safety of 150 and 200 kHz TTFields when delivered to torsos of healthy rats, where there are normal tissues with high cellular proliferation rates. Overall, TTFields delivery to the torso demonstrated safety and feasibility for the treatment of thoracic and other abdominal and pelvic cancers. TTFields are currently being investigated in clinical studies for the treatment of solid tumors located in the torso, including locally advanced pancreatic cancer (PANOVA-3 Study, NCT03377491), ovarian cancer (INNOVATE-3 Study, NCT03940196), lung cancer (LUNAR Study, NCT02973789), hepatocellular carcinoma (HEPANOVA Study, NCT03606590) and gastric cancer.
•TTFields are cytotoxic to pleural mesothelioma cells at optimal frequency of 150 kHz.•TTFields attenuate MPM tumor progression in murine models.•TTFields increase DNA damage in MPM, as previously ...seen in glioblastoma and NSCLC.•TTFields impair DNA damage repair in MPM, specifically the FA-BRCA pathway.•TTFields demonstrated synergism with cisplatin and additivity with pemetrexed.
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.
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.
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.
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.
Abstract
INTRODUCTION
Glioblastoma (GBM) is the most common malignant brain tumor. Tumor Treating Fields (TTFields) therapy is an approved treatment for GBM, delivered continuously by two array pairs ...placed on the skin surrounding the tumor region. Currently, most GBM animal models are based on mice. Since arrays for TTFields application to the mouse head are lacking, GBM-TTFields in vivo studies are limited. The small dimensions and specific geometries of the mouse head make the development of such arrays challenging.
METHODS
We tested different array layouts to identify one that will optimally accommodate the geometries of the mouse head while minimally restricting head movement. Additionally, we investigated adhesive tapes for array attachment to the skin, to allow good adherence and hence provide efficient treatment delivery. To validate that the selected array layout delivers therapeutic field intensity to the desired region, we performed simulations and fields measurements.
RESULTS
To overcome the issue of small head size we developed a layout in which the arrays on the head were divided into two smaller disks, with the opposing arrays situated on the mouse torso. We also identified a thin and transparent adhesive tape that facilitated the correct positioning of the arrays on the mouse head, offered good tackiness, and allowed for easy removal without leaving residual adhesive on the skin. The arrays met the minimum treatment requirements: field intensity ≥ 1 V/cm RMS, current ≥ 50 mA, usage ≥ 75%.
CONCLUSIONS
Our newly developed mouse head arrays are a flexible construct, that adheres strongly to the skin to enable efficient electric field delivery. By facilitating TTFields delivery to the heads of mice, we will expand the scope of GBM treatment research and will contribute to further advance this field.
Abstract
Tumor Treating Fields (TTFields) are an antineoplastic treatment modality targeting dividing cancer cells, approved by the FDA for treatment of glioblastoma and malignant pleural ...mesothelioma. TTFields are delivered to the patient continuously, using a portable signal generator and 2 pairs of transducer arrays attached to the skin. While in vitro research tools for TTFields have been available, animal experiments were so far limited due to the lack of a dedicated TTFields in vivo delivery system. The main challenges associated with TTFields application to mice are the absolute requirement for adequate and continuous contact between array electrodes and animal skin throughout treatments; and the need to deliver the electric fields through multiple wired conductors connected to electrodes, which limit animal movement thus imposing stress. This work reports on development of an in vivo system to facilitate continuous delivery of TTFields to mice bearing cancer tumors in the torso or flank that addresses the aforementioned challenges. Three major developments were introduced to the system: 1) transducer array electrodes composed of delicate, flexible inner layer to improve skin adherence and a breathable, durable, outer layer to secure electrodes to the skin; 2) a novel electric swivel to prevent cable coiling; and 3) a cage constructed to house 2 mice separately while still allowing socializing. During a 1-week study, the new inovivo system demonstrated improvement compared to previous systems in the following areas: increased animal treatment compliance, less weight loss, and fewer number of array replacements. In conclusion, the new inovivo system is a comprehensive tool for continuous, 2 directional TTFields delivery to tumors in the torso or flank of mice for conducting TTFields experiments with minimal animal stress, thus allowing further elucidation of the effects of TTFields on the whole animal.
Abstract
INTRODUCTION
Tumor Treating Fields (TTFields) therapy is an approved anti-cancer treatment for glioblastoma and mesothelioma. TTFields are delivered to patients continuously by two sets of ...arrays placed on opposite sides of the body at the tumor region to generate two perpendicular electric fields. Previously, in vivo studies of TTFields were limited due to the lack of a dedicated system that could maintain continuous and adequate contact of the arrays with the animal’s skin as well as the stress imposed on the animals by individual housing and the motility limitations they experience during treatment.
METHODS
Different electrode layouts were explored to optimize the intensity of the electric fields delivered to the target locations (therapeutic threshold >1 V/cm). The ability of various adhesive materials and wire coiling prevention strategies to increase TTFields device usage was examined. Stress reduction with different housing methods was evaluated via clinical examination of the animals.
RESULTS
Optimal array layouts were identified based on simulation data for TTFields delivery to the torso or the head of the mouse. Compacting conductors into a single printed circuit cable connected to a novel electric swivel machine resulted in fewer wire entanglements, and the improved adhesives resulted in fewer array replacements, overall elevating device usage. Improved cage design permitted pairs of mice to maintain social interactions while individually housed. Less weight loss was seen for animals housed in the dyadic relative to the standard solitary cages, indicating reduced stress.
CONCLUSIONS
The inovivo system provides means for continuous delivery of therapeutic levels of TTFields to the head and torso of mice while minimizing animal stress and increasing device usage. The new head arrays enable application of TTFields to the head of mice for the first time, allowing expansion of glioblastoma treatment research.
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