Aviran Itzhaki

Alternating electric fields arrest cell proliferation in animal tumor models and human brain tumors

We have recently shown that low intensity, intermediate frequency, electric fields inhibit by an anti-microtubule mechanism of action, cancerous cell growth in vitro. Using implanted electrodes, these fields were also shown to inhibit the growth of dermal tumors in mice. The present study extends these findings to additional cell lines [human breast carcinoma; MDA-MB-231, and human non-small-cell lung carcinoma (H1299)] and to animal tumor models (intradermal B16F1 melanoma and intracranial F-98 glioma) using external insulated electrodes. These findings led to the initiation of a pilot clinical trial of the effects of TTFields in 10 patients with recurrent glioblastoma (GBM). Median time to disease progression in these patients was 26.1 weeks and median overall survival was 62.2 weeks. These time to disease progression and OS values are more than double the reported medians of historical control patients. No device-related serious adverse events were seen after >70 months of cumulative treatment in all of the patients. The only device-related side effect seen was a mild to moderate contact dermatitis beneath the field delivering electrodes. We conclude that TTFields are a safe and effective new treatment modality which effectively slows down tumor growth in vitro, in vivo and, as demonstrated here, in human cancer patients.

Disruption of Cancer Cell Replication by Alternating Electric Fields

Low-intensity, intermediate-frequency (100-300 kHz), alternating electric fields, delivered by means of insulated electrodes, were found to have a profound inhibitory effect on the growth rate of a variety of human and rodent tumor cell lines (Patricia C, U-118, U-87, H-1299, MDA231, PC3, B16F1, F-98, C-6, RG2, and CT-26) and malignant tumors in animals. This effect, shown to be nonthermal, selectively affects dividing cells while quiescent cells are left intact. These fields act in two modes: arrest of cell proliferation and destruction of cells while undergoing division. Both effects are demonstrated when such fields are applied for 24 h to cells undergoing mitosis that is oriented roughly along the field direction. The first mode of action is manifested by interference with the proper formation of the mitotic spindle, whereas the second results in rapid disintegration of the dividing cells. Both effects, which are frequency dependent, are consistent with the computed directional forces exerted by these specific fields on charges and dipoles within the dividing cells. In vivo treatment of tumors in C57BL/6 and BALB/c mice (B16F1 and CT-26 syngeneic tumor models, respectively), resulted in significant slowing of tumor growth and extensive destruction of tumor cells within 3-6 days. These findings demonstrate the potential applicability of the described electric fields as a novel therapeutic modality for malignant tumors.

Chemotherapeutic treatment efficacy and sensitivity are increased by adjuvant alternating electric fields (TTFields)

BackgroundThe present study explores the efficacy and toxicity of combining a new, non-toxic, cancer treatment modality, termed Tumor Treating Fields (TTFields), with chemotherapeutic treatment in-vitro, in-vivo and in a pilot clinical trial.MethodsCell proliferation in culture was studied in human breast carcinoma (MDA-MB-231) and human glioma (U-118) cell lines, exposed to TTFields, paclitaxel, doxorubicin, cyclophosphamide and dacarbazine (DTIC) separately and in combinations. In addition, we studied the effects of combining chemotherapy with TTFields in an animal tumor model and in a pilot clinical trial in recurrent and newly diagnosed GBM patients.ResultsThe efficacy of TTFields-chemotherapy combination in-vitro was found to be additive with a tendency towards synergism for all drugs and cell lines tested (combination index ≤ 1). The sensitivity to chemotherapeutic treatment was increased by 1–3 orders of magnitude by adjuvant TTFields therapy (dose reduction indexes 23 – 1316). Similar findings were seen in an animal tumor model. Finally, 20 GBM patients were treated with TTFields for a median duration of 1 year. No TTFields related systemic toxicity was observed in any of these patients, nor was an increase in Temozolomide toxicity seen in patients receiving combined treatment. In newly diagnosed GBM patients, combining TTFields with Temozolomide treatment led to a progression free survival of 155 weeks and overall survival of 39+ months.ConclusionThese results indicate that combining chemotherapeutic cancer treatment with TTFields may increase chemotherapeutic efficacy and sensitivity without increasing treatment related toxicity.

Mitotic Spindle Disruption by Alternating Electric Fields Leads to Improper Chromosome Segregation and Mitotic Catastrophe in Cancer Cells

Tumor Treating Fields (TTFields) are low intensity, intermediate frequency, alternating electric fields. TTFields are a unique anti-mitotic treatment modality delivered in a continuous, noninvasive manner to the region of a tumor. It was previously postulated that by exerting directional forces on highly polar intracellular elements during mitosis, TTFields could disrupt the normal assembly of spindle microtubules. However there is limited evidence directly linking TTFields to an effect on microtubules. Here we report that TTFields decrease the ratio between polymerized and total tubulin, and prevent proper mitotic spindle assembly. The aberrant mitotic events induced by TTFields lead to abnormal chromosome segregation, cellular multinucleation, and caspase dependent apoptosis of daughter cells. The effect of TTFields on cell viability and clonogenic survival substantially depends upon the cell division rate. We show that by extending the duration of exposure to TTFields, slowly dividing cells can be affected to a similar extent as rapidly dividing cells.

Alternating Electric Fields (Tumor-Treating Fields Therapy) Can Improve Chemotherapy Treatment Efficacy in Non-Small Cell Lung Cancer Both In Vitro and In Vivo

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related deaths worldwide. Common treatment modalities for NSCLC include surgery, radiotherapy, chemotherapy, and, in recent years, the clinical management paradigm has evolved with the advent of targeted therapies. Despite such advances, the impact of systemic therapies for advanced disease remains modest, and as such, the prognosis for patients with NSCLC remains poor. Standard modalities are not without their respective toxicities and there is a clear need to improve both efficacy and safety for current management approaches. Tumor-treating fields (TTFields) are low-intensity, intermediate-frequency alternating electric fields that disrupt proper spindle microtubule arrangement, thereby leading to mitotic arrest and ultimately to cell death. We evaluated the effects of combining TTFields with standard chemotherapeutic agents on several NSCLC cell lines, both in vitro and in vivo. Frequency titration curves demonstrated that the inhibitory effects of TTFields were maximal at 150 kHz for all NSCLC cell lines tested, and that the addition of TTFields to chemotherapy resulted in enhanced treatment efficacy across all cell lines. We investigated the response of Lewis lung carcinoma and KLN205 squamous cell carcinoma in mice treated with TTFields in combination with pemetrexed, cisplatin, or paclitaxel and compared these to the efficacy observed in mice exposed only to the single agents. Combining TTFields with these therapeutic agents enhanced treatment efficacy in comparison with the respective single agents and control groups in all animal models. Together, these findings suggest that combining TTFields therapy with chemotherapy may provide an additive efficacy benefit in the management of NSCLC.

Mitotic disruption and reduced clonogenicity of pancreatic cancer cells in vitro and in vivo by tumor treating fields

OBJECTIVES Tumor Treating Fields (TTFields) are a non-invasive cancer treatment modality approved for the treatment of patients with recurrent glioblastoma. The present study determined the efficacy and mechanism of action of TTFields in preclinical models of pancreatic cancer. METHODS The effect of TTFields in vitro was assessed using cell counts, clonogenic assays, cell cycle analysis and analysis of mitotic figures. The effect in vivo effect was studied in the PC1-0 hamster pancreatic cancer model. RESULTS Application of TTFields in vitro showed a significant decrease in cell count, an increase in cell volume and reduced clonogenicity. Further analysis demonstrated significant increase in the number of abnormal mitotic figures, as well as a decrease in G2-M cell population. In hamsters with orthotopic pancreatic tumors, TTFields significantly reduced tumor volume accompanied by an increase in the frequency of abnormal mitotic events. TTFields efficacy was enhanced both in vitro and in vivo when combined with chemotherapy. CONCLUSIONS These results provide the first evidence that TTFields serve as an effective antimitotic treatment in preclinical pancreatic cancer models and have a long term negative effect on cancer cell survival. These results make TTFields an attractive candidate for testing in the treatment of patients with pancreatic cancer.

Alternating electric fields (TTFields) in combination with paclitaxel are therapeutically effective against ovarian cancer cells in vitro and in vivo.

Long‐term survival rates for advanced ovarian cancer patients have not changed appreciably over the past four decades; therefore, development of new, effective treatment modalities remains a high priority. Tumor Treating Fields (TTFields), a clinically active anticancer modality utilize low‐intensity, intermediate frequency, alternating electric fields. The goal of this study was to evaluate the efficacy of combining TTFields with paclitaxel against ovarian cancer cells in vitro and in vivo. In vitro application of TTFields on human ovarian cancer cell lines led to a significant reduction in cell counts as compared to untreated cells. The effect was found to be frequency and intensity dependent. Further reduction in the number of viable cells was achieved when TTFields treatment was combined with paclitaxel. The in vivo effect of the combined treatment was tested in mice orthotopically implanted with MOSE‐LTICv cells. In this model, combined treatment led to a significant reduction in tumor luminescence and in tumor weight as compared to untreated mice. The feasibility of effective local delivery of TTFields to the human abdomen was examined using finite element mesh simulations performed using the Sim4life software. These simulations demonstrated that electric fields intensities inside and in the vicinity of the ovaries of a realistic human computational phantom are about 1 and 2 V/cm pk‐pk, respectively, which is within the range of intensities required for TTFields effect. These results suggest that prospective clinical investigation of the combination of TTFields and paclitaxel is warranted.

Abstract B79: Translational study of tumor treating fields in combination with paclitaxel in ovarian cancer.

Tumor Treating Fields (TTFields), a clinically active anticancer modality, are based on low intensity intermediate frequency alternating electric fields that exert their cytotoxicity by disrupting mitosis. The present study examines whether concomitant paclitaxel and TTFields have a beneficial impact on ovarian cancer growth both in vitro and in vivo. Moreover, on the basis of the preclinical observations, an open-label pilot clinical study evaluating the effect of the combined modalities in 30 patients with recurrent ovarian cancer was initiated. Preclinical studies: To investigate the inhibitory effect of TTFields on ovarian cancer cell growth in vitro and determine optimal therapeutic frequency of TTFields in ovarian cancer, human ovarian cancer cell lines were treated with TTFields (100-400 kHz) for 72 hours using the inovitro system (Novocure, Haifa, Israel). To assess whether adding TTFields to paclitaxel increases the response of ovarian cancer cells to paclitaxel, we treated these cell lines with paclitaxel alone and in combination with TTFields. In vivo efficacy of the combined treatment was tested in female C57Bl/6 mice, orthotopically implanted with MOSE-L FFL luciferase positive cells. The feasibility of effective regional delivery of TTFields therapy to the ovaries, pelvis and abdomen of human subjects was examined using Finite Element Mesh (FEM) simulations performed using the Sim4life software. The FEM simulations demonstrated effective distribution of fields at intensities of 1-2 V/cm, which is above the minimal threshold required for TTFields response. The INNOVATE Trial (NCT02244502): Based on positive preclinical studies demonstrating the combined efficacy of TTFields and paclitaxel in different ovarian cancer models, a pilot clinical trial was initiated to evaluate this therapeutic combination in recurrent ovarian carcinoma patients. In this prospective, pilot, single arm study, 30 patients will receive bi-directional TTFields at 200 kHz applied to the ovaries and surrounding intra-abdominal tissues using 4 transducer arrays located on the surface of the lower abdominal region. In addition, patients will receive concomitant paclitaxel at a standard regimen and dose. The combined treatment will be administered until further radiological progression. Inclusion criteria include ECOG score of 0-1 and no serious co-morbidities. The trial9s primary endpoint is adverse events frequency and severity. The study will also collect preliminary efficacy data through the analysis of progression-free survival, 1-year survival rate and overall survival. Compliance data will be analyzed as an additional secondary endpoint. The INNOVATE study started to enroll patients in October 2014, and is currently accruing patients in Switzerland, Belgium and Spain. So far the trial has enrolled half of the planned 30 patients. In summary, we present the first preclinical evidence in ovarian cancer of the combined efficacy of paclitaxel and TTFields, a new anticancer treatment modality. Our results suggest that it may represent a novel, effective therapeutic strategy against ovarian cancer. Pilot clinical testing is ongoing. Citation Format: Mijal Munster, Roni Blat, Paul C. Roberts, Eva M. Schmelz, Moshe Giladi, Rosa S. Schneiderman, Yaara Porat, Zeev Bomzon, Noa Urman, Aviran Itzhaki, Tali Voloshin, Shay Cahal, Eilon D. Kirson, Uri Weinberg, Yoram Palti. Translational study of tumor treating fields in combination with paclitaxel in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B79.

Abstract 3665: Tumor Treating Fields (TTFields) plus anti-PD-1 therapy induce immunogenic cell death resulting in enhanced antitumor efficacy

Tumor Treating Fields (TTFields) are an effective anti-neoplastic treatment modality delivered via noninvasive application of low intensity, intermediate frequency, alternating electric fields. TTFields is approved for the treatment of both newly diagnosed and recurrent glioblastoma. TTFields interrupt cancer cell mitosis by disrupting microtubules and septin filaments, which play key roles in mitosis. The mitotic effects of TTFields include abnormal chromosome segregation and ER stress, which trigger different forms of cell death. We evaluated the in vitro and in vivo effects of TTFields combined with an immune checkpoint inhibitor (anti-PD1) on immunogenic cell death. Murine Lewis lung carcinoma (LLC) and ovarian surface epithelial (MOSE) cells were treated with TTFields using the inovitroTM system. Levels of calreticulin (CRT) on the surface of treated cells and intracellular ATP levels were evaluated using flow cytometry. High mobility group box 1 (HMGB1) secretion was measured using an ELISA assay. Mice were implanted with LLC cells were treated with TTFields, anti-PD-1, or a combination of the two modalities. Tumor volume was monitored; flow cytometry analysis was performed for phenotypic characterization of infiltrating immune cells. TTFields induced elevated cell surface expression of CRT, decreased intracellular ATP levels, and promoted HMGB1 secretion. In vivo, the combined treatment of lung tumor-bearing mice with TTFields plus anti-PD-1 led to a significant decrease in tumor volume compared to anti-PD-1 alone or to the control group. Significant increases in CD45+ tumor infiltrating cells were observed in the TTFields plus anti-PD-1 group. Infiltrating cells demonstrated a significant upregulation of surface PD-L1 expression. Both F4/80+CD11b+ cells and CS11c+ cells exhibited higher tumor infiltration and elevated PD-L1 expression as compared to infiltrating immune cell in the control group. Our results demonstrate that TTFields treatment potentiates immunogenic cell death in cancer cells. Combining TTFields with specific immunotherapies such as anti-PD-1 may enhance antitumor immunity and result in increased tumor control. Citation Format: Tali Voloshin, Orna Tal-Yitzhaki, Noa Kaynan, Moshe Giladi, Anna Shteingauz, Mijal Munster, Roni Blat, Yaara Porat, Rosa S. Schneiderman, Shay Cahal, Aviran Itzhaki, Eilon D. Kirson, Uri Weinberg, Yoram Palti. Tumor Treating Fields (TTFields) plus anti-PD-1 therapy induce immunogenic cell death resulting in enhanced antitumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3665. doi:10.1158/1538-7445.AM2017-3665

Abstract 5569: Tumor Treating Fields inhibit the growth of pancreatic and ovarian cancer in preclinical models .

Tumor Treating Fields (TTFields) therapy is an established anti-mitotic treatment modality. The Novo TTF-100A system, which delivers TTFields to the brain received FDA approval for the treatment of patients with Recurrent Glioblastoma (GBM) Brain Tumors. The goal of the present study is to test whether TTFields therapy is effective as a treatment in pre-clinical models of pancreatic and ovarian cancer. TTFields of various frequencies were applied for 72h to cancerous tumor cells using two pairs of perpendicular insulated electrodes. Pancreatic adenocarcinoma and ovarian carcinoma culture growth was significantly reduced compared to controls (65%+10% and 21%+13%, respectively) with a maximal inhibitory effect seen at 150 and 200 kHz (respectively). The surviving cells exhibited increased cell volume (35%+11% and 61%+43% for pancreatic and ovarian cell lines, respectively) and a reduced viability. Fluorescence microscopy and FACS analysis revealed abnormal mitotic figure in the treated cells cultures and an increase in the 4N population, suggesting these cells failed to complete mitosis. Yet, the increase in the 4N cells fraction was too small to explain for the observed increase in cell volume in the entire remaining cell population. Combining TTFields with gemcitabine and paclitaxel commonly used for the treatment of pancreatic and ovarian cancer, enhanced treatment efficacy and led to a further increase in cell volume. The efficacy of TTFields either alone or in combination with 5FU or gemcitabine was tested in hamsters bearing syngeneic, orthotopic pancreatic tumors. In vivo imaging as well as post mortem analysis demonstrated a significant decrease in tumor weight and volume. Compared to chemotherapy treatment alone, TTF had a sensitizing effect and increased tumor response to chemotherapy. Based on previous reports, the observed increase in cell volume is expected to shift the peak response to TTFields to a lower frequency, allowing some of the cells to escape the effect of TTFields. While this is the first proposed mechanism of resistance to TTFields it also opens the possibility of overcoming that resistance using serial application of several frequencies. Citation Format: Moshe Giladi, Rosa S. Schneiderman, Yaara Porat, Mijal Munster, Aviran Itzhaki, Daniel Mordechovich, Shay Cahal, Uri Weinberg, Eilon D. Kirson, Yoram Palti. Tumor Treating Fields inhibit the growth of pancreatic and ovarian cancer in preclinical models . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5569. doi:10.1158/1538-7445.AM2013-5569