Effects Of TTFields Research Articles

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

ObjectivesTumor 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. MethodsThe 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. ResultsApplication 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. ConclusionsThese 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.

Overcoming cell size escape from tumor treating fields using a varying frequency treatment paradigm in vitro.

e22134 Background: TTFields Therapy at 200 kHz received FDA approval for the treatment of patients with recurrent glioblastoma (GBM) based on the results of a phase III clinical trial. Radiological responses were observed in 14% of the treated patients. The lack of radiological response in the remaining patients suggests that GBM cells may escape the effect of TTFields over time. The goals of the present study were to identify possible routes by which cancer cells can escape the antimitotic effect of TTFields and to explore ways to overcome such escape mechanisms. Methods: Measurements of cell size before and after 72 hours TTFields application revealed a significant volume increase in 10 cancer cell lines (17% - 101%). A2780 ovarian cancer cultures (cell volume 2.0+0.2 pL) exhibit maximal inhibition by TTFields at 200 kHz. Twenty four hour application of 200 kHz TTFields led to a 70+29% increase in average cell volume (p<0.05). FACS analysis showed that TTFields application to A2780 cells led to a 44% increase in the G2 population due to the antimitotic effect of TTFields. However, the increase in the G2 proportion is not sufficient to explain the increase in cell volume seen after TTFields application. Furthermore, after an additional 24 hours of treatment, the fraction of cells in the G2 population returned to its original percentage (30%), while the increase in cell volume remained high (67+26%). Results: Previous publications demonstrated an inverse relationship exists between cell size and optimal TTFields frequency. Thus an increase in cell size may allow cells to avoid the maximal TTFields effect. To attempt to overcome such cell size escape we decreased the frequency of the TTFields from 200 to 150 kHz after 24 hours treatment to coincide with the increase in cell volume. We found that varying the frequency from 200 to 150 kHz increased the inhibitory effect of TTFields compared to continuous treatment at 200Khz (37+10% and 57+16% decrease in cell count, respectively; p=0.043). Conclusions: This is the first evidence of an escape mechanism from the antimitotic effect of TTFields. The results presented provide a simple way to restore treatment efficacy by varying TTFields frequency in parallel to the change in cell volume.

Meta-analysis of cancer cell lines genomes based on their response to ttfields.

e22125 Background: Tumor Treating Fields (TTFields) therapy is an established anti-mitotic treatment modality. The inhibitory effect of TTFields was demonstrated in numerous cancer cell lines with some cell lines exhibiting higher treatment sensitivity than others. The goal of the present study is to compare genomic characteristics of cell lines based on their response pattern to TTFields. Methods: Fifteen different human cancerous cell lines were treated using TTFields at their optimal frequency with the same nominal intensity (1.8 V/cm). The distribution of the response to TTFields was found to be bimodal with inhibitions of 30-40% and 50-60% compared to controls. Cell lines were divided into regular and high sensitivity groups according to their response to TTFields. Both groups were characterized based upon the distribution of mutated genes using the Cancer Cell Line Encyclopedia (CCLE) database. Results: Based on the distribution of mutated genes we identified 1 gene with significantly higher probability (P=0.017) to be present in the regular than the high sensitivity group. This gene (NEK3) is a never-in-mitosis (NIMA) related kinase 3 responsible for deacetylation of microtubules. In addition, 2 genes were found with significantly higher probability (P<0.05) to be present in the high- than the regular sensitivity group; ASPH, an aspartate beta-hydroxylase involved in negative regulation of cell proliferation through modulation of calcium homeostasis and TEC, a protein tyrosine kinase involved in immunological signaling pathways. Conclusions: The possible involvement of NEK3 mutations in determining sensitivity to TTFields is interesting in view of the known interference of TTFields with spindle microtubule assembly. ASPH mutations could release cells from negative regulation of proliferation, increasing their replication rate, and thus increasing their sensitivity to TTFields. In order to further test this hypothesis, a comparison of the effect of TTFields needs to be performed on wild type and knockout cell lines for these genes. A comparison of the mutation of these genes in tissue samples from patients whose tumors show high sensitivity to TTFields, may lead to a personalized approach to the use of TTFields clinically.

Abstract 709: Mitosis interference of cancer cells by NovoTTF-100A causes decreased cellular viability.

Abstract Cancer cell death from traditional cytotoxic chemotherapy and radiation is a result of triggering cell cycle checkpoint with p53 playing a prominent role in orchestrating this effect. Unlike these traditional approaches, the NovoTTF-100A device, which has been approved for the treatment of recurrent glioblastomas, kills tumor cells by a novel mechanism that interferes with the proper progression through mitosis. As is the case with earlier therapeutic interventions, only a subset of patients shows significant response to TTFields treatment. Our research reveals that the tumor-treating fields (TTFields) affects processes proximal to the metaphase-to-anaphase transition, leading to cell cycle arrest in anaphase and eventual cell death. Tissue culture cells exposed to the TTFields during mitosis exhibit a variety of outcomes, including cell arrest in anaphase, cytokinesis with cytokinetic furrow regression, and slippage out of mitosis without division. Time-lapse movies of cells stained with the DNA stain DRAQ5 and exposed to the TTFields revealed membrane blebbing and oscillation, as well as failed segregation of sister chromatids from the metaphase plate, both of which were initiated near the time of metaphase-to-anaphase transition. Immunoflourescence of cells after short-term treatment with TTFields during mitosis showed disordered separation of chromosomes from metaphase plates accompanied by lagging chromosomes, dispersion of chromosomes from the metaphase plate, chromosomes de-condensation in the absence of cytokinesis, and daughter cells with asymmetric chromosomal segregation. Flow cytometry also revealed the presence of cells with abnormal DNA content in cultures treated with TTFields. In contrast to the p53 mutant cell lines, MDA-MB-231 and HCT 116, U87 cells with wild-type p53 exhibit increased Annexin V binding, Caspase activation and increased markers of endoplasmic reticulum (ER) stress after brief exposure during mitosis. This suggests the TTFields induced cellular damage is able to trigger p53-dependent cell death in cells that escape killing during M-phase. These data show that the effects of TTFields on cellular damage during M-phase and provide evidence for the notion that the differential responses to treatment seen in patients may be rooted in the underlying genetic alterations in their tumors. Citation Format: Sze Xian Lee, Eric T. Wong, Kenneth D. Swanson. Mitosis interference of cancer cells by NovoTTF-100A causes decreased cellular viability. [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 709. doi:10.1158/1538-7445.AM2013-709

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

Abstract 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

Tumor treating fields: concept, evidence and future

Introduction: Local control is fundamental, both for the curative as well as the palliative treatment of cancer. Tumor treating fields (TTFields) are low intensity (1 – 2 V/cm), intermediate frequency (100 – 200 kHz) alternating electric fields administered using insulated electrodes placed on the skin surrounding the region of a malignant tumor. TTFields were shown to destroy cells within the process of mitosis via apoptosis, thereby inhibiting tumor growth. TTFields have no effect on non-dividing cells.Areas covered: This article reviews in vitro and in vivo preclinical studies, demonstrating the activity of TTFields both as a monotherapy as well as in combination with several cytotoxic agents. Furthermore, it summarizes the clinical experience with TTFields, mainly in two indications: one in recurrent glioblastoma multiforme: in a large prospective randomized Phase III trial TTFields was compared with best standard care (including chemotherapy): TTFields significantly improved median overall survival (OS) compared with standard therapy (7.8 vs 6.1 months) for the patients treated per protocol. Importantly, quality of life was also better in the TTFields group. The second indication was a Phase II study in second-line non-small cell lung cancer, where TTFields was administered concomitantly with pemetrexed. This combination resulted in an excellent median OS of 13.8 months. Interestingly, the progression-free survival (PFS) within the area of the TTFields was 28, however, outside the TTFields the PFS was only 22 weeks.Expert opinion: The proof of concept of TTFields has been well demonstrated in the preclinical setting, and the clinical data seem promising in various tumor types. The side effects of TTFields were minimal and in general consisted of skin reaction to the electrodes. There are a number of ways in which TTFields could be further evaluated, for example, in combination with chemotherapy, as a maintenance treatment, or as a salvage therapy if radiotherapy or surgery is not possible. While more clinical data are clearly needed, TTFields is an emerging and promising novel treatment concept.

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.