Aurora B Kinase Inhibition by AZD1152 Concomitant with Tumor Treating Fields Is Effective in the Treatment of Cultures from Primary and Recurrent Glioblastomas.

Tumor Treating Fields (TTFields) were incorporated into the treatment of glioblastoma, the most malignant brain tumor, after showing an effect on progression-free and overall survival in a phase III clinical trial. The combination of TTFields and an antimitotic drug might further improve this approach. Here, we tested the combination of TTFields with AZD1152, an Aurora B kinase inhibitor, in primary cultures of newly diagnosed (ndGBM) and recurrent glioblastoma (rGBM). AZD1152 concentration was titrated for each cell line and 5-30 nM were used alone or in addition to TTFields (1.6 V/cm RMS; 200 kHz) applied for 72 h using the inovitro™ system. Cell morphological changes were visualized by conventional and confocal laser microscopy. The cytotoxic effects were determined by cell viability assays. Primary cultures of ndGBM and rGBM varied in p53 mutational status; ploidy; EGFR expression and MGMT-promoter methylation status. Nevertheless; in all primary cultures; a significant cytotoxic effect was found following TTFields treatment alone and in all but one, a significant effect after treatment with AZD1152 alone was also observed. Moreover, in all primary cultures the combined treatment had the most pronounced cytotoxic effect in parallel with morphological changes. The combined treatment of TTFields and AZD1152 led to a significant reduction in the number of ndGBM and rGBM cells compared to each treatment alone. Further evaluation of this approach, which has to be considered as a proof of concept, is warranted, before entering into early clinical trials.

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.

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.

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.

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.

TTFields alone and in combination with chemotherapeutic agents effectively reduce the viability of MDR cell sub-lines that over-express ABC transporters.

BACKGROUND: Exposure of cancer cells to chemotherapeutic agents may result in reduced sensitivity to structurally unrelated agents, a phenomenon known as multidrug resistance, MDR. The purpose of this study is to investigate cell growth inhibition of wild type and the corresponding MDR cells by Tumor Treating Fields--TTFields, a new cancer treatment modality that is free of systemic toxicity. The TTFields were applied alone and in combination with paclitaxel and doxorubicin. METHODS: Three pairs of wild type/MDR cell lines, having resistivity resulting from over-expression of ABC transporters, were studied: a clonal derivative (C11) of parental Chinese hamster ovary AA8 cells and their emetine-resistant sub-line EmtR1; human breast cancer cells MCF-7 and their mitoxantrone-resistant sub lines MCF-7/Mx and human breast cancer cells MDA-MB-231 and their doxorubicin resistant MDA-MB-231/Dox cells. TTFields were applied for 72 hours with and without the chemotherapeutic agents. The numbers of viable cells in the treated cultures and the untreated control groups were determined using the XTT assay. Student t-test was applied to asses the significance of the differences between results obtained for each of the three cell pairs. RESULTS: TTFields caused a similar reduction in the number of viable cells of wild type and MDR cells. Treatments by TTFields/drug combinations resulted in a similar increased reduction in cell survival of wild type and MDR cells. TTFields had no effect on intracellular doxorubicin accumulation in both wild type and MDR cells. CONCLUSIONS: The results indicate that TTFields alone and in combination with paclitaxel and doxorubicin effectively reduce the viability of both wild type and MDR cell sub-lines and thus can potentially be used as an effective treatment of drug resistant tumors.

AMPK-dependent autophagy upregulation serves as a survival mechanism in response to Tumor Treating Fields (TTFields).

Tumor Treating Fields (TTFields), an approved treatment modality for glioblastoma, are delivered via non-invasive application of low-intensity, intermediate-frequency, alternating electric fields. TTFields application leads to abnormal mitosis, aneuploidy, and increased cell granularity, which are often associated with enhancement of autophagy. In this work, we evaluated whether TTFields effected the regulation of autophagy in glioma cells. We found that autophagy is upregulated in glioma cells treated with TTFields as demonstrated by immunoblot analysis of the lipidated microtubule-associated protein light chain 3 (LC3-II). Fluorescence and transmission electron microscopy demonstrated the presence of LC3 puncta and typical autophagosome-like structures in TTFields-treated cells. Utilizing time-lapse microscopy, we found that the significant increase in the formation of LC3 puncta was specific to cells that divided during TTFields application. Evaluation of selected cell stress parameters revealed an increase in the expression of the endoplasmic reticulum (ER) stress marker GRP78 and decreased intracellular ATP levels, both of which are indicative of increased proteotoxic stress. Pathway analysis demonstrated that TTFields-induced upregulation of autophagy is dependent on AMP-activated protein kinase (AMPK) activation. Depletion of AMPK or autophagy-related protein 7 (ATG7) inhibited the upregulation of autophagy in response to TTFields, as well as sensitized cells to the treatment, suggesting that cancer cells utilize autophagy as a resistance mechanism to TTFields. Combining TTFields with the autophagy inhibitor chloroquine (CQ) resulted in a significant dose-dependent reduction in cell growth compared with either TTFields or CQ alone. These results suggest that dividing cells upregulate autophagy in response to aneuploidy and ER stress induced by TTFields, and that AMPK serves as a key regulator of this process.

Determining the Optimal Inhibitory Frequency for Cancerous Cells Using Tumor Treating Fields (TTFields).

Tumor Treating Fields (TTFields) are an effective treatment modality delivered via the continuous, noninvasive application of low-intensity (1-3 V/cm), alternating electric fields in the frequency range of several hundred kHz. The study of TTFields in tissue culture is carried out using the TTFields in vitro application system, which allows for the application of electric fields of varying frequencies and intensities to ceramic Petri dishes with a high dielectric constant (Ɛ > 5,000). Cancerous cell lines plated on coverslips at the bottom of the ceramic Petri dishes are subjected to TTFields delivered in two orthogonal directions at various frequencies to facilitate treatment outcome tests, such as cell counts and clonogenic assays. The results presented in this report demonstrate that the optimal frequency of the TTFields with respect to both cell counts and clonogenic assays is 200 kHz for both ovarian and glioma cells.

Tumor treating fields (TTFields) delay DNA damage repair following radiation treatment of glioma cells.

BACKGROUND: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

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.

Alternating electric fields (TTFields) inhibit metastatic spread of solid tumors to the lungs.

Tumor treating fields (TTFields) are low intensity, intermediate frequency, alternating electric fields used to treat cancerous tumors. This novel treatment modality effectively inhibits the growth of solid tumors in vivo and has shown promise in pilot clinical trials in patients with advanced stage solid tumors. TTFields were tested for their potential to inhibit metastatic spread of solid tumors to the lungs in two animal models: (1) Mice injected with malignant melanoma cells (B16F10) into the tail vein, (2) New Zealand White rabbits implanted with VX-2 tumors within the kidney capsule. Mice and rabbits were treated using two-directional TTFields at 100-200 kHz. Animals were either monitored for survival, or sacrificed for pathological and histological analysis of the lungs. The total number of lung surface metastases and the absolute weight of the lungs were both significantly lower in TTFields treated mice then in sham control mice. TTFields treated rabbits survived longer than sham control animals. This extension in survival was found to be due to an inhibition of metastatic spread, seeding or growth in the lungs of TTFields treated rabbits compared to controls. Histologically, extensive peri- and intra-tumoral immune cell infiltration was seen in TTFields treated rabbits only. These results raise the possibility that in addition to their proven inhibitory effect on the growth of solid tumors, TTFields may also have clinical benefit in the prevention of metastatic spread from primary tumors.

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

BACKGROUND: The 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. METHODS: Cell 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. RESULTS: The 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