TTFields Application Research Articles

CSIG-09. ASSESSMENT OF TUMOR TREATING FIELDS (TTFields) COMBINED WITH TRICHOSTATIN A (TSA) IN PATIENT-DERIVED GLIOBLASTOMA (GBM) CELLS

Abstract BACKGROUND TTFields therapy is a non-invasive biophysical approach for GBM patient treatment. TTFields produce an anti-mitotic effect by destabilizing microtubule polymerization and interrupting cell division. However, epigenetic modifications that may be induced by TTFields remain unknown. To address this, TTFields were applied to patient-derived GBM cells ± the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) and cellular proliferation was assessed. HDAC activity was also measured after TTFields application. METHODS GBM cells were isolated from a newly-diagnosed patient tumor (IDH-WT, unmethylated MGMT promoter). Cells were grown in DMEM/F12 media with 10% FBS and gentamicin. Cells were plated on plastic coverslips (1×104cells/coverslip) and incubated overnight. TTFields were applied at 200 kHz (field intensity of ~1.6 V/cm) for 6 days. For the last 2 days of TTFields, cells were incubated with DMSO or 250 nM TSA. Cells were harvested and counted to assess proliferation (n=4-5/group). To determine if TTFields have a direct effect on HDAC activity, TTFields were applied as described and nuclei were extracted. Nuclear HDAC activity was measured using a commercial kit (n=2-3/group). Cell counts were compared with one-way ANOVA and Tukey multiple comparisons with p< 0.05 considered significant. RESULTS Cell counts for the DMSO control group, TSA only, TTFields only, and the combination were: 643,400 ± 111,384, 233,800 ± 144,200, 90,775 ± 45,209, and 41,520 ± 36,168, respectively (mean ± SD; ANOVA p< 0.0001). There was no statistical difference between the TTFields only and TSA only or TTFields + TSA groups, with all other comparisons statistically significant. For the HDAC activity measurements, control nuclei were 1068 ± 135 pmol/min/mg and TTFields-treated nuclei were 1301 ± 313.6 pmol/min/mg and were not statistically different. CONCLUSIONS The addition of TSA to TTFields did not alter cellular proliferation more than TTFields alone and TTFields did not alter HDAC activity in these GBM cells.

EXTH-74. INCREASING CANCER CELL MEMBRANE PERMEABILITY THROUGH APPLICATION OF TUMOR TREATING FIELDS (TTFIELDS)

Abstract INTRODUCTION Tumor Treating Fields (TTFields) are intermediate frequency, alternating electric fields with anti-mitotic effects on cancerous cells. TTFields are delivered non-invasively through arrays placed on the skin at the tumor region. TTFields therapy is approved in several territories for treatment of glioblastoma (GBM) and mesothelioma. Recently, TTFields have been shown to increase GBM cell membrane permeability. The current study aimed to explore this effect in multiple cell lines and examine the potential benefits of combining TTFields with other anticancer agents. METHODS TTFields were delivered to GBM (U-87 MG), uterine sarcoma (MES-SA), and breast adenocarcinoma (MCF-7) cell lines for 72hr across a range of frequencies (50-500kHz). Cytotoxicity of TTFields was examined by cell counts, and intracellular accumulation of 7-aminoactinomycin D (7-AAD) was measured by flow cytometry. Exposing the cells to 7-AAD at different time points relative to TTFields application cessation was used to determine the kinetics of cell membrane permeability. The potential of TTFields to facilitate intracellular accumulation of anthracycline chemotherapeutics was tested in chemotherapy-sensitive and chemotherapy-resistant cells. RESULTS Elevated intracellular accumulation of 7-AAD was observed in all examined cell lines treated with TTFields, at an optimal frequency that differed from that for maximal TTFields-induced cytotoxicity. No intracellular accumulation of 7-AAD was seen for measurements performed after termination of TTFields application, indicating that increased cell membrane permeability by TTFields was temporary and reversible. Lastly, the accumulation of chemotherapeutic agents in chemotherapy-resistant cancer cells was elevated to the same extent as in matched chemotherapy-sensitive cells when TTFields were delivered concomitant with chemotherapy. CONCLUSIONS TTFields increased cancer cell permeability in a transient and reversible manner across multiple cancer cell types. The increased permeability enhanced intracellular accumulation of chemotherapeutics, even within chemotherapy-resistant cells.

DDRE-46. REDUCED CANCER CELL SENSITIVITY TO TUMOR TREATING FIELDS (TTFields) THROUGH ACTIVATION OF THE PI3K/AKT/mTOR SIGNALING PATHWAY CAN BE MITIGATED USING PI3K INHIBITORS OR PI3K/mTOR DUAL INHIBITORS

Abstract INTRODUCTION Tumor Treating Fields (TTFields) therapy is an approved anti-cancer treatment modality applied non-invasively and loco-regionally to the tumor region. TTFields have been demonstrated to extend life, however, most patients do eventually progress. The current study aimed to identify potential molecular mechanisms involved in reduced cellular sensitivity to TTFields. METHODS Cancer cells that exhibit reduced sensitivity to TTFields were generated by continuous long duration application of TTFields (7 or 13 days, depending on the cell line). Changes in cellular signaling pathways were evaluated in ovarian A2780 and glioblastoma U-87 MG cancer cells exposed to long-term relative to short-term (3 or 7 days, depending on the cell line) treatment with TTFields using Luminex multiplex assay followed by Western blot validation. The relevance of the affected pathways was confirmed through evaluation of the response to long-term application of TTFields in combination with pharmacological pathway inhibitors by measuring cell counts, apoptosis, and clonogenicity. Relevant pathway markers in tumor sections from tumor-bearing rats treated with TTFields were examined using immunohistochemistry. RESULTS Continuous long-term application of TTFields reduced cellular sensitivity to TTFields and was accompanied by increased levels of phosphorylated AKT, mTOR and additional proteins from the PI3K/AKT/mTOR signaling pathway. Increased phosphorylation of AKT was also evident in tumor sections from rats treated with TTFields. Concomitant use of TTFields with PI3K inhibitors or PI3K/mTOR dual inhibitors sensitized A2780 cells to long-term TTFields application. CONCLUSION Our study demonstrates that decreased cancer cell sensitivity to long-term TTFields application is mediated by activation of the PI3K/AKT/mTOR signaling pathway and provides a rationale for further examining the potential benefit of combining TTFields with PI3K or PI3K/mTOR dual inhibitors.

Abstract 1382: Targeting Akt signaling pathway potentiates the antitumor effect of Tumor Treating Fields (TTFields) in vitro

Abstract Introduction Tumor Treating Fields (TTFields) therapy is a clinically applied anti-neoplastic treatment modality, approved for the treatment of glioblastoma and malignant pleural mesothelioma. TTFields are low intensity (1-3 V/cm) alternating electric fields within the intermediate frequency range (100-300 kHz), that are applied loco-regionally and non-invasively to the tumor site. Although TTFields therapy has demonstrated improved progression free survival and overall survival, tumor recurrence still occurs in some patients. To evaluate the cellular mechanisms of treatment resistance to TTFields, we analyzed changes in signaling pathway mediators in treated human glioma (U-87 MG) and ovarian (A2780) cancer cell lines. Also, we examined the in vitro efficacy of TTFields in combination with therapies that target molecular candidates that may confer resistance to TTFields. Methods To establish TTFields resistant glioma and ovarian cancer cell lines in vitro, 2 approaches were tested: 1) cyclic repetitions, where TTFields treatment was repeatedly applied for 72 hours with 24 hours breaks between treatment cycles; and 2) continuous, long-duration application of TTFields (1 or 2 weeks, depending on the cancer cell line type) without treatment breaks. Luminex assay followed by Western blot analysis were used to study changes in signaling pathways following TTFields treatment. Finally, we examined the in vitro efficacy of continuous, long-duration application of TTFields in combination with BGT226 (a phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dual inhibitor). Results Application of TTFields using cyclic repetition significantly decreased proliferation of human glioma and ovarian cancer cells, whereas cells treated using continuous, long-duration application of TTFields showed reduced sensitivity to TTFields. Luminex analysis clearly showed activation of the PI3K/mTOR/AKT signaling pathway in response to TTFields treatment. To clarify the importance of this signaling pathway on regulating treatment resistance to TTFields, we examined the effect of TTFields, alone and in combination, with a PI3K/mTOR inhibitor (BGT226) on cancer cell proliferation, apoptosis, and activation of AKT. While long-duration application of TTFields led to Akt activation, TTFields in combination with BGT226 resulted in reduction of p-AKT S473, with a corresponding blockade of proliferation, induction of apoptosis, and decrease in the clonogenic potential. Conclusions We propose here a new mechanism of resistance to prolonged TTFields treatment mediated by the PI3K/mTOR/AKT signaling pathway in glioma and ovarian cancer cells. We demonstrate that combination therapy of TTFields and a targeted PI3K/mTOR dual inhibitor (BGT226) inhibited AKT S473 phosphorylation and sensitized cancer cells to “long-duration” TTFields application. Citation Format: Anat Klein-Goldberg, Tali Voloshin, Efrat Zemer-Tov, Rom Paz, Lilach Koren, Alexandra Volodin, Boris Brant, Moshe Giladi, Uri Weinberg, Yoram Palti. Targeting Akt signaling pathway potentiates the antitumor effect of Tumor Treating Fields (TTFields) in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1382.

Abstract 1037: Valproic acid (VPA) combined with Tumor Treating Fields (TTFields) in vitro decreases cellular proliferation and increases clonogenic potential of glioblastoma (GBM) cells

Abstract Background: VPA is an antiepileptic drug used to treat tumor-related seizures. TTFields is an antimitotic therapy that is FDA-approved for GBM. Since GBM patients may use both therapies, we examined the combination of TTFields and VPA in vitro using GBM cells isolated from a newly-diagnosed GBM patient. Cells proliferation and clonogenic potential were assessed after cells were exposed to VPA during TTFields application. Methods: A single-cell suspension was generated from a patient tumor (male, age 64, IDH-WT, no 1p19q deletion, EGFR not amplified, unmethylated MGMT promoter). Cells were cultured in DMEM/F12 media with 10% FBS and gentamicin. Cells were plated on plastic coverslips (1×104cells/coverslip) and incubated overnight. Coverslips were divided into 8 groups (Control, 250 µM, 500 µM, 1 mM VPA, ± TTFields; n=5/group). TTFields were applied at 200 kHz with a field intensity of ~1.6 V/cm for 14 days with VPA in the media as indicated. Cells were harvested, stained and counted with a Cellometer K2 (Nexcelom Bioscience, Lawrence, MA) that provides viable cell counts. For each group, 2000 cells each from 3 coverslips were plated for the clonogenic assay (CA). After 2 weeks, cells were crystal violet stained, imaged and counted. Groups were compared by ANOVA (GraphPad Prism v9, GraphPad Software, San Diego, CA) with significance at P<0.05. Results: Cell count and CA data are in the Table. TTFields alone reduced cell counts by >50%, and reduced colony counts and area in the CA. VPA alone was only effective at the 1 mM concentration and reduced the cell count by 40%. Combination of TTFields and 1 mM VPA reduced the cell counts by 95%. However, VPA alone and combined with TTFields increased colony number and area in the CA. Conclusions: Although the combination of VPA and TTFields (200 kHz) produced a greater decrease in GBM cell proliferation, VPA increased the clonogenic potential and may hinder the response to TTFields. Cell counts and clonogenic assay dataCell countsControl250 μM VPA500 μM VPA1 mM VPATTFieldsTTFields + 250 μM VPATTFields + 500 μM VPATTFields + 1 mM VPAANOVA P-valueMean2061002121002314001234007722042160452909193<0.0001Std. Deviation73065706377136621001387367754359108910Clonogenic assay colony countsMean31.2943.8940.5663.5618.3311.6730.5636.33<0.0001Std. Deviation29.0617.6919.5514.948.6610.8220.2516.56Clonogenic assay colony area (pixels)Mean1177249419773070394.8202.9787.6854.7<0.0001Std. Deviation148512501371987.5225.9206.9548.3438.8 Citation Format: Sharon K. Michelhaugh, Susan Coombe, Katherine E. Degen, Sandeep Mittal. Valproic acid (VPA) combined with Tumor Treating Fields (TTFields) in vitro decreases cellular proliferation and increases clonogenic potential of glioblastoma (GBM) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1037.

Abstract 1007: Patient-derived metastatic renal carcinoma cells are highly sensitive to in vitro Tumor Treating Fields (TTFields)

Abstract Background: Until the advent of checkpoint inhibitor (CI) therapies, renal cell carcinoma (RCC) was clinically challenging to treat given its radio- and chemotherapy resistance. Despite the advances with CIs, patients with brain metastases derived from RCC still face challenges with treatment due to limited brain penetration of CI compounds. Anti-mitotic TTFields therapy is FDA-approved for the treatment of glioblastoma and mesothelioma, and clinical trials are underway for the application of TTFields therapy to primary lung cancer as well as lung cancer brain metastases. In this study, in vitro TTFields were applied to patient-derived brain metastasis RCC cells to determine the potential efficacy of this emerging treatment modality. Methods: This study was approved by the Institutional Review Board. A single-cell suspension was generated from tumor tissues obtained from a patient with newly-diagnosed RCC brain metastasis (male, age 52, mutant p53). Cells were cultured in DMEM/F12 media with 10% fetal bovine serum and gentamicin. Prior to TTFields initiation, cells were plated on plastic coverslips (1×104cells/coverslip; n=8/group) and incubated overnight. Then, TTFields were applied at 200 kHz with a field intensity of ~1.6 V/cm for 14 days, with one group of coverslips maintained as untreated controls. At the conclusion of the experiment, cells were harvested, stained with acridine orange/propidium iodide and counted with a Cellometer K2 (Nexcelom Bioscience, Lawrence, MA) that provides viable cell counts and average cell diameter. Groups were compared with a two-tailed t-test (GraphPad Prism v9, GraphPad Software, San Diego, CA) with significance set at P<0.05. Results: Viable cell count per coverslip for the control group was 149,863 ± 85644 (mean ± SD; n=8) and for the TTFields (200 kHz) group was 1,426 ± 1345 (n=6, 2 coverslips had no cell recovery). Average cell diameter was 9.713 ± 0.6312 for the control group and 16.92 ± 2.903 for the TTFields group. TTFields application significantly reduced cell count (P=0.0012) and increased the average cell diameter (P<0.0001) compared to the controls. Conclusions: TTFields application (200 kHz) demonstrated efficacy by markedly reducing cell proliferation (i.e., decreased cell counts) in RCC. The increase in average cell diameter observed with TTFields is consistent with previously reported findings of other research groups also showing increases in cell volume with TTFields in multiple cancer types (brain, lung, pancreas, breast, ovarian, and cervical). This study is the first to demonstrate the efficacy of in vitro TTFields treatment in patient-derived RCC and suggests that patients with renal carcinoma brain metastases may benefit from TTFields therapy. Citation Format: Sharon K. Michelhaugh, Blake C. Walker, Susan Coombe, Sandeep Mittal. Patient-derived metastatic renal carcinoma cells are highly sensitive to in vitro Tumor Treating Fields (TTFields) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1007.

Abstract 1692: A novel immunoregulatory role of tumor treating fields (TTFields) on macrophage polarization

Abstract Introduction: Tumor Treating Fields (TTFields) therapy is a clinically applied anti-neoplastic treatment modality delivered via noninvasive application of low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields. In this study we investigated the potential of TTFields for regulating macrophage phenotype skewing, with or without lipopolysaccharide (LPS) and interferon-γ (IFN-γ) or IL-4 activation. Methods: Bone marrow cells were flushed from the femurs and tibias of 5-8 week old Balb\C mice to generate bone marrow-derived macrophages (BMDMs). To study individual macrophage responses to TTFields, we profiled macrophages that were stimulated with LPS+IFN-γ (M1 polarization) or IL-4 (M2 polarization), followed by treatment with TTFields (150 kHz) for 24 hours. Flow cytometry was used to assess surface expression of F4/80 (a macrophage biomarker) and the activation markers that included major histocompatibility complex class II (MHC II), CD80, and inducible nitric oxide synthase (iNOS). A multiplexed secretion assay was used to measure the heterogeneity of the stimulated macrophages. We captured the secretion of 13 different proteins including CXCL1 (KC), IL-18, IL-23, IL-12p70, IL6, TNF-α, IL-12p40, free active TGF-β1, CCL22 (MDC), IL-10, IL-6, G-CSF, CCL17 (TARC) and IL-1β. Results: TTFields treatment polarized IL-4 stimulated M2 macrophages to the M1 phenotype, as indicated by a significant increase in the percentage of CD80+MHC IIhigh cells. Treatment with TTFields of BMDMs that were either stimulated with IFN-γ and LPS (M1 phenotype) or had remained unstimulated increased the percentage of CD80+MHC IIhigh cells to a lesser degree. Moreover, treatment with TTFields significantly increased intracellular iNOS levels in M1 polarized BMDMs. In unstimulated BMDMs, application of TTFields resulted in significant increases in secretion of the pro-inflammatory cytokines CXCL1, IL-18, IL-23, IL-12p70, TNF-α, IL-12p40, CCL22, G-CSF, CCL17 and IL-1β. Also, a similar pattern was observed in cell supernatants of M1 and M2 stimulated BMDMs after TTFields application. Conclusions: These results elucidate a novel immunoregulatory role of TTFields on macrophage polarization. Future studies will aim to focus on the mechanism governing phenotypic skewing of macrophages treated with TTFields and to explore the effect of TTFields on macrophage in the tumor microenvironment. Citation Format: Boris Brant, Tali Voloshin, Alexandra Volodin, Lilach Koren, Anat Klein-Goldberg, Efrat Zemer-Tov, Rom Paz, Moshe Giladi, Uri Weinberg, Yoram Palti. A novel immunoregulatory role of tumor treating fields (TTFields) on macrophage polarization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1692.

Abstract 279: Transient opening of the blood brain barrier by Tumor Treating Fields (TTFields)

Abstract Introduction Alternating electric fields of intermediate frequency and low intensity, known as Tumor Treating Fields (TTFields), are an effective and clinically approved approach for treatment of glioblastoma (GBM). The optimal frequency for treatment of glioma cells based on the cytotoxic response is at 200 kHz. Combination of TTFields with chemotherapy appears to be synergistic with further increase in overall survival of patients with GBM, beyond that with chemotherapy alone. The blood brain barrier (BBB) limits delivery of a majority of drugs to the brain thus limiting treatment options for GBM patients. Recent in vitro studies suggest that TTFields applied at 100 kHz can disturb the BBB. In this study, we investigated the potential use of TTFields to transiently disturb the BBB in animal models. Methods BBB permeation was tested in healthy rats subsequently to 100 kHz TTFields or sham (heat) application to the rat head. BBB permeability was analyzed by several staining agents: (1) Evans Blue (EB) that was quantified at 610 nm in brain homogenates; (2) 4 kDa TRITC-dextran (TD) that was quantified based on fluorescence intensity in brain cryosections; and (3) the MRI contrast agent Gd-DTPA. Accumulation and clearance of Gd-DTPA were tracked by serial dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). In addition, brain sections were stained for claudin-5, occludin, PECAM-1 and immunoglobulin G (IgG). BBB permeation induced by TTFields was further evaluated in rats orthotopically bearing F98 glioma cells and treated with TTFields at 100 kHz in combination with the chemotherapeutic drug paclitaxel (PTX) for a duration of 72 h. Tumor cell proliferation was assessed by Ki67 staining and the tumor volume was measured by T2 weighted MRI. Results BBB permeation of EB and TD staining agents was observed in the brains of healthy rats after TTFields application. Moreover, brain cryosections displayed delocalization of claudin-5 and occludin, but not of PECAM-1. Accumulation of IgG in the brain parenchyma was also noted. Confirming these observations, DCE-MRI post-TTFields treatment showed accumulation of Gd in the brain. Return to normal BBB integrity was detected 96 h after TTFields treatment cessation, indicating the effect was transient and reversible. In GBM-induced rats, the combination of PTX (a drug which normally does not cross the BBB) with TTFields significantly decreased tumor cell proliferation and tumor volume compared to animals treated with TTFields alone, sham alone, or sham combined with PTX. Conclusions Administration of 100 kHz TTFields to the brain of rats led to transient alterations in BBB integrity and permeability, allowing increased uptake of combination chemotherapy. These data indicate that TTFields treatment may be a feasible, novel clinical strategy for transient opening of the BBB to allow for enhanced and more effective delivery of permeable and non-permeable anticancer drugs to the brain. Citation Format: Catherine Tempel Brami, Ellaine Salvador, Almuth F. Kessler, Malgorzata Burek, Tali Voloshin, Moshe Giladi, Ralf-Ingo Ernestus, Mario Löhr, Carola Förster, Carsten Hagemann. Transient opening of the blood brain barrier by Tumor Treating Fields (TTFields) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 279.

Abstract 1064: Antiproliferative effects of Tumor Treating Fields in human mesothelioma cell lines

Abstract Despite the increasing knowledge of the biology of malignant pleural mesothelioma (MPM), patients with this diagnosis still have a poor prognosis with a 5-year survival rate of lower than 10%. The effectiveness demonstrated by Tumor Treating Fields (TTFields) in combination with chemotherapy in preclinical experiments and in MPM patients (NCT02397928) affirms TTFields as a promising therapeutic tool. Knowledge of the mechanism of action of TTFields and its interaction with chemotherapeutic agents may contribute to the design of more effective clinical trials for MPM patients. A panel of primary MPM cells was isolated from pleural effusion and/or lavages of patients' thoracic cavity, before administration of therapeutic treatment, obtaining six cell lines from the 3 types of MPM cells: epithelioid CD473 and CD484, sarcomatoid CD60 and CD432, and biphasic CD487 and CD491. RNAseq analysis revealed that the transcriptional profiles of CD473 and CD60 cells were comparable with those derived from patient biopsies of the same histotype, representing clinically relevant MPM models. Antiproliferative effects induced by TTFields alone (1.12 V/cm; 150 KHz; for 96h duration) were studied in these MPM cell lines. Cell survival and cell cycle perturbations analyses during and after treatment were performed. Consistent with clinical observations, epithelioid MPM cells demonstrated greater sensitivity to TTFields than biphasic and sarcomatoid MPM cells. Cell cycle analysis revealed that the former cell type was blocked in G2M phase and was followed by formation of polyploid cells; while the latter cell types were only slightly affected by TTFields with a general delay in all cell cycle phases. Apoptotic cells were present in all treated samples, but while epithelioid and biphasic cell death was already observed during the first 24h of treatment, sarcomatoid cells needed to be treated for at least 24h before starting apoptotic pathways. The antiproliferative effect observed in epithelioid cells was persistent even after treatment cessation, whereas biphasic and sarcomatoid cells were able to reinitiate growth upon TTFields cessation. RNAseq experiments were performed in CD473 and CD60 cells at different times during TTFields application to explore transcriptional modifications that may explicate these varied MPM cell responses. Preliminary data suggested that TTFields induced a transcriptional response already detectable at earlier time points of treatment (8h; maximum effect at 24h). The number of differentially expressed genes was higher in CD473 relative to CD60 cells (1951 in CD473 and 336 in CD60 at 24h), involving several pathways, that need to be further investigated. These data demonstrate that TTFields induce specific effects on cell proliferation in varied subsets of mesothelioma cells and provide a mechanistic rationale for future therapies and combinations with other anticancer drugs for MPM treatment. Citation Format: Monica Lupi, Federica Mirimao, Nicolò Panini, Greta Piazza, Lara Paracchini, Laura Mannarino, Sergio Marchini, Federica Grosso, Serena Penpa, Antonio Maconi, Giovanni L. Ceresoli, Maurizio D'Incalci. Antiproliferative effects of Tumor Treating Fields in human mesothelioma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1064.

Abstract 2011: Tumor treating fields induce cellular and morphologic changes including disruption of intercellular communication networks in malignant pleural mesothelioma

Abstract Tumor Treating Fields (TTFields) is a novel therapeutic strategy that uses alternating electric fields to disrupt mitosis in actively dividing cells through exertion of dielectrophoretic force and dipole alignment on microtubule subunits. However, the additional effects of TTFields on cellular morphology and communication remain unclear. Tunneling nanotubes (TNTs) are ultrafine F-actin-based protrusions that facilitate intercellular communication through cell-cell contact, including efficient transport of molecular cargo that accelerate invasive potential and chemoresistance. We hypothesized that by creating dielectrophoretic force on polar actin subunits, treatment with TTFields would lead to sustained disruption or prevention of formation of MPM TNTs. TTFields (200 kHz) were applied at 0.5 or 1.0 V/cm to VAMT and MSTO MPM cell lines using the Inovitro system (Novocure). TNT index (average # of TNTs/cell) was determined at 0, 24, 48, and 72 hours of TTFields application. At the 72 hour period, TTFields were discontinued and assessment for recovery of TNT formation was performed after an additional 24 hours. Cell viability was determined by staining with NucGreen 488 dye. We also used time-lapse microscopy with concurrent application of TTFields and the chemotherapeutic agents cisplatin and pemetrexed to analyze effects on TNTs and functional cargo transfer. Application of continuous TTFields at 1.0 V/cm, but not at 0.5 V/cm, suppressed TNT formation by 48.9% in MSTO (p=0.005). This suppression was achieved at the 48-hour time point and was independent of cell proliferation. No significant differences in TNT index were noted for VAMT. Cell viability was consistently above 95% at all time points for both cell lines at the stated frequency and intensities. Cargo transfer rates were lower in experimental groups treated with TTFields and either cisplatin, pemetrexed, or both. Here, we show that treatment with TTFields suppresses formation of TNTs between MSTO cells, but not VAMT, suggesting additional factors that may determine susceptibility to TTFields treatment. Additionally, TTFields treatment of MSTO decreased the function of TNTs in these cells, as demonstrated by lower cargo transfer rates. In sum, these data identify effects of TTFields on TNTs as a novel mechanism for this therapeutic modality. Citation Format: Akshat Sarkari, Sophie Korenfeld, Katherine Ladner, Phillip Wong, Antonia Martinez, Eyal Dor-On, Moshe Giladi, Amrinder Nain, Emil Lou. Tumor treating fields induce cellular and morphologic changes including disruption of intercellular communication networks in malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2011.

Abstract 1317: inovivo: a dedicated system for delivery of therapeutic level Tumor Treating Fields (TTFields) to mice

Abstract Purpose/objective: Tumor Treating Fields (TTFields) therapy is a noninvasive antineoplastic treatment modality that is FDA approved for treatment of glioblastoma and malignant pleural mesothelioma. TTFields are delivered to the patient continuously by 2 pairs of transducer arrays attached to the skin. In vivo TTFields experiments were so far limited due to the lack of a dedicated animal delivery system. Two main challenges are associated with TTFields application to the animal: 1) there is an absolute requirement for adequate and continuous contact between the electrodes and the animal skin throughout treatment; and 2) the wires connected to the electrodes require individual housing and limit animal movement, thus imposing stress. This work aimed to develop an in vivo system for continuous TTFields delivery to mice bearing cancer tumors in the torso or flank while addressing these challenges. Materials/Methods: To tackle the challenges and develop a viable in vivo system, several solutions were tested: 1) various electrode layouts; 2) a variety of adhesive materials; and 3) devices for preventing wire entanglement. Results: The final design of the transducer array electrodes included 2 adhesive layers, an inner layer for improved adherence, and an outer layer for securing the electrodes to the skin. Conductors were compacted into a single printed circuit cable connected to a novel electric swivel machine, that prevented cable coiling by sensing and rotating according to animal movement. These improvements resulted in fewer electrode entanglements and replacements, and thus in higher compliance (continuity) and less need for animal handling. To further reduce the impact of stress factors on the mice, a new cage was developed, that allows for 2 mice to be housed separately while still maintaining an interaction with one another. Indeed, animals treated with the inovivo system for 1-week displayed lower weight loss than animals treated with the previous non-dedicated system, indicative of reduced stress. Simulation were performed to ensure electric fields were indeed generated at the desired locations, showing above threshold TTFields intensities around the tumor for the flank subcutaneous model. For the torso orthotopic model, TTFields were shown to generate effective electric fields in the lung, liver, and pancreas, suggesting tumors in these organs may be treated effectively using the inovivo system. Conclusion: The new inovivo system provides means for continuous, 2 directions TTFields delivery to tumors in the torso or flank while minimizing stress on the mice. The inovivo system thus provides a tool for conducting TTFields experiments in mice, facilitating further in vivo studies for gaining additional mechanistical insight. The development of mouse head arrays to allow further research of the effect TTFields on glioblastoma, an application of widespread interest, is currently underway. Citation Format: Shiri Davidi, Roni Blatt, Mijal Munster, Anna Shteingauz, Yaara Porat, Adel Zeidan, Tal Marciano, Zeev Bomzon, Moshe Giladi, Uri Weinberg, Yoram Palti. inovivo: a dedicated system for delivery of therapeutic level Tumor Treating Fields (TTFields) to mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1317.

In Vivo Safety of Tumor Treating Fields (TTFields) Applied to the Torso.

BackgroundTumor 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.MethodsThe 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).ResultsNo 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.ConclusionsThis 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.

Cancer cell permeability is increased after Tumor Treating Fields (TTFields) application

Cancer cell permeability is increased after Tumor Treating Fields (TTFields) application

SS-3 Tumor Treating Fields: from the Petri dish to the patient

Tumor Treating Fields (TTFields) are a non-invasive, loco-regional, antineoplastic treatment modality targeting rapidly dividing cancer cells using low intensity, alternating electric fields at cell-type-specific intermediate frequencies (100–500 kHz). TTFields therapy is approved for the treatment of newly-diagnosed and recurrent glioblastoma as well as malignant pleural mesothelioma, following clinical trials demonstrating efficacy and a favorable safety profile. Using novel in vitro and in vivo systems for TTFields application, research activities are being conducted to extend the understanding of the underlying mechanisms of action (MoA) and to assess additional means to improve treatment outcomes. The demonstrated antimitotic effects of TTFields discerned the positive combinatorial potential of TTFields with other agents targeting the division process. Subsequent to elucidation of anti-mitotic effects, other downstream effects of TTFields include induction of endoplasmic reticulum stress, up-regulation of autophagy and cell death, thus driving immunogenic cell death. Indeed, in several preclinical models, combining TTFields with immunotherapeutics demonstrated enhanced efficacy. Recently, additional novel effects of TTFields were characterized, including inhibition of DNA damage repair responses and induction of transient and reversible permeabilization of the blood brain barrier (BBB). These new findings offer potentially innovative means to optimize treatment outcomes by combining TTFields with radiation therapy and DNA damaging agents, as well as improved delivery of impermeant agents across the BBB. These scientific findings were instrumental in advancing the clinical pipeline of TTFields, which includes conduct of ongoing trials combining TTFields with a variety of modalities, in approved indications and in other solid malignant tumor types. The aim of this talk is to describe TTFields’ preclinical research activities and tools, and to specify how these study outcomes have defined and advanced the clinical pipeline.

TAMI-04. TUMOR TREATING FIELDS (TTFIELDS) HINDER GLIOMA CELL MOTILITY THROUGH REGULATION OF MICROTUBULE AND ACTIN DYNAMICS

Abstract The ability of glioma cells to invade adjacent brain tissue remains a major obstacle to therapeutic disease management. Therefore, the development of novel treatment modalities that disrupt glioma cell motility could facilitate greater disease control. Tumor Treating Fields (TTFields), encompassing alternating electric fields within the intermediate frequency range, is an anticancer treatment delivered to the tumor region through transducer arrays placed non-invasively on the skin. This novel loco-regional treatment has demonstrated efficacy and safety and is FDA-approved in patients with glioblastoma and malignant pleural mesothelioma. TTFields are currently being investigated in other solid tumors in ongoing trials, including the phase 3 METIS trial (brain metastases from NSCLC; NCT02831959). Although established as an anti-mitotic treatment, the anti-metastatic potential of TTFields and its effects on cytoskeleton rapid dynamics during cellular motility warrant further investigation. Previous studies have demonstrated that TTFields inhibits metastatic properties of cancer cells. Identification of a unifying mechanism connecting the versatile TTFields-induced molecular responses is required to optimize the therapeutic potential of TTFields. In this study, confocal microscopy, computational tools, and biochemical analyses were utilized to show that TTFields disrupt glioma cellular polarity by interfering with microtubule assembly and directionality. Under TTFields application, changes in microtubule organization resulted in activation of GEF-H1, which led to an increase in active RhoA levels and consequent focal adhesion formation with actin cytoskeleton architectural changes. Furthermore, the optimal TTFields frequency for inhibition of invasion in glioma cells was 300 kHz, which differed from the optimal anti-mitotic frequency leading to glioma cell death of 200 kHz. The inhibitory effect of TTFields on migration was observed at fields intensities of 0.6 V/cm RMS (below the threshold of 1 V/cm RMS previously reported for cytotoxic effects). Together, these data identify discrete TTFields effects that disrupt processes crucial for glioma cell motility.

EXTH-76. THE INOVIVO SYSTEM: A NOVEL PRECLINICAL TOOL FOR IN VIVO DELIVERY OF TUMOR TREATING FIELDS (TTFIELDS)

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.

EXTH-33. VALPROIC ACID ENHANCES THE ANTI-PROLIFERATIVE EFFECT OF TUMOR-TREATING FIELDS ON PATIENT-DERIVED GLIOSARCOMA CELLS IN VITRO

Abstract BACKGROUND Valproic acid (VPA) is an anti-epileptic drug often prescribed for glioma-associated seizures. VPA is also investigated as a potential anti-proliferative cancer treatment due to its histone deacetylase inhibitor activity. Tumor-treating fields (TTFields) are an FDA-approved therapy that reduce proliferation of tumor cells by interfering with microtubule formation during mitosis. In this study, we determined whether the combination of VPA with TTFields in comparison to each treatment alone would alter proliferation or cytotoxicity in patient-derived gliosarcoma cells. METHODS Gliosarcoma cells were grown in DMEM/F12 media with 10% fetal bovine serum and gentamicin. Prior to TTFields application and VPA incubation, cells were plated on plastic coverslips (1×104cells/coverslip) and incubated overnight. TTFields were applied to cells at 200 kHz optimal frequency (field intensity of ~1.6 V/cm) for 14 days and/or incubated with VPA at concentrations of 0, 0.25, 0.5, or 1mM. Proliferation was assessed by quantifying total RNA from each coverslip (n=3–4/group). Cytotoxicity was assessed by measuring lactate dehydrogenase (LDH) released into the media (n=8/group). Groups were compared by t-tests. RESULTS RNA yields from cells treated with only VPA were unchanged compared to control. TTFields treatment alone reduced RNA yield from 14.85±4.23 to 8.92±1.39 µg (mean± SD; P< 0.05). The addition of 1mM VPA to TTFields further reduced RNA yield to 5.48±1.5 µg (P< 0.03), although VPA alone had no effect. Additionally, 1mM VPA increased media LDH from 4.00±1.3 to 6.68±1.7 AU (mean± SD; P< 0.01), while TTFields alone also increased media LDH to 5.67±0.99 AU (P< 0.02). Media LDH levels due to combined TTFields and 1mM VPA treatment was 5.86±1.2 AU (not statistically different from either alone). CONCLUSIONS In patient-derived gliosarcoma cells, TTFields and VPA individually elicited modest cytotoxicity. However, TTFields plus VPA combination treatment reduced proliferation more than TTFields alone, though VPA treatment alone did not induce an anti-proliferative effect.

Effectiveness and Safety of Tumor Treating Fields (150 kHz) plus Cisplatin or Pemetrexed Combination in Mesothelioma: in Vitro and in Vivo Findings

Malignant pleural mesothelioma (MPM) is an aggressive thoracic cancer linked to asbestos exposure with a poor prognosis and limited treatment options. Tumor Treating Fields (TTFields) are a noninvasive, antineoplastic treatment delivered locoregionally to the tumor bed via low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields. TTFields are FDA-approved as a first line therapy with pemetrexed plus platinum-based agent for unresectable MPM. TTFields with this combination was evaluated in the phase 2 STELLAR clinical trial (Ceresoli et al, Lancet Oncology 2019) in patients with unresectable MPM, demonstrating a promising median overall survival of ∼18.2 months (vs ∼12 months; historical control) without increases in systemic toxicity. Utilizing in vitro and in vivo models, we investigated the effects of TTFields alone and in combination with cisplatin or pemetrexed in MPM. Using the inovitroTM system, cells from human MPM cell lines (NCI-H2052 and MSTO-211H) were treated at various frequencies of TTFields for 72 hours. The combined treatment of TTFields with cisplatin or pemetrexed was tested by applying TTFields at the 150 kHz optimal frequency with various concentrations of cisplatin (80–5120 nM) or pemetrexed (2–4096 nM). Cell counts, clonogenic potential, and apoptosis induction were determined. TTFields (150 kHz) were applied to Fisher rats with IL-45 intrapleural tumors cells for 8 days and to C57BL/6 mice with RN5 S.C. tumors for 7 days. Tumor volume was determined using MRI. To determine treatment safety, TTFields (2–3 V/cm) were applied to the torsos of Sprague-Dawley rats at 150 kHz for 2 weeks and all major internal organs were histologically compared. The optimal TTFields frequency was 150 kHz in both NCI-H2052 and MSTO-211H human MPM cell lines. TTFields (150 kHz; 1.1 V/cm) application for 72 hours led to a 45-51% reduction in cell counts and a 46-64% additional decrease in clonogenic potential. TTFields plus cisplatin or pemetrexed significantly reduced the cell count, the clonogenic potential and induced apoptosis versus each treatment alone (P <0.0001). Tumor volume was significantly decreased in the TTFields plus pemetrexed plus cisplatin combination group versus the control group in both tumor models. Histological analysis of major internal organs showed no increase in pathological findings when TTFields were applied to the rat torso. These in vitro and in vivo data demonstrate that the optimal TTFields frequency to inhibit MPM is 150 kHz. TTFields alone showed effectiveness and safety, and combined with cisplatin or pemetrexed demonstrated enhanced effectiveness with no increased safety concerns in the torso. Findings were consistent with efficacy and safety findings in the STELLAR trial.

Tumor Treating Fields (TTFields) Hinder Cancer Cell Motility through Regulation of Microtubule and Acting Dynamics.

Simple SummaryTumor Treating Fields (TTFields), encompassing alternating electric fields within the intermediate frequency range, is an anticancer treatment delivered to the tumor region through transducer arrays placed non-invasively on the skin. Although established as an anti-mitotic treatment modality, the anti-metastatic potential of TTFields and their effect on rapid cytoskeletal dynamics during cellular motility warrant further investigation. In this study, we report that TTFields application induces changes in microtubule organization leading to interference with the directionality and robustness of cancer cell migration. We show that these changes in microtubule organization result in activation of GEF-H1/RhoA/ROCK signaling pathway, and the consequent formation of focal adhesions and changes in actin cytoskeleton architecture. Together, these results propose a novel mechanism by which TTFields induce changes in microtubule and actin organization and dynamics, thereby disrupting processes important for polarity generation and motility in cancer cells.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.

Abstract 572: In vitro and in vivo evidence for the safety and efficacy of Tumor Treating Fields (TTFields) in combination with sorafenib

Abstract Objective: Hepatocellular carcinoma (HCC) is the third cause of cancer related mortality. Sorafenib, an oral multikinase inhibitor, is approved for patients with advanced HCC, however its survival benefit is limited. Tumor Treating Fields (TTFields) therapy is an effective, anti-neoplastic treatment modality delivered via noninvasive application of low intensity, intermediate frequency, alternating electric fields. The aim of this work is to explore the potential use of TTFields alone and in combination with sorafenib as a treatment for HCC. Methods: HepG2 and Huh-7D12 HCC cells were treated with various TTFields frequencies (100-400 kHz) for 72 hours using the inovitroTM system. Efficacy of the combined treatment of TTFields and sorafenib was tested by applying TTFields at the optimal frequency together with various sorafenib concentrations. Cell counts, induction of apoptosis, and clonogenic potential were determined. Moreover, N1S1 HCC cells were injected into the left lobe of the liver of Sprague Dawley rats. After 1 week, TTFields (1.2 V/cm) and sorafenib (10 mg/kg) were applied for 6 days and tumor growth was evaluated, using MRI. Healthy rats were used to study the safety of TTFields (150 kHz) application to the abdomen. Results: The optimal frequency of TTFields was 150 kHz for both HCC cell lines. TTFields application (1.0 - 1.7 V/cm, 72 hours) at 150 kHz led to a 53-55% reduction in cell counts and to an additional reduction (65-69%) in clonogenic potential. The combination of TTFields and sorafenib led to a significant reduction in cell count (2-way ANOVA, P &amp;lt;0.05) as compared to either treatment alone. HCC tumor growth was significantly reduced in the combined group compared to the control group (student t-test, P &amp;lt;0.01). On average, the HCC tumor volume (fold-increase) in the combination treatment group (1.6-times) was significantly lower than in the control group (5.9-times, P &amp;lt;0.0001), TTFields alone group (3.3-times, P &amp;lt;0.01), and sorafenib alone group (2.3-times, P &amp;lt;0.05). Histological analysis of the KI67 proliferation marker in HCC tumors showed reduced proliferation in all treated groups. Based on preliminary analysis of autophagy marker (LC3) in tumors, we hypothesized the involvement of autophagy as 1 of the mechanisms underlying increased treatment efficacy. Safety studies did not reveal any adverse events associated with TTFields application to the rat abdomen. Conclusions: These results demonstrate that TTFields can be a safe and effective in the treatment of HCC, and that the combination with sorafenib leads to further enhancements in treatment effectiveness. Based on these results, a Phase 2 clinical trial evaluating the effects of TTFields and sorafenib treatment in patients with HCC is planned (HEPANOVA; NCT03606590). Citation Format: Shiri Davidi, Catherine Tempel-Brami, Mijal Munster, Anna Shteingauz, Einav Zeevi, Rosa Schneiderman, Tali Voloshin, Moshe Giladi, Adrian Kinzel, Uri Weinberg, Yoram Palti. In vitro and in vivo evidence for the safety and efficacy of Tumor Treating Fields (TTFields) in combination with sorafenib [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 572.