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.
Resumen El mesotelioma maligno es un tumor poco frecuente pero está asociado a una alta mortalidad y se considera por tanto un tumor muy agresivo con una incidencia que va aumentando en todo el mundo. Se origina más frecuentemente en las membranas serosas pleurales, seguido del peritoneo, pericardio y la túnica vaginalis,suponiendo los mesoteliomas peritoneales un 10-15% de todos los casos de mesotelioma. La TAC es la prueba diagnóstica de elección pero para el diagnóstico definitivo es necesaria la inmunohistoquímica. El tratamiento del mesotelioma peritoneal maligno precisa un tratamiento multimodal por lo que es necesario un comité multidisciplinar. El tratamiento estándar para pacientes seleccionados con mesotelioma maligno incluye cirugía citorreductora y quimioterapia hipertérmica intraperitoneal, mientras que los mesoteliomas irresecables se pueden beneficiar de tratamiento combinado con quimioterapia, radioterapia e inmunoterapia. Actualmente hay varios ensayos clínicos para conocer mejor las características de dicha neoplasia.
Introduction: Malignant pleural mesothelioma (MPM) is an aggressive thoracic cancer with a poor prognosis and limited treatment options. Tumor Treating Fields (TTFields) are a noninvasive, locoregionally, antineoplastic treatment, delivering low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields, that has demonstrated a promising median overall survival in patients with MPM without increases in systemic toxicity (STELLAR clinical trial). Accordingly, TTFields with pemetrexed and a platinum-based chemotherapy agent received FDA-approval as first line therapy for MPM. While efficacy of TTFields for MPM treatment is well-established, the underlying mechanism of action needs further elucidation. Methods: Human MPM cell lines (NCI-H2052 and MSTO-211H) were treated using various TTFields frequencies to assess the most effective frequency. The effect of optimal frequency TTFields on levels of DNA double strand breaks (DSB) was examined by fluorescent microscopy detection of γH2AX foci, and the levels of DNA damage repair proteins was evaluated by immunoblotting. The combined cytotoxic effect of TTFields with cisplatin or pemetrexed was tested in vitro, and efficacy of TTFields in combination with both chemotherapeutic agents was examined in C57BL/6 mice injected subcutaneously with RN-5 cells, by measuring tumor volume and through detection for DNA damage within the tumor. Results: The optimal TTFields frequency in both MPM cell lines was 150 kHz, demonstrating significant cytotoxicity and increases in formation of DNA DSB. These effects were associated with reduced expression of proteins from the Fanconi Anemia (FA) repair pathway for DNA repair - FANCA, FANCD2, FANCJ, and BRCA1. Co-treatment of TTFields with cisplatin or pemetrexed significantly increased treatment efficacy versus each treatment alone, with an additive effect shown by the TTFields-pemetrexed combination, and a tendency towards synergism displayed for TTFields-cisplatin co-administration. In animal models, tumor volume fold increase was significantly decreased for co-treatment with TTFields and chemotherapy (cisplatin + pemetrexed) versus the control, showing also increased DNA damage within the tumor bed in comparison to control or chemotherapy alone. Conclusions: The results presented here demonstrate that the efficacy of TTFields for treatment of MPM is associated with reduced expression of FA pathway proteins and increased DNA DSB. This effect may account for the synergistic effect seen for TTFields-cisplatin co-treatment, as cisplatin is known to cause DNA damage that requires the FA pathway for repair. This research provides further insights on the mechanism of action of TTFields in MPM, a treatment already approved against this malignancy. Citation Format: Helena Mumblat, Antonia Martinez, Ori Braten, Mijal Munster, Eyal Dor-On, Rosa S. Schneiderman, Yaara Porat, Tali Voloshin, Shiri Davidi, Roni Blatt, Anna Shteingauz, Catherine Tempel-Brami, Einav Zeevi, Carolina Lajterer, Yuval Shmueli, Shiri Danilov, Adi Haber, Moshe Giladi, Adrian Kinzel, Uri Weinberg, Yoram Palti. Efficacy of Tumor Treating Fields (TTFields) in mesothelioma is associated with reduced capacity for DNA damage repair [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 1186.
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