Tumor-Treating Fields (TTFields) Suppress Tunneling Nanotube Formation in Malignant Mesothelioma

Abstract

Tumor Treating Fields (TTFields) have emerged as an effective therapeutic modality through application of alternating electric fields, which exert dipole alignment and dielectrophoretic force to induce an anti-mitotic effect. TTFields is approved for use in glioblastoma, following exposure to ionizing radiation concurrent with chemotherapy. Therapeutic targets of TTFields include mitotic activity but could also affect other cellular processes essential to cancer cell viability, such as DNA damage repair. Tunneling nanotubes (TNTs) are actin-based membrane protrusions that act as bridges of communication and transport of molecular signals and organelles that stimulate invasion and drug resistance. The goal of this study was to examine the ability of TTFields to disrupt or prevent the formation of TNTs in malignant pleural mesothelioma. The inovitro system was used to generate TTFields (0.5 V/cm; 200 kHz) for in vitro study of malignant mesothelioma cell lines (VAMT and MSTO-211H). Cells were seeded in ceramic dishes and were allowed to adhere overnight prior to initiation of continuous TTFields. The average # of TNTs/cell was determined at 24, 48, and 72 hours of TTFields application, and cell viability was quantified using Trypan Blue staining. TUNEL assays were also performed at each time point to verify DNA damage. In addition, we examined the effects of complete cell cycle inhibition on TNTs using the compound AZD 5438 (Tocris Biosciences) with or without TTFields. Separately, TTFields either alone or in combination were used to treat cells along with chemotherapeutic drugs cisplatin (range 0-10 μM) or pemetrexed (0-512 nM) and assessed using colony formation assays. Application of TTFields suppressed TNT formation in both cell lines by 60% over a 72-hour period (p=0.0196). This suppression was achieved by the 24-hour timepoint and maintained over the 72 hours. Assessment of cell viability showed 65% viability of MSTO by 48 hours. Full inhibition of the cell cycle stimulated a significantly higher number of TNTs. The addition of cisplatin or pemetrexed to TTFields significantly decreased the cell count and resulted in enhanced treatment efficacy. TTFields suppress formation of TNTs in malignant mesothelioma, and lead to enhanced treatment efficacy when combined with chemotherapeutic drugs cisplatin and pemetrexed. The rise in TNTs following cell cycle inhibition is a potential cellular stress response to cancer-directed treatment, consistent with our prior studies. Adding TTFields concurrently to chemotherapy may stem this cellular stress response, resulting in suppression of cell growth and TNT-mediated intercellular communication. Disrupting cellular communication networks represents a novel therapeutic strategy for mesothelioma, glioblastoma, and other invasive cancers.