Enhancing Cancer Cell Membrane Permeability by Application of Tumor Treating Fields (TTFields)

Abstract

<h3>Purpose/Objective(s)</h3> Tumor Treating Fields (TTFields) are alternating electric fields that display anti-mitotic effects on cancer cells. TTFields therapy is approved for treatment of glioblastoma (GBM) and unresectable malignant pleural mesothelioma. It has recently been shown that treatment of GBM cells with TTFields increases cell membrane permeability. The current study aimed to further explore this effect, testing the potential of TTFields to facilitate cellular accumulation of the anticancer agent doxorubicin (DOX) <i>in vitro</i> and <i>in vivo</i>. <h3>Materials/Methods</h3> Breast mammary carcinoma (4T1), breast adenocarcinoma (MCF-7), uterine sarcoma (MES-SA), and lung fibroblast (MRC-5) cell lines were treated with TTFields for 72 h at an intensity of 1.7 V/cm RMS across a range of frequencies (100-400 kHz). Membrane permeability was determined by 7-aminoactinomycin D (7-AAD) intracellular accumulation, and cytotoxicity was examined by cell counts, both measured by flow cytometry. TTFields at the frequency inducing highest permeability were applied together with DOX to DOX-sensitive and matched DOX-resistant 4T1 cells, followed by flow cytometry measurements of intracellular DOX accumulation and cytotoxicity. Mice orthotopically inoculated with 4T1 cells were treated with TTFields for 72 h, with DOX injected 24 h before end of treatment. DOX florescence was measured using flow cytometry for detection at the single-cell level and <i>in vivo</i> imaging system (IVIS) for whole tumor assessment. <h3>Results</h3> While TTFields increased 7-AAD intracellular accumulation in 4T1, MCF-7, and MES-SA cancer cells, there was no such effect when TTFields were applied to the non-cancer MRC-5 cells. Maximal permeability was induced in 4T1 cells with 300 kHz TTFields, whereas highest TTFields-induced cytotoxicity was observed at 150 kHz. TTFields application allowed for DOX accumulation in DOX-resistant cells to the same extent as in DOX-sensitive cells. TTFields delivery also sensitized both cell types to DOX, with cytotoxicity observed at low drug concentrations. <i>In vivo</i>, DOX accumulation was elevated by 2- to 3-fold in tumors isolated from mice treated with TTFields relative to control mice. <h3>Conclusion</h3> TTFields elevated the permeability of 4T1 breast cancer cells, allowing for enhanced intracellular accumulation of DOX and improved drug efficacy, even in cells resistant to DOX. TTFields-induced accumulation of DOX was also demonstrated <i>in vivo</i>.