Abstract 1738: Sensitizing cancer cell to doxorubicin by tumor treating fields (TTFields)-induced, elevated membrane permeability
Authors: Koltun Bella , Voloshin Tali , Kan Tal , David Cfir , Koren Lilach , Porat Yaara , Volodin Alexandra , Kaynan Noa , Klein-Goldberg Anat , Paz Rom , Brant Boris , Barsheshet Yiftah , Zemer-Tov Efrat , Haber Adi , Giladi Moshe , Weinberg Uri , Palti Yoram 
Published: April 2023

Abstract
Background: Tumor Treating Fields (TTFields) are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression, ultimately leading to cell death. In addition, application of TTFields to glioblastoma cells has been shown to increase cell membrane permeability. The aim of the current study was to examine whether this mechanism is relevant in other tumor types, and may be leveraged to facilitate cellular internalization of the anticancer agent doxorubicin (DOX).
Methods: Lung fibroblast (MRC-5), brain endothelial (HBMVEC), and several cancer cell lines - breast mammary carcinoma (4T1), breast adenocarcinoma (MCF-7), and uterine sarcoma (MES-SA) - were treated with TTFields (100-400 kHz, 1.7 V/cm RMS) using the inovitroTM system. Intracellular accumulation of 7-aminoactinomycin D (7-AAD) was measured to determine membrane permeability, and cell counts were examined to evaluate cytotoxicity. To examine the kinetics and reversibility of the phenomenon, 7-AAD was added at different time points following TTFields application initiation or termination. TTFields were also applied together with DOX to DOX-sensitive and matched DOX-resistant 4T1 cells, followed by flow cytometry examination of DOX accumulation and cytotoxicity measurements. Mice orthotopically inoculated with 4T1 cells were treated with TTFields for 72 h and concomitant DOX injected 24 h before treatment cessation. DOX florescence was measured by flow cytometry in single-cell tumor suspension and by whole tumor in vivo imaging system (IVIS).
Results: TTFields increased intracellular accumulation of 7-AAD specifically in the cancer cell lines, with no such effect seen on the non-cancer MRC-5 and HBMVEC cells. In 4T1 cells, maximal TTFields-induced cellular permeability was recorded with 300 kHz TTFields, whereas highest TTFields-induced cytotoxicity was observed at 150 kHz. TTFields application allowed for DOX accumulation to the same extent in both DOX-resistant and DOX-sensitive cells, and sensitized both cell types to DOX cytotoxicity. In vivo, a 2- to 3-fold higher DOX accumulation was seen in tumors isolated from mice treated with TTFields relative to control mice.
Conclusions: TTFields elevated cancer cell permeability, resulting in enhanced cell accumulation of DOX and improved drug efficacy, even in DOX resistant cells. TTFields-induced accumulation of DOX was also demonstrated in vivo.
Citation Format: Bella Koltun, Tali Voloshin, Tal Kan, Cfir David, Lilach Koren, Yaara Porat, Alexandra Volodin, Noa Kaynan, Anat Klein-Goldberg, Rom Paz, Boris Brant, Yiftah Barsheshet, Efrat Zemer-Tov, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Sensitizing cancer cell to doxorubicin by tumor treating fields (TTFields)-induced, elevated membrane permeability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1738.