Abstract 1305: Tumor Treating Fields (TTFields) promote a pro-inflammatory phenotype in macrophages

Introduction: Tumor Treating Fields (TTFields) are low intensity (1-3 V/cm), intermediate frequency (100-500 kHz), alternating electric fields that are delivered non-invasively to tumors. TTFields therapy is approved in the US and Europe for the treatment of patients with glioblastoma or mesothelioma. In addition to an established anti-mitotic mechanism of action, previous studies have shown that TTFields-induced cell death stimulates anti-tumor immunity and promotes the maturation of dendritic cells. In the current research, the effect of TTFields on the polarization of unstimulated macrophages and the phenotypic regulation of M1 and M2 macrophages was investigated. Methods: Bone marrow-derived macrophages (BMDMs) were generated from the femurs and tibias of 5-8-week-old Balb\C mice. TTFields (150 kHz) were applied for 24 h to unstimulated (M0 phenotype) BMDMs and BMDMs stimulated with LPS+IFN-γ (M1 polarization) or IL-4 (M2 polarization). Flow cytometry was used to identify surface expression of the macrophage biomarker F4/80 and activation markers CD80, major histocompatibility complex class II (MHC II), inducible nitric oxide synthase (iNOS), CD206, and ARG-1. Multiplexed secretion assays were conducted to quantify 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 as to examine the heterogeneity of the stimulated macrophages. Results: The percentage of cells expressing the pro-inflammatory M1 markers CD80+ and MHC IIhigh was significantly increased following application of TTFields to polarized (M1 or M2) or unpolarized BMDMs, while expression of the M2 markers CD206 and ARG-1 was significantly decreased. A pro-inflammatory secretion pattern, with increased levels of CXCL1, IL-18, IL-23, IL-12p70, TNF-α, IL-12p40, CCL22, G-CSF, CCL17 and IL-1β was observed in cell supernatants of M1 and M2 stimulated BMDMs, and unstimulated M0 BMDMs, following delivery of TTFields. Taken together, TTFields polarized unstimulated BMDMs to the M1 phenotype, and induced phenotype skewing of M2 polarized BMDMs to the M1 phenotype. Conclusions: TTFields therapy displays a novel immunoregulatory role in macrophage polarization and promotes a pro-inflammatory phenotype. Future investigations will focus on defining the underlying mechanism of this phenotypic skewing. Citation Format: Yiftah Barsheshet, Boris Brant, Tali Voloshin, Alexandra Volodin, Lilach Koren, Bella Koltun, Anat Klein-Goldberg, Efrat Zemer-Tov, Tal Kan, Rom Paz, Moshe Giladi, Uri Weinberg, Yoram Palti. Tumor Treating Fields (TTFields) promote a pro-inflammatory phenotype in macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1305.