Abstract 2255: Pro-inflammatory macrophage phenotype skewing induced by tumor treating fields (TTFields)
Authors: Tal Kan, Yiftah Barsheshet, Boris Brant, Tharwat Haj Khalil, Tali Voloshin, Lilach Koren, Simona Zisman-Rozen, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti
Journal: Cancer Research (2025)

Abstract Introduction: Tumor treating fields (TTFields) therapy is an approved cancer treatment based on the delivery of electric fields that disrupt cellular processes critical for cancer cell viability. TTFields have been shown to induce immunogenic cell death in cancer cells, elicit a systemic anti-cancer immune response in preclinical models, and are approved for concomitant treatment with PD-1/PD-L1 inhibitors or docetaxel in patients with metastatic non-small cell lung carcinoma after platinum-based therapy. The current study examined the possible effects of TTFields on macrophage phenotype modulation. Methods: Bone marrow-derived macrophages (BMDMs), flushed from femurs and tibias of 8-10-week-old Balb\C or C57bl/6 mice, were cultured in the presence of granulocyte-colony stimulating factor (GM-CSF) for 7 days. BMDMs were then exposed to either IFN-γ alone or IFN-γ with LPS for M1 polarization or IL-4 for M2 polarization. The polarized BMDMs were next treated with TTFields (150 kHz) for 24 h, followed by flow cytometry examination of surface expression of the F4/80 macrophage marker and activation markers including CD80, major histocompatibility complex class II (MHC-II), inducible nitric oxide synthase (iNOS), CD206, and ARG-1. Lysates of TTFields-treated macrophages were examined for RhoA activation using an appropriate kit and by western blot for phosphorylation of GEF-H1, c-Jun, and the NF-κB subunit p65. iNOS expression was also examined in cells treated with IFN-γ in the presence of TJ-M2010-5, an inhibitor of the TLR component myeloid differentiation primary response 88 (MyD88). Results: BMDMs exposed to TTFields demonstrated elevated expression of the pro-inflammatory M1 markers CD80 and MHC-II and decreased expression of the anti-inflammatory M2 markers CD206 and ARG-1. BMDMs treatment with TTFields also induced RhoA activation and GEF-H1, c-Jun, and p65 phosphorylation. TTFields treatment of BMDMs that were exposed to IFN-γ markedly elevated iNOS expression to a level similar to that of BMDMs that were exposed to IFN-γ together with LPS, while blockage of TLR signaling substantially reduced iNOS expression. Conclusions: TTFields demonstrate immunomodulatory effects, promoting pro-inflammatory macrophage polarization and activation in vitro. TTFields exhibit LPS-like activity by acting as a second signal to polarize M1 macrophages, possibly via involvement of the TLR4 signaling pathway and the transcriptional response driven by the GEF-H1/RhoA/ROCK/NF-κB pathway. Citation Format: Tal Kan, Yiftah Barsheshet, Boris Brant, Tharwat Haj Khalil, Tali Voloshin, Lilach Koren, Simona Zisman-Rozen, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Pro-inflammatory macrophage phenotype skewing induced by tumor treating fields (TTFields) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2255.