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Background Tumor Treating Fields (TTFields) are electric fields that disrupt cellular processes critical for cancer cell viability and tumor progression. Cancer cells treated with TTFields may undergo immunogenic cell death, eliciting a systemic anti-cancer immune response. Optune Lua, a TTFields device, received FDA approval for concurrent use with PD-1/PD-L1 inhibitors or docetaxel in metastatic NSCLC progressing after platinum therapy. The current study examined the direct effects of TTFields on modulating macrophage phenotypes.Methods Bone marrow-derived macrophages (BMDMs) were flushed from femurs and tibias of 8-10-week-old BALB/c or C57BL/6 mice and cultured for 7 days in the presence of granulocyte- macrophage colony-stimulating factor (GM-CSF). BMDMs were then polarized to the M1 phenotype by exposure to IFN-γ, with or without LPS, and to the M2 phenotype by IL-4 treatment. Next, TTFields (150 kHz) were applied for 24 h. Surface expression of the macrophage marker F4/80 and activation markers including CD80, major histocompatibility complex class II (MHC-II), inducible nitric oxide synthase (iNOS), CD206, and ARG-1 was examined by flow cytometry. Western blot was performed on lysates of TTFields-treated BMDMs for determining phosphorylation of GEF-H1, c-Jun, and the NF-κB subunit p65. NF-κB phosphorylation and iNOS expression were also examined in cells treated with TTFields and IFN-γ, in the presence or absence of TJ-M2010-5, an inhibitor of the toll-like receptor (TLR) component myeloid differentiation primary response 88 (MyD88).Results Exposure to TTFields elevated expression of the M1 pro-inflammatory markers CD80 and MHC-II and decreased expression of the M2 anti-inflammatory markers CD206 and ARG-1 in BMDMs. TTFields treatment also activated RhoA and induced phosphorylation of GEF-H1, c-Jun, and p65. When BMDMs were exposed to IFN-γ with TTFields, iNOS expression levels elevated to a level similar to when treated with IFN-γ with LPS. The inhibition of MyD88 prevented the induction of NF-κB phosphorylation and iNOS expression in TTFields-treated BMDMs that were exposed to IFN-γ.Conclusions TTFields promote pro-inflammatory polarization and activation of macrophages in vitro. TTFields provide a co-stimulatory signal that potentiates IFN-γ-driven M1 polarization, mimicking LPS-like activation, possibly via MyD88-dependent TLR signaling and transcriptional response via the GEF-H1/RhoA/ROCK/NF-κB axis. Future in vivo validations are needed.