Low-frequency transcranial electric field stimulation reprograms the immunosuppressive microenvironment and suppresses lung cancer brain metastasis

Background: Brain metastasis (BrM) from non-small cell lung cancer (NSCLC) is driven by a profoundly immunosuppressive tumor microenvironment (TME) characterized by M2-polarized tumor-associated macrophages (TAMs) and microglia, T-cell exhaustion, and programmed cell death (PD) signaling, which jointly limit the efficacy of current therapies. Transcranial electric field stimulation (EFS) has emerged as a potential neuromodulatory strategy to regulate the TME of brain cancers. Nonetheless, its impact on the BrM TME, optimal stimulation parameters, and functional mechanisms remain unclear. Materials and methods: This preclinical investigation utilized an integrated translational platform comprising patient-derived NSCLC BrM tumor organoids ( n = 8), an immunocompetent syngeneic Lewis lung cancer (LLC) BrM mouse model, and in vitro organotypic brain slice cultures. Low-frequency EFS at 2–15 and 75 Hz was applied, and its efficacy was compared with tumor-treating fields (TTF; 150 kHz) and sham treatment. TME phenotypes were characterized by immunofluorescence, flow cytometry, Western blotting, and enzyme-linked immunosorbent assay (ELISA). Mechanistic studies were conducted in TAMs using calcium chelation, ion channel blockade, and protein kinase C (PKC) inhibition. Results: Among all EFS regimens tested, 75 Hz EFS significantly reduced BrM bioluminescence, tumor cross-sectional area (a 56% reduction vs. controls), and Ki-67 proliferation index, and prolonged survival in LLC BrM mice, whereas 2–15 Hz EFS had minimal effect and TTF showed only modest tumor control. 75 Hz EFS shifted TAMs from an M2-like CD206 + phenotype to an M1-like inducible nitric oxide synthase (iNOS)⁺/CD86⁺ phenotype, increased IL-1β and tumor necrosis factor (TNF)-α, and suppressed Janus kinase (JAK)2–signal transducer and activator of transcription (STAT3), IL-6, IL-10, and transforming growth factor (TGF)-β. It also reduced PD-1⁺ CD8⁺ exhausted T cells and PD-L1 expression in the BrM TME. These effects were recapitulated in patient-derived BrM tumor organoids and BrM-conditioned macrophage and microglia cultures, and were abrogated by calcium chelation, PKCα/β inhibition, or systemic dexamethasone. Conclusion: In this preclinical investigation, low-frequency transcranial EFS at 75 Hz represents a promising neuro-immunomodulatory strategy that reprograms the NSCLC BrM TME, restores anti-tumor immunity, and restrains tumor progression. Further clinical trials are required to validate the efficacy of low-frequency transcranial EFS in NSCLC BrM.