Non-Temperature-Induced Antitumor Effects of Amplitude-Modulated Radiofrequency: Molecular and Functional Synergies with Radiotherapy

Background/Objectives: Amplitude-modulated radiofrequency (AMRF) fields have emerged as promising non-thermal strategies in oncology. While conventional hyper- thermia (HT) relies on thermal stress, the biological impact of AMRF, particularly in combination with radiotherapy (RT), remains insufficiently characterized. Methods: We assessed RF and AMRF, alone or with RT, using phenotypic analyses of proliferation, apop- tosis, and necrosis across four cancer cell lines (HT29, SW620, U343, U138). Transcriptomic profiling with Kyoto Encyclopedia of Genes and Genomes (KEGG), GO:BP and Reactome enrichment was performed in SW620 and U138 cells, selected for their strong phenotypic responses. Results: AMRF generally triggered broader cytotoxic effects than RF or HT, with AMRF+RT exhibiting the strongest necrotic responses. In SW620 cells, AMRF induced extensive transcriptional reprogramming involving immune modulation, extracellular matrix remodeling, and cell cycle regulation, whereas RF alone exhibited narrower and delayed effects. In contrast, U138 cells showed elevated apoptosis and necrosis but limited transcriptional changes, suggesting post-transcriptional or metabolic mechanisms rather than large-scale gene regulation. Conclusions: AMRF and AMRF+RT emerge as promising non-thermal anticancer modalities, expanding the biological impact of RF-based treatments and setting the grounds for further investigation in mechanistic and translational studies.