Non-temperature-induced anti-tumor effects of amplitude-modulated radiofrequency: Molecular and functional synergies with radiotherapy.

In oncology, radiofrequency electromagnetic fields (RF-EMFs) are used for hyperthermia. This method heats tissue to temperatures above 41°C. Recently, more attention has been given to non-thermal field effects, which are poorly understood. This study examines whether amplitude modulation can enhance these effects in oncology and whether it can be combined with radiation therapy (RT). The authors compared the effects of amplitude-modulated radiofrequency radiation (AMRF) and unmodulated radiofrequency radiation (RF) under strict temperature control in vitro, both alone and in combination with RT.

Two colorectal cell lines (HT29 and SW620) and two glioblastoma cell lines (U138 and U343) were exposed using an Oncotherm system operating at 13.56 MHz (AMRF was used as the band-limited 1/f noise and the modulation index was set to 50%). The cells were isothermally exposed at 42°C for 65 minutes to achieve a SAR value between 200 and 240 W/kg. The temperature was controlled via water flow and continuously monitored. To eliminate the influence of thermal effects when comparing to the 42°C control group, each exposed group was paired with an isothermal water bath control group at 37, 42, or 44°C. To simulate co-exposure to ionizing radiation (radiotherapy), the cells were irradiated with 8 Gy of gamma radiation immediately before exposure. Proliferation, apoptosis (Annexin V), and necrosis (YOYO-3) were assessed over 96 hours using live-cell imaging. We also performed a transcriptome analysis using RNA sequencing for the SW620 and U138 cell lines.

The cytotoxicity of the exposure combinations depended on the cell line. For the HT29 cell line, the combination of RT and AMRF was the most effective at combating tumor cells. Compared to the 42°C control group, there was a statistically significant decrease in cell proliferation, while apoptosis and necrosis increased significantly. The increase in apoptosis was greater than that observed in the suprathermal 44°C control group. AMRF and RF+RT did not have a statistically significant effect on proliferation. However, RF alone increased proliferation, thereby having an antitherapeutic effect. For the SW620 cell line, necrosis increased significantly above the suprathermal control level in all four treatment scenarios. Cell proliferation decreased only under the RF+RT combination. For the U138 cell line, only AMRF and RF+RT influenced all three parameters of cytotoxicity (proliferation, apoptosis, and necrosis) in a manner conducive to tumor regression. Neither RF alone nor AMRF+RT had a statistically significant effect on proliferation. For the U343 cell line, AMRF monotherapy was the most effective, with all three parameters confirming cytotoxicity. All other combinations were either completely ineffective or significantly less effective. At the transcriptomic level, which was examined only in the SW620 and U138 cell lines, an even more pronounced cell line-specific response was observed. In SW620 cells, AMRF triggered the strongest response, peaking immediately after exposure and subsiding after 24 hours. RF and RF+RT had only moderate effects, and AMRF+RT fell somewhere in between. Compared to the hyperthermia control, AMRF activated gene expressions that heat alone did not trigger. These included metal detoxification, mitochondrial respiration, oxidative phosphorylation, amino acid starvation, and eIF2-mediated stress responses. U138, on the other hand, showed hardly any changes in gene expression despite strong phenotypic cell death responses.

According to the authors, AMRF and AMRF+RT exert a biological, non-thermal cytotoxic effect on tumor cell lines. They favor a mechanism of field-induced rectification at ion channels. The radiofrequency field is rectified by ion channels, which generates a sustained direct current (DC) voltage across the membrane. This drives ion fluxes, particularly calcium influx, which could trigger apoptosis and necrosis without transcriptional changes. Since glioblastoma cells have altered "oncochannels" (Kir4.1, KCa3.1, and TRPV4), this effect may be amplified in U138 cells. This could explain the discrepancy between phenotype and transcriptome.

Editor’s note:

The design of the constant temperature water bath appropriately separates RF and thermal effects. The fact that AMRF alone induces more severe necrosis than the suprathermal control suggests that a thermal mechanism of action is unlikely. The authors present their ion channel mechanism as a falsifiable hypothesis rather than a confirmed finding. However, the lack of blinding in the analysis and in the experimental setup provided by the device manufacturer is concerning. Caution should be exercised when applying the results of this study to non-ionizing radiation protection. The effective SAR value of 200 to 240 W/kg is orders of magnitude higher than the exposure one receives from everyday Wi-Fi or cellular networks. A cytotoxic effect at these intensities does not speak to the subtle effects of everyday fields. Nevertheless, evidence of non-thermal RF effects is relevant. (RH)