Intensity‑ and time‑dependent anticancer effects of alternating electric fields in non‑small cell lung cancer cell lines

Introduction

Non-small cell lung cancer (NSCLC) accounts for 85–87% of all lung cancer cases and recent studies indicated that 30–50% of patients present with distant metastases at the time of diagnosis (1,2). Although immune checkpoint inhibitors and targeted therapies have extended survival in selected patient groups, long-term treatment efficacy remains limited by the emergence of drug resistance, immune escape and tumor heterogeneity (3). In advanced or metastatic NSCLC, conventional drug-based regimens often fail to achieve sufficient responses, underscoring the need for non-pharmacological, non-invasive therapeutic strategies that can complement existing modalities.

Against this background, alternating electric fields (AEF), commonly referred to as tumor treating fields (TTFields), have gained increasing attention (4). TTFields deliver low-intensity AEF within the 100–300 kHz range to tumor tissues, selectively targeting dividing cells. By disrupting microtubule polymerization, impairing mitotic spindle assembly and increasing dielectrophoretic forces on intracellular organelles, TTFields induce mitotic catastrophe and apoptosis in actively proliferating tumor cells (5). Since the mechanism of action is physical rather than molecular, TTFields are not affected by conventional resistance mechanisms and can be combined seamlessly with existing anticancer therapies (6,7).

Clinically, TTFields have demonstrated efficacy in glioblastoma (GBM) and malignant pleural mesothelioma, leading to Food and Drug Administration (FDA) approvals in both indications (8,9). Based on the results of the LUNAR phase III trial published in 2024, Optune Lua subsequently received FDA approval in metastatic NSCLC in combination with immune checkpoint inhibitors or docetaxel following platinum-based chemotherapy (10). This notable event positions TTFields as an established therapeutic option for NSCLC and further highlights the need to understand tumor-specific electrical responses and optimize TTFields parameters for this cancer type in the future.

Previous studies have indicated that TTFields efficacy is governed by several biophysical parameters, including field intensity and exposure duration, with even small parameter variations producing non-linear amplification of mitotic disruption (11–13). In clinical practice, prolonged daily wear time (typically ~18 h per day) is required to maximize therapeutic benefit; however, long-term use is frequently challenged by skin irritation, discomfort associated with transducer arrays and reduced patient compliance (14). These limitations have created a growing clinical interest in identifying intensity-duration combinations capable of maintaining antitumor efficacy while reducing total wear time. Nevertheless, in NSCLC, quantitative studies that systematically evaluate the interaction between field intensity and exposure duration (dose-schedule dependency) remain scarce (15). Therefore, in the present study, a multilayered profiling of both short-term (48 h) and long-term (7-day) responses to TTFields in NSCLC cell lines (H460 and A549) was performed to elucidate the relationship between field intensity, exposure duration and antitumor efficacy. This approach provides quantitative and functional insight into TTFields response patterns in NSCLC, which may potentially serve as a basis for future investigations of parameter-dependent effects.

Materials and methods

Cell culture

H460 and A549 NSCLC cell lines were obtained from the Korean Cell Line Bank (cat. nos. 30096 and 10185, respectively; Korean Cell Line Research Foundation). Cells were cultured in RPMI-1640 medium (Welgene, Inc.) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Thermo Fisher Scientific, Inc.). Cultures were maintained at 37°C in a humidified 5% CO2 incubator. For routine maintenance, cells were seeded at an initial density of 5×105 cells and subcultured every 3–4 days. All experiments were performed using healthy cells in the logarithmic growth phase.

TTFields setup

AEF were generated using a custom-built system consisting of a function generator (cat. no. AFG-2112; Good Will Instrument Co., Ltd.), a high-voltage amplifier (cat. no. A303; A.A. Lab Systems Ltd.) and a sterilized parallel-plate electrode assembly. Prior to cell seeding, electrode wires were sterilized, fully air-dried and attached to culture dishes inside a biosafety cabinet. The assembled dish-wire system was then UV-sterilized for at least 30 min. Two electrodes were positioned 3 cm apart at the center of the dish to ensure uniform field distribution. A sine-wave signal at 150 kHz was used for all experiments.

Electric field measurement and verification

The applied electric field intensity (V/cm) was experimentally measured and verified to ensure accurate and reproducible field delivery. Voltage measurements were performed using an OWON HDS1022M-N digital oscilloscope (OWON Technology, Inc.) equipped with a Keysight N2791A 25 MHz differential probe (Keysight Technologies, Inc.). The probe tips were positioned at the center region between the insulated electrode wires with a fixed inter-probe distance of 1 cm, corresponding to the area where cells were cultured. The measured voltage difference was used to calculate the electric field intensity (V/cm) and measurements were repeated to confirm consistency across experimental conditions.

TTFields treatment

To assess the intensity-time dependency of TTFields, cells were exposed to one of three conditions: Protocol (P)1, 0.4 V/cm for 24 h; P2, 0.8 V/cm for 6 h; or P3, 1.6 V/cm for 3 h. These conditions represent progressively higher field intensities with proportionally shorter exposure durations. Control groups were cultured under identical conditions without electric field stimulation. All treatments were performed under carefully controlled thermal conditions. During preliminary testing, high-intensity TTFields exposure was associated with a modest temperature increase (~1°C) under standard incubator settings. To minimize potential Joule heating effects, incubator temperature settings were adjusted such that the effective culture temperature was maintained at 37°C across all experimental conditions. Medium temperature was monitored during electric field exposure using a non-contact infrared thermometer (FLUKE 62 MAX; Fluke Corporation), which was employed to avoid physical disturbance of the culture environment and interference with the electric field setup. For detailed temperature monitoring experiments presented in Fig. S1, temperature was recorded at 30-min intervals over a 3-h period using a contact-type data logger thermometer (model TES-1384; TES Electrical Electronic Corp.). The temperature probe was fixed at the center of the culture dish and positioned ~1.5 cm away from the electrode surface. Representative temperature profiles before and after thermal compensation are provided in Fig. S1.

Cell imaging

At 48 h after TTFields exposure, overall cell density and cluster formation patterns were examined using an inverted phase-contrast microscope (ECLIPSE TS100; Nikon Corporation) equipped with a digital camera (IMTcam3; i-Solution, Inc.). Representative images were captured to document qualitative morphological differences across treatment groups.

Cell viability assessment

At 48 h after TTFields exposure, cells were harvested using trypsin-EDTA and viable cell numbers were determined by trypan blue exclusion. Equal numbers of viable cells from each group were re-seeded into 96-well plates (500–1,000 cells per well) and allowed to attach overnight. The following day, the medium was replaced with fresh medium containing WST-8 reagent (Quanti-MAX™ WST-8 Cell Viability Assay Kit; cat. no. QM2500; Biomax) and plates were incubated for 1 h at 37°C. Absorbance at 450 nm was measured using a microplate reader (PHOmo; Autobio Labtec Instruments). For direct viability assessment, a portion of harvested cells was stained with trypan blue and counted manually using a hemocytometer (DHC-N01-5; INCYTO). All experiments were performed in triplicate.

Colony formation assay

After TTFields exposure, cells were harvested and seeded into 6-well plates at low density (500–1,000 cells per well). Cells were cultured in complete medium without soft agar and maintained for 10–14 days at 37°C in a humidified incubator with 5% CO2, with the medium replaced every 3–4 days. Colonies were washed with phosphate-buffered saline (PBS), fixed with 100% methanol for 15 min at room temperature (20–25°C) and stained with 0.5% crystal violet for 20 min at room temperature. Plates were then rinsed with distilled water and air-dried. After drying, colonies containing ≥50 cells were counted by visual inspection of the entire well surface. Survival fraction=(colonies in treated group)/(colonies in control group). All experiments were conducted in triplicate.

Migration assay

Cell migration was evaluated using Transwell chambers (8-µm pore size; Corning, Inc.). After TTFields treatment, 4,000 cells in 200 µl of serum-free medium were seeded into the upper chamber and complete medium containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) was added to the lower chamber as a chemoattractant. Cells were incubated for 18–24 h at 37°C in a humidified incubator with 5% CO2. Following incubation, the upper chamber medium was removed by gentle pipetting and non-migrated cells on the upper membrane surface were carefully removed with PBS. Migrated cells on the lower surface were fixed with 100% methanol for 10–15 min at room temperature and stained with 0.5% crystal violet for 20 min at room temperature. Representative images of stained membranes were captured using a light microscope. All experiments were performed in triplicate.

Apoptosis analysis by Annexin V/propidium iodide (PI) staining

Apoptosis was assessed using an Annexin V-FITC Apoptosis Detection Kit (BioVision, Inc.). H460 and A549 cells were treated under the indicated experimental conditions. After treatment, cells were harvested, washed twice with cold PBS and resuspended in 1X binding buffer (provided in the kit). Cells were stained with Annexin V-FITC and PI according to the manufacturer's instructions and incubated for 15 min at room temperature in the dark. The staining mixture contained 5 µl Annexin V-FITC and 5 µl PI per 100-µl cell suspension. Flow cytometric analysis was performed using a BD FACSAria™ flow cytometer (BD Biosciences). At least 10,000 events per sample were acquired. Annexin V-FITC and PI fluorescence signals were detected using a 488 nm excitation laser and appropriate emission filters. Apoptotic cell populations were classified as viable cells (Annexin V−/PI−), early apoptotic cells (Annexin V+/PI−), late apoptotic or secondary necrotic cells (Annexin V+/PI+), and necrotic cells (Annexin V−/PI+). The percentage of total apoptotic cells, defined as the sum of early and late apoptotic populations, was calculated for quantitative analysis. Data were analyzed using FlowJo software (version 10; BD Biosciences).

Western blotting analysis

Cells were collected after TTFields exposure and washed with cold PBS. Total protein was extracted using RIPA buffer (Thermo Fisher Scientific, Inc.) containing protease and phosphatase inhibitors, incubated on ice and centrifuged at 12,000 × g for 15 min at 4°C. Protein concentrations were determined using a BCA assay. Equal amounts of protein (20–30 µg) were denatured at 95°C for 5 min and separated on 12% SDS-polyacrylamide gels, followed by transfer onto PVDF membranes. Membranes were blocked with 5% BSA in TBST (0.1% Tween-20) for 1 h at room temperature and incubated overnight at 4°C with primary antibodies against poly (ADP-ribose) polymerase (PARP; cat. no. 9542; Cell Signaling Technology, Inc.) and β-actin (cat. no. 3700; Cell Signaling Technology, Inc.) (both diluted 1:1,000). After washing, membranes were incubated with HRP-conjugated secondary antibodies (cat. nos. rabbit IgG #7074; mouse IgG #7076; Cell Signaling Technology, Inc.; diluted 1:1,000) for 1 h at room temperature. PARP was detected using Atto Ultimate Sensitivity ECL (cat. no. 34579; Thermo Fisher Inc.) and β-actin was detected using standard ECL. Images were acquired using an iBright CL750 imaging system (Thermo Fisher Scientific, Inc.).

Statistical analysis

Statistical analyses were performed using GraphPad Prism software (version 8.0; Dotmatics). For comparisons between two groups, an unpaired two-tailed Student's t-test was used. For comparisons involving more than two experimental groups, one-way analysis of variance was applied, followed by Tukey's multiple comparisons post hoc test to account for familywise error rate inflation. Data are presented as mean ± standard deviation from at least three independent experiments. P<0.05 was considered to indicate a statistically significant difference.

Results

TTFields disrupt early morphology and reduce short-term cell viability in NSCLC cells

Phase-contrast imaging performed after 2 days of treatment revealed clear intensity-dependent morphological alterations in both H460 and A549 cells following TTFields exposure. Compared with untreated controls, TTFields-treated cells exhibited reduced monolayer confluence (Fig. 1A). These disruptions were most apparent at 0.8 and 1.6 V/cm, where sparsely populated regions and diminished cellular clustering were prominent. Consistent with these morphological changes, direct cell counting at the 2-day experimental endpoint demonstrated a proportional reduction in viable cell numbers across all TTFields conditions (Fig. 1B). Both H460 and A549 demonstrated significantly decreased viability at endpoint, with the most notable reduction observed at 1.6 V/cm, indicating a progressively stronger inhibitory effect with increasing field intensity.

Figure 1. Early morphological alterations and reduced cell viability in H460 and A549 cells following TTFields exposure. (A) Representative phase-contrast images acquired at the 48 h endpoint (maximum active exposure of 24 h/day) illustrate relative changes in monolayer confluence, cellular morphology and adherence following TTFields treatment. These images are presented to highlight qualitative differences among conditions and are not intended to reflect absolute cell numbers. (B) Quantitative analysis of viable cell numbers at the 48 h endpoint was performed by direct cell counting across multiple independent fields and experiments. The analysis demonstrates a reduction in cell number under TTFields exposure, with the strongest inhibitory effect observed at 1.6 V/cm. Scale bar, 250 µm. Data are presented as mean ± SD. *P<0.05 and ***P<0.001. TTFields, tumor treating fields.

TTFields reduce long-term clonogenic survival, with similar inhibitory effects at 0.4 and 0.8 V/cm

Colony formation assays revealed that TTFields markedly impaired the clonogenic capacity of both H460 and A549 cells (Fig. 2A). Compared with untreated controls, all TTFields-treated groups exhibited reduced colony numbers and smaller colony clusters. Notably, 0.4 and 0.8 V/cm conditions produced similar levels of inhibition, indicating a similar extent of colony reduction and diminished cluster formation. By contrast, 1.6 V/cm resulted in the most notable suppression, with sparse or nearly absent colonies in both cell lines. Survival fraction analysis performed in three independent experiments consistently demonstrated this trend, indicating a plateau in inhibitory effect between 0.4 and 0.8 V/cm, followed by a significant decline at 1.6 V/cm (Fig. 2B).

Figure 2. Effects of TTFields on clonogenic growth in non-small cell lung cancer cell lines. (A) Representative colony formation images of H460 and A549 cells following TTFields exposure. Both cell lines exhibited reduced colony number and colony size compared with untreated controls. The 0.4 and 0.8 V/cm groups exhibited similar inhibitory patterns, whereas 1.6 V/cm resulted in the strongest suppression, with markedly fewer surviving colonies. (B) Quantitative analysis of clonogenic survival fraction calculated from colony formation assays. Data are presented as mean ± SD from three independent experiments. *P<0.05 and ***P<0.001. TTFields, tumor treating fields.

Long-term TTFields exposure induces mild inhibition under P1 and 2 and marked suppression under P3

To evaluate sustained TTFields effects, cells were cultured for 7 days under three stimulation protocols (P1-3), each representing distinct intensity-duration combinations (Fig. 3A). Cell counting on day 7 revealed that both P1 and 2 produced mild reductions in total cell number, whereas P3 led to a significant decrease, leaving only a small population of surviving cells (Fig. 3B). This indicates that long-term cytoreduction becomes notable only under the strongest stimulation condition. WST-8 analysis supported these observations (Fig. 3C). Metabolic activity exhibited a modest decline under P1, followed by a stronger reduction under P2 and the lowest activity under P3. These findings demonstrated differential inhibitory patterns across the three stimulation protocols, with mild long-term suppression under P1 and 2 and a marked loss of proliferative capacity under the P3 protocol.

Figure 3. Long-term inhibitory effects of TTFields under three stimulation protocols. (A) Schematic representation of the three TTFields protocols (P1-3), each corresponding to a distinct intensity-duration combination. (B) Total cell counts after 7 days of culture demonstrating mild inhibition under P1 and 2, and a marked reduction in surviving cells under P3. (C) WST-8 metabolic activity demonstrates a mild decrease under P1, with significantly stronger suppression under P2 and 3. Data are presented as mean ± SD from three independent experiments. *P<0.05, **P<0.01 and ***P<0.001. Con, control; TTFields, tumor treating fields; P1, 0.4 V/cm for 24 h; P2, 0.8 V/cm for 6 h; P3, 1.6 V/cm for 3 h.

TTFields induce differential apoptosis-associated signaling and migration responses across intensity-duration conditions

Western blotting analysis revealed that cleaved PARP was detectable at low levels in control cells, likely reflecting basal stress associated with culture conditions (Fig. 4A). When normalized to total PARP, the cleaved PARP/total PARP ratio exhibited no marked change under lower intensity-duration conditions (P1 and 2), whereas a marked increase was observed under the highest condition (P3), indicating a threshold-dependent enhancement of PARP cleavage. This effect was more evident in H460 cells compared with that in A549 cells, suggesting cell-line-specific differences in apoptotic sensitivity. By contrast, Annexin V/PI staining (Fig. 4B) revealed a more gradual apoptotic response, in which P1 induced only minimal changes, P2 increased early apoptosis and P3 produced the highest levels of both early and late apoptotic cells. Consistent with these trends, Transwell migration analysis (Fig. 4C) revealed that P1 and 2 were associated with only mild reductions in cell motility, whereas P3 was associated with a more notable decrease in migration in both H460 and A549 cells.

Figure 4. Effects of long-term TTFields exposure on apoptosis-associated signaling and migration in non-small cell lung cancer cells. (A) Western blotting analysis of total PARP and cleaved PARP in H460 and A549 cells after long-term TTFields exposure. The ratio of cleaved PARP to total PARP was quantified by densitometric analysis and is presented as a bar graph. (B) Annexin V/PI flow cytometry demonstrating a progressive increase in apoptotic populations, with P3 exhibiting the highest levels of early and late apoptosis. (C) Transwell migration assay revealing migration of H460 and A549 cells under different TTFields intensity-duration conditions. Representative crystal violet-stained images and quantification of overall stained area are presented for control, P1, 2 and 3. Scale bar, 250 µm. *P<0.05, **P<0.01 and ***P<0.001. Con, control; TTFields, tumor treating fields; PARP, poly (ADP-ribose) polymerase; PI, propidium iodide; P1, 0.4 V/cm for 24 h; P2, 0.8 V/cm for 6 h; P3, 1.6 V/cm for 3 h.

Discussion

TTFields have been extensively investigated in GBM, whereas research in lung cancer remains comparatively limited (9). Since NSCLC exhibits biological behaviors and treatment responses that differ markedly from those of GBM, stimulation parameters optimized for GBM cannot be assumed to translate directly to NSCLC (16,17). Nonetheless, majority of NSCLC studies have adopted GBM-derived settings despite marked biophysical differences between the two tumor types (18,19), underscoring the need to evaluate NSCLC-specific responses. In the present study, two NSCLC cell lines across a range of short-term and long-term exposure schedules were examined to explore their responsiveness to AEF delivered at different intensities. This experimental framework was designed to assess how NSCLC cells integrate field intensity and exposure duration, providing a structured basis in interpreting their combined influence on cellular responses.

Notably, these findings hold practical relevance for clinical translation. Current treatment protocols emphasize prolonged daily exposure, typically ~18 h, to maximize therapeutic efficacy (20). However, extended wear time often leads to skin irritation, discomfort associated with transducer arrays and notable restrictions in daily activities, ultimately compromising patient adherence (21–23). Within this clinical context, the present study observations indicated that similar inhibitory effects can be achieved through alternative combinations of field intensity and exposure duration, rather than through prolonged stimulation alone. This finding is consistent with the concept of an intensity-duration trade-off and aligns with growing interest in parameter configurations that may improve treatment tolerability while maintaining antitumor activity (24–28). However, these implications should be viewed as preliminary and interpreted with appropriate caution.

A key consideration is that the interpretation of TTFields efficacy should account for the physical and dosimetric principles governing energy deposition within conductive biological media. Since the power density dissipated by an alternating electric field scales with the square of the field intensity (P ∝ σE2), the time-integrated power density and thus, total delivered energy, can be expressed as a function of field intensity and exposure duration (∝ σE2t) (29). Within this framework, treatment regimens that differ in intensity and duration are not necessarily energy-equated, yet biological responses may converge within certain effective dose ranges. In the present study, the similar biological responses observed under the 0.4 V/cm for 24 h and 0.8 V/cm for 6 h conditions are consistent with the notion that partially overlapping ranges of effective energy exposure can produce similar levels of growth inhibition, even when regimens are not strictly energy-matched. By contrast, the enhanced cytotoxic effects observed under the highest-intensity condition (1.6 V/cm for 3 h) are more parsimoniously explained by increased total energy delivery, although the contribution of intensity-dependent biological thresholds cannot be fully excluded. Taken together, and in line with previous studies describing diminishing returns with prolonged exposure, these observations underscored that TTFields efficacy is best interpreted within an integrated dosimetric framework reflecting combined interactions between intensity and duration, rather than dominance of individual parameters in isolation (30–32).

From a biological and therapeutic perspective, these dosimetric considerations also warrant cautious interpretation of high-intensity, short-duration TTFields regimens. Abbreviated exposure windows may permit tumor cell repopulation during off-treatment intervals and could limit synergistic interactions with cell-cycle-dependent therapies, such as chemotherapy, which are known to benefit from prolonged or continuous TTFields exposure (33,34). Accordingly, modulation of field intensity should be considered within a broader therapeutic context that accounts for treatment scheduling and combination strategies, rather than as a standalone optimization approach.

Lastly, the present study has inherent limitations associated with its in vitro experimental design. The simplified culture system does not capture the complexity of the tumor microenvironment, including tissue architecture, stromal interactions and immune components that may influence electric field distribution and treatment response in vivo. Furthermore, the electric-field geometry applied here differs from clinical conditions and the mechanistic pathways underlying dosimetry-dependent TTFields responses were only partially explored. Future studies incorporating in vivo tumor models, detailed electric field and thermal simulations and expanded molecular analyses will therefore be key to validate the biological relevance and translational feasibility of alternative intensity-duration configurations.

Supplementary Material

Supporting Data

Acknowledgements

Not applicable.

Funding

The present study was supported by National Research Foundation of Korea (NRF) grants funded by the Korean government (MSIT; grant nos. NRF-2021R1A2C2008695 and NRF-2022R1A2C1010337). The present study was also supported by a Korea Medical Device Development Fund grant funded by the Korean Government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare and the Ministry of Food and Drug Safety; grant nos. 1711196423 and RS-2023-00254868).

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

JH conceived and performed the present study, carried out all experiments, analyzed the data and wrote the manuscript. MY contributed to the conception and interpretation of the study, provided supervision and critically revised the manuscript. JH and MY confirm the authenticity of all the raw data. Both authors have read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References