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.

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.

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.

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.

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.

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.

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.

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 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).

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 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.

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.

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).

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.

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.