Cell lines and culture conditions

Human liver cancer cell lines (HepG2 and Huh7) and normal human liver cell line (THLE-2) were purchased from Meisen Cell (Zhejiang, China). All cells were cultured in Dulbecco’s modified eagle medium (DMEM). The cell cultures were maintained at 37 °C in a humidified incubator with 5% CO2.

Cell treatment

LFMFs were generated using OM-100. THLE-2 cells were treated with 1.066 mT LFMF at 0 kHz, 10 kHz, 25 kHz, 50 kHz, 100 kHz, and 150 kHz for 0, 24, 48, and 72 h; HepG2 and Huh7 cells were treated with 1.066 mT LFMF at 0 kHz, 10 kHz, 25 kHz, 50 kHz, and 100 kHz for varying durations (0, 24, 48, and 72 h).

Cell viability assay

To evaluate cell viability, we employed the CCK-8 assay. Totally 100 µL cells (2 × 103 cell/well) were seeded in 96-well plates and exposed to LFMF for different frequency and different duration as mentioned before. Subsequently, 10 µL of CCK-8 solution was introduced to each well, and the cells were incubated for another 2 h at 37 °C. The optical density was then ascertained at a wavelength of 450 nm utilizing a microplate reader.

Apoptotic activity assay

Apoptotic cells were identified using an Annexin V-FITC/PI apoptosis detection kit. Post exposure to OM-100 for 72 h, cells were collected, washed with cold PBS, and then labeled with 5 µL Annexin V-FITC and 10 µL PI as per the kit guidelines.

Cell proliferation assay

Colony formation was employed for cell proliferation assay. Cells were dispersed in 6-well plates. Following a 14-day incubation, the colonies were set with methanol for 15 min and tinted with crystal violet for additional 20 min. The colony count was done using microscopic analysis.

Cell migration and invasion assay

The Transwell apparatus was utilized to evaluate both cellular migration and invasion. For the migration assay, the cells were treated with OM-100 for 72 h, and then 200 µL cells (1 × 105 cell/mL) were plated in non-coated upper chamber. For invasiveness, chambers were pre-treated with Matrigel. After incubation for 24 h, cells adhering to the upper membrane surface were eradicated, while the cells that had migrated or invaded through to the lower membrane surface were dyed with crystal violet for a span of 20 min. Three fields of view per group were randomly selected to observe the cells and take photos. The cell number was calculated using Image J software.

ROS quantification

For the detection of intracellular ROS, a DCFH-DA probe was employed. After treatment with OM-100, cells were exposed to 10 µM DCFH-DA for a half-hour at 37 °C. The resultant fluorescence intensity was then quantified via flow cytometry.

In vivo tumor xenograft model

All experimental setup were shown in Fig. 2A. BALB/c nude mice (6-week-old, male, n = 12) were purchased from Yangzhou University Laboratory Animal Center. Mice were subcutaneously injected with 100 µL 5 × 105 HepG2 cells to establish a liver cancer xenograft model. Once tumor volume reached approximately 100 mm3, the mice were randomly divided into the model group (LC: liver cancer) and the OM-100-treated group (LC + 24 d). Tumor growth was monitored every three days, and the tumor volume was calculated using the formula V = 0.5 × L × W2, where L is the length and W is the width of the tumor. OM-100-treated mice received a treatment of 1.066 mT magnetic fields at 100 kHz for 24 days with 2 h at morning (7:00 to 9:00), noon (12:00 to 14:00), and evening (17:00 to 19:00). Mice without any treatment were used as controls.

Upon completion of the treatment, 800 µL of blood was collected from the orbital sinus of a mouse. EDTA-K2 anticoagulant was added to 150 µL of this whole blood, and then the levels of blood routine indicators, including granulocytes (Gran), hematocrit (HCT), hemoglobin (HGB), lymphocytes (Lymph), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), platelets (PLT), red blood cells (RBC), platelet distribution width (PDW), mean platelet volume (MPV), and white blood cells (WBC), were analyzed using an automatic hematology analyzer. The remaining 650 µL of blood was allowed to stand for 30 min, followed by centrifugation at 10,000 r/min for 2 min. The serum was then taken and tested for biochemical indicators, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (T-BIL), creatinine (CREA), triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-c), and low-density lipoprotein cholesterol (LDL-c), using an automatic biochemistry analyzer.

At last, mice were anesthetized using 10% sodium pentobarbital solution, and euthanized by cervical dislocation. The tumors were excised, photographed, and weighed. The animal experiments were approved by Yangzhou University Laboratory Animal Center (No. 202311014).

Fig. 2

OM-100 inhibits cell proliferation and promotes apoptosis in liver cancer cell lines. A The animal experiment. B Cell viability of normal human liver cells (THLE-2) under varying LFMF frequencies (0, 10, 25, 50, 100, and 150 kHz) at 0, 24, 48, and 72 h. C Cell viability of liver cancer cell lines (HepG2, Huh7) under varying LFMF frequencies (0, 10, 25, 50, and 100 kHz) at 0, 24, 48, and 72 h. . D Apoptosis rates in HepG2 and Huh7 cells following OM-100 treatment at a 100 kHz frequency for 72 h. E Colony formation capability of HepG2 and Huh7 cells after OM-100 treatment at a 100 kHz frequency for 72 h. *P < 0.05, **P < 0.01. HepG2 and Huh7 cells were treated with 100 kHz and 1.066 mT of OM-100 for 72 h

Histopathological analysis

Heart, liver, spleen, lung, and kidney were harvested and fixed in 10% formalin. These tissues were then embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histological examination as previous described [15].

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assessment

TUNEL assay was conducted to detect cell apoptosis in tumor tissues of the xenograft model. After deparaffinization and rehydration, tumor sections were treated with proteinase K for 30 min. Then, slides were incubated with the TUNEL reaction mixture for 60 min. The nuclei were counterstained with DAPI for 10 min. Apoptotic cells were visualized and quantified under a fluorescence microscope. Percentage of TUNEL-positive cells was calculated to assess the extent of apoptosis.

Statistical analysis

All data are presented as the mean ± standard deviation. Differences between two groups were analyzed using Student’s t-test, those among multiple groups were evaluated through a one-way analysis of variance followed by Tukey’s test. P < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad 8.0.