All cell culture reagents, including Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin, were sourced from HyClone (GE Healthcare Life Sciences, Logan, UT, USA). Trypsin-EDTA was acquired from Gibco (Thermo Fisher Scientific, Waltham, MA, USA).

Eribulin mesylate (1 mg/vial) was generously provided by Eisai Co., Ltd. (Tokyo, Japan), and cisplatin was obtained from JW Pharmaceutical (Seoul, Korea). The half-maximal inhibitory concentration (IC₅₀) of eribulin in MDA-MB-231 cells was determined via CCK-8 assay after 72 h of treatment and found to be 40.12 µM. Based on this, a concentration of 60 µM-approximately 1.5 times the IC₅₀- was selected for subsequent combination experiments. This supra-physiological dosing strategy aligns with previous mechanistic synergy studies, such as Ko et al (17) in TNBC cells combining eribulin and cisplatin and Swami et al (19) demonstrating similar effects in SK-BR-3 cells, as well as the in vitro/in vivo work by Terashima et al (20) on EMT/MET modulation in TNBC treated with eribulin plus S-1.

PD98059 (a MAPK inhibitor) and crystal violet dye were procured from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Primary antibodies against LC3-I/II (cat. no. 4108), phospho-ERK1/2 (Thr202/Tyr204; cat. no. 9101), and β-actin (cat. no. 4967) were purchased from Cell Signaling Technology. Additional antibodies against ERK (SC-94) and p62 (sc-48389) were acquired from Santa Cruz Biotechnology (Dallas, TX, USA). Secondary antibodies conjugated with horseradish peroxidase (anti-mouse: cat. no. 7076; anti-rabbit: cat. no. 7074) were also from Cell Signaling Technology. Chemiluminescence reagents (Super Signal^® West Pico) and DAPI stain were provided by Thermo Fisher. CCK-8 kits were sourced from Dojindo Molecular Technologies (Japan), and autophagy assays (ab139484) were performed using kits from Abcam (Cambridge, MA, USA). Annexin V-FITC apoptosis detection kits were obtained from Koma Biotech (Seoul, Korea).

MDA-MB-231, a human breast cancer cell line, was obtained from the Korean Cell Line Bank. Cells were grown in DMEM supplemented with 10% FBS, antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin), sodium pyruvate (1 mM), sodium bicarbonate (1.5 g/l), and glucose (4.5 g/l). Cultures were maintained in a humidified 5% CO₂ incubator at 37°C.

To evaluate cytotoxicity, cells were plated at 5,000 cells per well in 96-well plates and allowed to adhere overnight. Treatments with test compounds were applied for 72 h. After incubation, CCK-8 reagent (100 µl) was added, and absorbance at 450 nm was measured after a 2-h incubation using a microplate reader.

Colony formation assays were conducted to evaluate long-term proliferative capacity. MDA-MB-231 cells were seeded at low density in 6-well plates and treated with eribulin, cisplatin, or their combination for 14 days. Colonies were fixed with methanol and stained with 0.5% crystal violet.

Colonies were defined as discrete clusters containing ≥50 cells. Due to the dispersed and small-sized colonies formed by MDA-MB-231 cells even after 14 days, manual quantification under a microscope was technically unfeasible. Thus, colony numbers and areas were quantified using ImageJ software (NIH, USA) following standard image analysis steps: conversion to 8-bit grayscale, threshold adjustment, watershed segmentation, and automated particle counting via the ‘Analyze Particles’ function.

Cells at approximately 80% confluency were treated and harvested. Cell lysis was performed using buffer containing Tris, NaCl, EDTA, Triton X-100, NP-40, PI, DTT, and PMSF. Lysates were centrifuged, and protein concentrations were measured using a BCA protein assay kit. Protein (20 µg/lane) was resolved by SDS-PAGE and transferred onto PVDF membranes. Blots were blocked with 5% milk in PBST and incubated with primary antibodies overnight. HRP-conjugated secondary antibodies were applied, and bands were visualized with enhanced chemiluminescence. Band intensities were quantified and normalized to the respective loading controls.

Apoptosis was quantified using an Annexin V-FITC/PI kit as per the manufacturer's instructions. Cells treated for 72 h were collected, stained with Annexin V-FITC and PI, and analyzed via flow cytometry. Histogram analysis was done using Kaluza software.

Autophagy flux was examined using Abcam's fluorescent dye-based assay (ab139484), which selectively labels autophagic compartments. Cells were cultured in 8-well chamber slides and treated with the indicated compounds (100 µM) for the specified time, fixed with paraformaldehyde, and stained with autophagy-specific dyes according to the manufacturer's instructions. Fluorescent signals were observed using a confocal microscope and quantified with ImageJ.

To assess drug synergy, the CI was computed using the Chou-Talalay method with CompuSyn software (version 1.0, Combosyn Inc., Paramus, NJ, USA). CI values were interpreted as follows: CI <1 (synergy), CI=1 (additivity), and CI >1 (antagonism).

All experiments were independently repeated at least three times. All statistical analyses were performed using SPSS version 29.0 for Windows (IBM Corp.). One-way ANOVA was used with Tukey's Honestly Significant Difference post hoc analysis. Data are shown as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

Cell viability was assessed using the CCK-8 assay (Fig. 1A), and the synergistic efficacy of the drug combination was further confirmed by colony formation assays (Fig. 1B and C). Table I summarizes the CI values derived for the two drugs in this cell model. Single treatment with eribulin mesylate (60 µM) decreased cell viability to 75.11±0.41% after 72 h of exposure. Cisplatin (60 µM) single treatment showed a reduction of 50.57±0.20%. The combination of these drugs significantly inhibited cell viability to 17.15±0.10% (Fig. 1A). Colony-forming assays were performed to test the ability of single cells to grow into colonies. This drug combination synergistically inhibited colony formation by MDA-MB-231 cells (Fig. 1B and C).

We observed that the ERK phosphorylation level increased more than 5.5-fold in the experimental cells compared to that in the control cells after the administration of 60 µM eribulin for 72 h (Fig. 2A and B). Moreover, 60 µM cisplatin increased ERK phosphorylation 6.0-fold (Fig. 2A and B). In addition, when cisplatin was added to eribulin, ERK activation increased by 14.8 times. When cisplatin was added to eribulin, ERK activation was elevated 14.8-fold compared to the control, reflecting a 2.7- and 2.5-fold increase relative to eribulin and cisplatin treatment alone, respectively. These findings confirmed that ERK1/2 activation increased synergistically with eribulin-cisplatin combination.

To evaluate autophagy induction, the LC3-I/II ratio and p62 expression were measured. As autophagy markers, the microtubule-associated protein LC3-I/II ratio and p62 expression were determined using western blot analysis (Fig. 3A). The LC3-I/II level increased 4.3-fold in the eribulin group compared to that in the control group. Cisplatin alone increased the expression of these parameters by 6.1-fold; the corresponding value for eribulin-cisplatin combination treatment was 13.9-fold, indicating a synergistic effect (Fig. 3B). A significant downregulation of p62 was observed following co-treatment with eribulin and cisplatin, suggesting promoted autophagic degradation (Fig. 3C). The corresponding values for eribulin alone and eribulin-cisplatin combination were 9.0 and 23.6%, respectively, indicating a significant increase in autophagic activity in the combination group compared with the eribulin-only group (Fig. 3D and E). Autophagic vacuole staining showed a slight increase in autophagic activity with either eribulin or cisplatin alone, whereas their combination resulted in a marked enhancement, indicating cumulative autophagic activation.

The apoptotic activity of the combination of eribulin and cisplatin was analyzed by flow cytometry after double staining with annexin V and propidium iodide (Fig. 4A). The apoptotic activity levels were 28.61, 64.77, and 99.26% when the cells were treated with eribulin alone, cisplatin alone, or a combination thereof, respectively. Upon co-treatment with 3-methyladenine, a known autophagy inhibitor, the apoptotic rate induced by the eribulin-cisplatin combination significantly decreased, suggesting that the observed cytotoxicity is dependent on autophagic mechanisms. When 3-methyladenine samples were treated with a combination of eribulin and cisplatin, the apoptotic cell volume was reduced to 13.68% compared with 99.26% in the eribulin-cisplatin combination group (Fig. 4A and B).

To assess the impact of ERK inhibition, cells were pretreated with PD98059 prior to exposure to eribulin and cisplatin, followed by a viability assessment using the CCK-8 assay. When PD98059 was combined with the two-drug combination, the cell viability increased significantly from 33.63 to 53.37% (Fig. 5A). Flow cytometry was used to quantify apoptotic cells following dual labeling with Annexin V and propidium iodide (Fig. 5B). Compared with the eribulin and cisplatin combination group, the apoptosis rate decreased from 92.72 to 18.01% when PD98059 was used (Fig. 5C).

Using the control value as the reference (100%), LC3-I/II expression was markedly increased in the eribulin plus cisplatin group (824.99%) but was reduced to 119.30% when PD98059 was additionally administered, whereas p62 expression increased from 38.70 to 72.59% with PD98059 treatment (Fig. 5D-F). Thus, ERK inhibition affected the expression of autophagy-related proteins.

An autophagy assay (ab139484, Abcam) confirmed that the levels of autophagosomes decreased when PD98059 was used in combination with eribulin and cisplatin (Fig. 5G). Quantitative analysis of fluorescence intensity showed consistent results, decreasing from 15.59 to 2.92% (P<0.05; Fig. 5H).