Optimum Serum Concentration Enhances Migration of MDA-MB-231 Triple-Negative Breast Cancer Cells and Promotes Intracellular Delivery of Proapoptotic Domain via Cell-Penetrating Peptides

Abstract

Breast cancer remains one of the most prevalent cancers among women, with triple-negative breast cancer (TNBC), lacking estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2, accounting for approximately 15–20% of all patients with breast cancer. TNBC is notably aggressive, with a high invasive, metastatic, and recurrence potential. In this study, we found that the migration and invasion capabilities of MDA-MB-231 cells, derived from human TNBC, were strongly influenced by the serum concentration. Transwell assays revealed that TNBC cell migration varied depending on the fetal bovine serum (FBS) level, with an optimal concentration that substantially enhanced migration and invasion. In contrast, non-TNBC MCF-7 cells exhibited no such serum-dependent migration pattern. In addition to data-independent acquisition (DIA) phosphoproteomic analysis for understanding the mechanisms, the cellular uptake of the flock house virus coat (35–49) peptide, a type of arginine-rich cell-penetrating peptide with serum-dependent cellular uptake efficacy, was significantly increased under the optimal serum conditions, which induces cell migration, leading to efficient delivery of apoptosis-inducible peptide and TNBC-killing activity. Our findings highlight the critical role of serum concentration in regulating TNBC behavior and offer insights into leveraging serum-responsive delivery systems for targeted breast cancer therapy.

1. Introduction

According to the World Health Organization, breast cancer affected 2.3 million individuals and caused 685,000 deaths in 2020, with 30–40% of cases progressing to metastatic disease. Triple-negative breast cancer (TNBC), which accounts for approximately 15–20% of all breast cancer subtypes, is characterized by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal receptor 2 (HER2). Because of this lack of targetable receptors, treatment options are limited, making chemotherapy the primary approach, despite its challenges such as inadequate response, drug resistance, and associated adverse effects. Consequently, TNBC is associated with a poor prognosis, with a median overall survival (OS) of approximately 14 months in metastatic cases. ,

TNBC has been further classified into six molecular subtypes: basal-like 1, basal-like 2, immunomodulatory, mesenchymal, mesenchymal stem-like, and luminal androgen receptor. ,

Survival outcomes vary widely depending on the subtype. Patients with hormone receptor (HR: ER and/or PR)+/HER2– subtype exhibit the highest 4-year survival rate (92.5%), followed by HR+/HER2+ subtype (90.3%) and HR–/HER+ subtype (82.7%). In contrast, patients with TNBC have the lowest survival rate (77.0%, including early- and advanced-stage patients), further dropping to 11.2% in those with stage IV TNBC. Hammershøi Madsen et al. reviewed progression-free survival (PFS) and OS data, finding limited improvement with most targeted therapies. However, some agents such as the antibody–drug conjugate sacituzumab govitecan (targeting TROP-2 and delivering the topoisomerase I inhibitor SN-38) showed higher PFS and OS than standard chemotherapy.

Migration, invasion, and metastatic potential of TNBC contribute to poor prognosis, and their complicated molecular mechanisms have been reported. Among these, recent studies have highlighted microRNAs, such as the miR-30 family, which regulates apoptosis and proliferation. These were markedly downregulated in tumor-initiating cells from patients with poor relapse-free survival, and ER–/PR– breast cancer cells expressed lower miR-30 family levels than their ER+/PR+ counterparts. , Restoration of miR-30 in ER–/PR– cells reduced cellular migration, invasion, and bone metastasis in vivo. , The transcription factor RGPR-p117, which binds the TTGGC motif in the regucalcin gene promoter region, plays a crucial role in suppressing TNBC growth. Specifically, it downregulates oncogenic signaling pathways (Ras, PI3-kinase, Akt, mitogen-activated protein kinase, and mTOR) while simultaneously upregulating tumor suppressors, including p53, Rb, p21, and regucalcin. These activities indicate that RGPR-p117 may inhibit both tumor growth and bone metastatic activity by modulating key signaling processes, including those related to epidermal growth factor. Furthermore, the WNT1/ROR2 signaling pathway has been implicated in TNBC progression. Downregulation of ROS2 expression slows tumor growth, and treatment with the WNT inhibitor pyrvinium pamoate shows efficacy in vivo.

Long noncoding RNA (lncRNA) RP3-340B19.3 has also been shown to enhance the TNBC cancer proliferation and metastasis. Exosomes from MDA-MB-231 cells enriched with RP3-340B19.3 promotes proliferation and migration of other breast cancer cells through complex formation with miR-4510 and activation of Wnt/β-catenin and NF-κB pathways. Other recently reported TNBC-related molecules include ankyrin receptor domain 1 (ANKRD1; a coactivator of p53 tumor suppressor protein and NF-κB regulator); ATP synthase subunit ATP5MF; Kindlin-2, which stabilizes β1-integrin:TGF-β receptor complexes; small-nucleolar RNA host gene 14 (SNHG14), which modulates ERK/MAPK signaling; regulation of HSP90AA1 by P-element-induced wimpy testis (PIWI)-interacting RNA (piRNA) piR-31115; and zinc finger protein ZNF703. These factors contribute to TNBC metastasis through various intricate signaling networks.

In this study, we demonstrated that the fetal bovine serum (FBS) concentration influences the migration and invasion efficacy of MDA-MB-231 TNBC cells in vitro. As the primary components of serum, it contains proteins such as albumin and immunoglobulins, nutrients like carbohydrates and lipids, electrolytes, and waste products. It also includes components involved in molecular transport and receptor stimulation related to cellular functions such as cell proliferation, differentiation, and apoptosis regulation. In our research identifying molecules involved in serum-mediated functional control of TNBC, we discovered that serum concentration significantly influences the migration and invasion of MDA-MB-231 cells, a type of TNBC, and that an optimal serum concentration exists, implying that variations in serum compositions in vivo may influence tumor progression and metastasis. Notably, a 1% FBS concentration significantly enhanced cell migration and invasion, coinciding with the formation of actin-based lamellipodia. Although a 10% FBS concentration promoted the highest cell proliferation efficacy, it was less effective in promoting migration and invasion than 1% FBS concentration. In contrast, a 0% FBS concentration led to robust invasion in Matrigel-based cell culture assay but showed minimal migration in Transwell assays (8 μm diameter pore size). In MCF-7 cells (non-TNBC), migration was FBS concentration dependent; however, their migration efficiency was markedly lower (approximately 160-fold) than that of MDA-MB-231 cells at 1% FBS. These findings indicate that serum concentration modulates TNBC behavior, potentially influencing metastatic progression in vivo.

In addition, under the 1% FBS concentration condition that significantly induces cellular migration and invasion, MDA-MB-231 cells efficiently internalized arginine-rich cell-penetrating peptides (CPPs), enabling the delivery of apoptosis-inducing peptides and promoting TNBC cell death under invasion-inducible FBS conditions.

2. Experimental Section

2.1. Peptide Synthesis

All peptides were synthesized using the 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase method on CLEAR-Amide-Resin (Peptide Institute, Osaka, Japan). Fmoc-amino acid derivatives (Peptide Institute) were coupled using 1-hydroxybenzotriazole (HOBt)/2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) (Peptide Institute)/N-methylmorpholine (NMM) (Nacalai Tesque, Kyoto, Japan) in dimethylformamide (DMF) (Nacalai Tesque), as previously reported. Peptide deprotection and cleavage were performed using trifluoroacetic acid (Watanabe Chemical Industries, Ltd., Hiroshima, Japan) and 1,2-ethanedithiol (Nacalai Tesque) (95:5) treatment for 3 h at 25 °C, followed by purification via reverse-phase high-performance liquid chromatography (HPLC). Purity (>97%) was confirmed by analytical HPLC. The molecular size of the synthesized peptides was assessed using the matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (Microflex, Bruker, Billerica, MA, USA).

FHV coat (35–49)-PAD-amide, NH₂-Arg-Arg-Arg-​Arg-Asn-​Arg-Thr-Arg-​Arg-Asn-​Arg-Arg-​Arg-Val-​Arg-Gly-Gys-_D​Lys-_D​Leu-_D​Ala-​_DLys-_D​Leu-_D​Ala-_D​Lys-_D​Lys-_D​Leu-_D​Ala-_D​Lys-_D​Leu-_D​Ala-_D​Lys)-amide:

MALDI-TOF-MS: 3783.9 (calculated for [M + H]⁺: 3784.9). HPLC retention time: 12.5 min (column: Cosmosil 5C18-AR-II [4.6 mm × 150 mm]; gradient: 5–95%, B in A [A = H₂O containing 0.1% CF₃COOH; B = CH₃CN containing 0.1% CF₃COOH] over 30 min; flow rate: 1 mL/min; detection: 220 nm). The yield from the starting resin was 2.7%.

To prepare fluorescently labeled peptides, the synthesized glycyl-cysteine-amide peptide was treated with 1.1 equiv of Alexa Fluor 488 (Alexa488) C5 maleimide sodium salt (Invitrogen, Eugene, OR, USA) in dimethylformamide (DMF) (Nacalai Tesque)/methanol (Nacalai Tesque) (1:1) for 3 h at 25 °C, followed by purification using HPLC, as previously reported.

FHV coat (35–49)-GC­(Alexa488)-amide, NH₂-Arg-​Arg-Arg-​Arg-Asn-Arg-​Thr-Arg-Arg-​Asn-Arg-Arg-Arg-​Val-Arg-Gly-​Cys­(Alexa488)-amide:

MALDI-TOF-MS: 3024.2 (calculated for [M + H]⁺: 3023.5). Retention time in HPLC: 12.6 min (column: Cosmosil 5C4-AR-300 [4.6 mm × 150 mm]; gradient: 5–85%, B in A [A = H₂O containing 0.1% CF₃COOH; B = CH₃CN containing 0.1% CF₃COOH] over 40 min; flow rate: 1 mL/min; detection: 215 nm). The yield from the starting resin was 66.3%.

2.2. Cell Culture

The human breast cancer cell lines MDA-MB-231 (TNBC) and MCF-7 (non-TNBC) were obtained from the European Collection of Cell Cultures (Salisbury, UK). Cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI 1640, Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated FBS (Gibco, Life Technologies Corporation, Grand Island, NY, USA) on a 100 mm dish (Iwaki, Tokyo, Japan) and incubated at 37 °C under 5% CO₂. Cells were subcultured every 2–3 days upon reaching approximately 80% confluence. The American Type Culture Collection (ATCC, Manassas, Virginia, USA) provided the normal mammary epithelial cells MCF-10A which were cultured in Mammary Epithelial Cell Basal Medium (MEBM) supplemented with Bovine Pituitary Extract (BPE), human Epidermal Growth Factor (hEGF), hydrocortisone, insulin and 100 ng/mL cholera toxin (Sigma-Aldrich, St. Lois, Missouri, USA) according to the manufacturer’s protocol. The cells were incubated in a standard humidified incubator at 37 °C with 5% CO₂.

For experiments involving varying FBS concentration conditions, the cell culture medium was removed from the 100 mm cell culture dishes, and the cells were washed three times with phosphate buffered saline (PBS) (1 mL). Cells were then incubated with either 0.1 g/L trypsin-EDTA (0.1 g/L trypsin and 0.106 mmol/L EDTA solution (Nakalai Tesque) or 2 mM EDTA (Nakalai Tesque) (2 mL/well) for 10 min at 37 °C under 5% CO₂. Following detachment, 3 mL of serum-free RPMI 1640 was added, and cells were collected by centrifugation at 200g for 5 min. The supernatant was then removed, and cells were resuspended in 1 mL of serum-free RPMI 1640 for counting before use in FBS concentration-dependent assays.

2.3. Three-Dimensional Tumorsphere Growth Assay in Matrigel

MDA-MB-231 TNBC cells were washed three times with Dulbecco’s PBS (Gibco) (2 mL) and incubated with 0.25% trypsin-EDTA (Gibco) (2 mL) for 3 min at 37 °C under 5% CO₂. A trypsin neutralizing solution [HEPES buffered type, Kurabo, Osaka, Japan (4 mL) and DPBS (3 mL)] was added, and cells were collected by centrifugation at 200g for 5 min. After the supernatant was discarded, 20 μL of the cell suspension was transferred to a 1.5 mL tube, and 1 μL of this suspension was mixed with 75 μL of Matrigel (Corning, NY, USA) and seeded into 24-well microplates at 37 °C for 30 min under 5% CO₂. Cultures were maintained in RPMI 1640 containing 0%, 1%, or 10% FBS [heat-inactivated FBS (Gibco, Life Technologies Corporation, Grand Island, NY, USA)] in a stage-top incubator at 37 °C for 10 min, with image acquisition every 20 min using a culture microscope (BZ-X710, Keyence, Osaka, Japan) equipped with a 4× magnification objective (Nikon Plan Fluor, Nikon, Tokyo, Japan).

For 3D imaging, spheroids were fixed with 4% paraformaldehyde (Wako Pure Chemical Industries, Ltd., Osaka, Japan) for 20 min at 25 °C and rinsed twice with PBS (10 min each). Cells were permeabilized with PBS containing 0.5% Triton X-100 for 10 min at 4 °C, followed by three 10 min washes in PBS containing 100 mM glycine. Samples were blocked with blocking solution (Nakalai Tesque) and incubated for 60 min at 25 °C. Nuclear and actin staining was performed using 300 nM DAPI and Alexa Fluor 488 Phalloidin (1:200, Invitrogen) in PBS for 20 min at 25 °C, followed by three 20 min rinses with PBS. Finally, three-dimensional images were acquired at 30 μm intervals over a total depth of 300 μm using the Keyence BZ-X710 microscope, and maximum projection images were generated.

2.4. Scratch Wound Migration Assay

MDA-MB-231 TNBC (1 × 10⁵ cells/well) were seeded on 35 mm glass-based dish with grid-marked glass bottom (Iwaki) and cultured in RPMI 1640 supplemented with 10% FBS [heat-inactivated FBS (Gibco, Life Technologies Corporation, Grand Island, NY, USA)] for 24 h at 37 °C under 5% CO_2. A scratch was created using a pipet tip, after which cells were washed with FBS-free RPMI 1640 and incubated in RPMI 1640 containing 1% or 10% FBS (100 μL/well) for 3 h at 37 °C under 5% CO₂. The cells were observed using a microscope equipped with a 4× objective lens (CKX53, Olympus, Tokyo, Japan).

2.5. Migration Assay Using Transwell Membrane

For the Transwell migration assay, MDA-MB-231 TNBC or MCF-7 non-TNBC cells (1 × 10⁵ cells/well) were seeded onto Transwell membrane inserts with 8 μm pores (Corning, NY, USA). The upper chamber contained 100 μL of RPMI 1640 with 0%, 1%, 5%, or 10% FBS [heat-inactivated FBS (Gibco, Life Technologies Corporation, Grand Island, NY, USA), except for heat-inactivated FBS (SAFC Biosciences, Sigma-Aldrich, Merck, Darmstadt, HE, Germany) in Figure S6A and heat-inactivated FBS (Corning, NY, USA) in Figure S6B], whereas the lower chamber contained 600 μL of the same medium. After incubation for 24 h at 37 °C under 5% CO₂, migrated cells on the underside of the membrane were counted using a microscope equipped with a 4× objective lens (CKX53, Olympus, Tokyo, Japan).

2.6. Confocal Laser Microscopy (Actin Staining)

MDA-MB-231 TNBC cells (1.0 × 10⁵ cells/well) were cultured on 35 mm glass-bottom dishes (Iwaki) in RPMI 1640 containing 0%, 1%, or 10% FBS [heat-inactivated FBS (Gibco, Life Technologies Corporation, Grand Island, NY, USA)] for 24 h at 37 °C under 5% CO₂. Cells were fixed with 4% paraformaldehyde (PFA) for 30 min at 25 °C, followed by PBS washes. Permeabilization was performed using 0.1% Triton X-100 (Nacalai Tesque, Kyoto, Japan) for 5 min at 25 °C, followed by PBS washes. For actin staining, rhodamine-phalloidin (2.5 μL [300 units]) (Invitrogen, Eugene, Or, USA) was diluted in PBS at 97.5 μL/well for 20 min at 37 °C. Cells were washed with PBS and visualized using an FV1200 confocal laser scanning microscope with a 20× objective lens (Olympus).

2.7. Confocal Laser Microscopy (JC-1, FITC-annexin V, and Propidium Iodide Staining)

MDA-MB-231 TNBC cells (1.0 × 10⁵ cells/well) were treated with FHV-PAD peptides (5 μM, 200 μL/well) in RPMI 1640 supplemented with 0%, 1%, or 10% FBS [heat-inactivated FBS (Gibco, Life Technologies Corporation, Grand Island, NY, USA)] for 24 h at 37 °C under 5% CO_2. After treatment, the medium was replaced with serum-free RPMI 1640. Cells were then stained with either JC-1 staining (4 μM, 100 μL/well) or FITC-annexin V/propidium iodide (Apoptosis Detection Kit, Sigma-Aldrich, Merck, Darmstadt, HE, Germany) for 15 min at 37 °C under 5% CO₂. Following three washes with FBS-free RPMI 1640 (200 μL) each, cells were visualized using an FV1200 confocal laser scanning microscope with a 20× objective lens (Olympus). In the FITC-annexin V staining (Figure S20), the cells were fixed with 4% paraformaldehyde (PFA) at 25 °C for 30 min and washed with PBS prior to observation using the confocal laser scanning microscope.

2.8. Cell Counting Assay (Cell Viability)

MDA-MB-231 TNBC or MCF-7 non-TNBC cells (2.0 × 10⁵ cells/well, 200 μL) were seeded in 24-well microplates (Iwaki) and cultured in RPMI 1640 containing 0%, 1%, or 10% FBS [heat-inactivated FBS (Gibco, Life Technologies Corporation, Grand Island, NY, USA)] for 24 h at 37 °C under 5% CO₂. Cells were washed three times with PBS (200 μL/well) and treated with 0.1 g/L trypsin-EDTA (200 μL/well) for 10 min at 37 °C under 5% CO₂. Trypan blue (0.4% w/v) (Gibco) was added, and cell viability was assessed using a Countess II Automated Cell Counter (Thermo Fischer Scientific, IL, USA).

2.9. Cell Counting Assay (FHV-PAD Treatment)

MDA-MB-231 TNBC cells (2.0 × 10⁵ cells/well) were treated with FHV-PAD peptides (5 μM, 200 μL/well) on a 24-well microplate (Iwaki) in RPMI 1640 containing 0%, 1%, or 10% FBS [heat-inactivated FBS (Gibco, Life Technologies Corporation, Grand Island, NY, USA)] for 24 h at 37 °C under 5% CO₂. Cells were washed three times with PBS (200 μL/wash) and incubated with 0.1 g/L trypsin-EDTA (200 μL/well) for 10 min at 37 °C under 5% CO₂. Trypan blue (0.4% w/v) (Gibco) was added, and cell viability was assessed using a Countess II Automated Cell Counter (Thermo Fischer Scientific, IL, USA).

2.10. Flow Cytometry (FITC-dextran)

MDA-MB-231 TNBC or MCF-7 non-TNBC cells (2 × 10⁵ cells/well, 200 μL) were treated with FITC-dextran (0.5 mg/mL, 200 μL, molecular weight: 70,000, Signa-Aldrich, St. Louis, MO, USA) in the corresponding FBS-containing medium [heat-inactivated FBS (Gibco, Life Technologies Corporation, Grand Island, NY, USA)] for 24 h at 37 °C under 5% CO₂ in 24-well microplates (Iwaki). Cells were washed three times with PBS (200 μL/well) and incubated with 0.1 g/L trypsin-EDTA (200 μL/well) for 10 min at 37 °C under 5% CO₂. Following trypsinization, PBS (400 μL/well) was added, and the cells were centrifuged at 200g for 5 min at 4 °C. After discarding the supernatant, the cells were washed again with PBS (400 μL), centrifuged, and resuspended in 400 μL of PBS. Fluorescence analysis was then performed on a Guava easyCyte flow cytometer (Merck Millipore, Billerica, MA, USA) with excitation at 488 nm and emission at 525 nm. A total of 10,000 cells/sample were analyzed for fluorescence intensity based on forward- and side-scattering analyses.

2.11. Flow Cytometry (Alexa488-Labeled Peptides)

MDA-MB-231 TNBC or MCF-7 non-TNBC cells (2 × 10⁵ cells/well, 200 μL) were seeded in 24-well microplates (Iwaki) and cultured in RPMI 1640 containing 1% or 10% FBS [heat-inactivated FBS (Gibco, Life Technologies Corporation, Grand Island, NY, USA)] for 24 h at 37 °C under 5% CO₂. After washing with RPMI 1640 containing 1% or 10% FBS, cells were treated with FHV-Alexa488 peptides (5 μM, 200 μL/well) in either 1% or 10% FBS-containing RPMI 1640 for 30 min at 37 °C under 5% CO₂. The cells were then washed three times with PBS containing heparin (0.5 mg/mL, 200 μL/wash) (Nacalai Tesque) and treated with 0.1 g/L trypsin-EDTA (200 μL/well) for 10 min at 37 °C under 5% CO₂, prior to the addition of PBS (200 μL/well) and centrifugation (200 g, 5 min, 4 °C). The washing step with PBS (400 μL/well) and centrifugation was repeated, and the final cell suspension was resuspended in 400 μL of PBS. Fluorescence intensity was measured using a Guava easyCyte flow cytometer (Merck Millipore, Billerica, MA, USA) with excitation at 488 nm and emission at 525 nm. A total of 10,000 cells/sample were analyzed for fluorescence intensity based on forward- and side-scattering analyses.

2.12. Protein Expression and Relative Quantification Detection by DIA Phosphoproteomic Analysis

MDA-MB-231 TNBC cells (5 × 10⁶ cells/well, 10 mL) were cultured in 100 mm dishes for 12 h at 37 °C under 5% CO₂. Cells were washed three times with FBS-free RPMI 1640 (3 mL), and subsequently incubated in RPMI 1640 containing either 1% or 10% FBS [heat-inactivated FBS (Gibco, Life Technologies Corporation, Grand Island, NY, USA)] (10 mL/well) for 3 h at 37 °C under 5% CO₂. Following incubation, cells were washed three times with cold PBS (3 mL) at 4 °C and samples were processed for DIA phosphoproteomic analysis by Promega Corporation (Tokyo, Japan) and Kazusa Genome Technologies Corporation, Kazusa DNA Research Institute (Chiba, Japan). After the identification and quantification of phosphorylated peptides in experiments with 1% and 10% FBS, and we calculated fold changes in Tables S1 and S2.

2.13. KEGG Pathway Analysis

KEGG pathway analysis was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). , Proteins with phosphopeptide counts (or scores) that changed by at least 2-fold in cells cultured in 1% FBS compared with 10% FBS (Tables S1 and S2) were included. The results were visualized using the KEGG mapper.

2.14. Statistical Analyses

All statistical analyses were performed using GraphPad Prism software (version 10.3.1; GraphPad Software, San Diego, CA). For multiple comparisons, one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test or Dunnett’s post hoc test was used. For experiments involving two independent variables, two-way ANOVA followed by Sidak’s or Tukey’s post hoc test was used. P values < 0.05 were considered statistically significant.

3. Results

3.1. Effect of FBS Concentration on Migration and Invasion Efficacy of MDA-MB-231 TNBC Cells

To evaluate the mechanism by which FBS concentration affects the capacity of MDA-MB-231 TNBCs cells, we performed a 3D-tumorsphere growth assay using Matrigel with varying FBS concentrations (0%, 1%, or 10%) in the cell culture medium (Figure A and Movies S1–S3). Time-lapse imaging over 74 h at 37 °C under 5% CO₂ showed that both 0% and 1% FBS conditions facilitated notable cellular migration and invasion, whereas 10% FBS preserved compact spheroid structures with minimal migration efficacy (Figure A and Movies S1–S3). Although all conditions initially exhibited spheroid shrinkage within approximately 10 h of incubation, cells cultured in 1% FBS exhibited the fastest and most extensive radial migration through the Matrigel matrix (Figure A and Movies S1–S3). Following the 74 h incubation, we stained the actin cytoskeleton and nuclei using Alexa488-labeled phalloidin and DAPI, respectively. Differences in actin and nuclear staining patterns reflected distinct cell morphologies: compact and condensed in 10% FBS versus more spread and diverse in 1% FBS (Figure S1). These results indicate that FBS concentration significantly affects the migratory behavior of MDA-MB-231 cells in the 3D-tumorsphere growth assay.

1.

Enhanced migration and invasion of MDA-MB-231 TNBC cells under 1% FBS concentration in vitro. (A) Microscopic observation of MDA-MB-231 TNBC spheroids incubated in Matrigel with various FBS concentrations (0%, 1%, or 10%) in a cell culture medium at 37 °C for 74 h under 5% CO₂. Representative images at 0 and 74 h are shown. Scale bar: 500 μm. Time-lapse videos under similar experimental conditions are available in Movies S1–S3. (B) Wound-healing assay of MDA-MB-231 TNBC cells cultured with 1% or 10% FBS for 3 h at 37 °C. Images at 0 and 3 h are shown. (C, D) Transwell migration assays of MDA-MB-231 TNBC (C) and MCF-7 non-TNBC cells (D) through an 8 μm pore membrane. Cells were incubated with varying FBS concentrations (0%, 1%, 5%, or 10%) for 24 h at 37 °C under 5% CO₂. The number of migrated cells was quantified. Data represent means (±SD) of three experiments. ***P < 0.001.

In addition, wound-healing assays further confirmed superior migratory activity of cells under 1% FBS after 3 h of incubation at 37 °C compared with cells cultured in 10% FBS conditions (Figures B, S2, and S3).

Next, we assessed the invasion efficacy of the MDA-MB-231 cells in different FBS concentrations using a Transwell membrane with 8 μm pores (Figures C, S4, and S5). Cells were seeded and incubated on the Transwell membrane at 37 °C for 24 h under 5% CO₂. Quantification of cells that migrated through the membrane pores and arrived at the opposite side of the membrane revealed that the 1% FBS condition yielded the highest number of migrated cells (Figure C, S4, and S5). Although the 0% FBS condition promoted migration and invasion in the Matrigel assay (Figure A), very low migrations were confirmed in the transmembrane assay (Figures C and S5). Conversely, a higher FBS concentration (Figure C) consistently correlated with reduced migration in both Matrigel and Transwell assays (Figure A).

Furthermore, regarding the migration assessment of MDA-MB-231 cells, similar experiments using Transwell were conducted with FBS from another supplier. As shown in Figure S6, similar evaluations were performed for FBS from different suppliers. The results confirmed that for all FBS samples the concentration at which migration was promoted, as indicated in Figure C, was present.

To preserve surface proteins potentially affected by trypsinization, we also used EDTA treatment for cell detachment prior to Transwell invasion assays (Figure S7). After the detachment of the MDA-MB-231 cells, the cells were incubated on the Transwell membrane at 37 °C for 24 h under 5% CO₂. Even with this modification, the 1% FBS condition produced the highest number of migrated MDA-MB-231 cells (Figure S7).

We further compared MDA-MB-231 TNBC cells with MCF-7 non-TNBC cells using the Transwell assay (Figure D). MCF-7 migration increased in an FBS-dependent manner; however, their migration under the 1% FBS concentration condition was approximately 160-fold lower than that of MDA-MB-231 TNBC cells, highlighting intrinsic differences in their migration and invasion mechanisms. We additionally compared MDA-MB-231 TNBC cells with MCF 10A nontumorigenic human breast epithelial cells using the Transwell assay (Figure S8). For MCF 10A cells, the cell migration was scarcely observed under any serum concentration condition (Figure S8).

Additional cell death and apoptosis assessments were performed (Figure S9). MDA-MB-231 (TNBC) cells were cultured at different FBS concentrations (0%, 1%, or 10%) for 8 h at 37 °C under 5% CO₂ conditions. Following this, the cells were stained with FITC-Annexin V [early apoptosis detection (phosphatidylserine)] or propidium iodide [cell membrane disruption (cell death)] (Figure S9). In this experiment, no apparent cell death, including apoptosis, was observed under any FBS concentration condition (Figure S9).

3.2. Assessment of Lamellipodia Formation under Different FBS Concentrations

Lamellipodia formation, driven by actin reorganization, plays a crucial role in cancer cell migration. , To assess lamellipodia dynamics, we visualized the actin cytoskeleton by staining with rhodamine-labeled phalloidin (Figure ). TNBC MDA-MB-231 cells were incubated at 37 °C for 24 h under different FBS concentrations (0%, 1%, or 10%), followed by fixation with 4% paraformaldehyde (PFA), membrane permeabilization with 0.1% Triton X-100, and staining. Our results showed that under 1% FBS, MDA-MB-231 cells exhibited prominent lamellipodia at the cell periphery and increased the intercellular distance (Figure A,B). Cells cultured in 0% FBS also induced lamellipodia formation but appeared more shrunken and tightly arranged (Figure A,B). In contrast, the 10% FBS condition induced prominent stress fiber formation with limited lamellipodia formations, and the cells were closely packed (Figure A,B).

2.

Induction of lamellipodia formation by 1% FBS in MDA-MB-231 TNBC cells. (A) Confocal laser microscopy of MDA-MB-231 TNBC and MCF-7 non-TNBC cells after incubation with varying FBS concentrations (0%, 1%, or 10%) for 24 h at 37 °C under 5% CO₂, followed by cellular fixation and rhodamine-phalloidin staining (red). Scale bar: 10 μm. (B) Magnified views of rhodamine-phalloidin-stained MDA-MB-231 TNBC cells-dotted white squares in (A).

Figure S10 shows the microscopic images of the MDA-MB-231 cells incubated for 24 h at 37 °C under different FBS concentrations (0%, 1%, or 10%). These images confirm these patterns, i.e., tight, compact cell morphology under 0% FBS; extended but tightly packed cells under the 10% FBS condition; and expanded intracellular space under 1% FBS (Figure S10). These results indicate that FBS concentration affects both cytoskeletal organization and cell–cell adhesion, contributing to differential migration and invasion behavior of the MDA-MB-231 TNBC cells.

The MCF-7 non-TNBC cells were also treated with different FBS experimental concentrations (0%, 1%, or 10%) at 37 °C for 24 h under 5% CO₂ before cellular staining with rhodamine-labeled phalloidin (Figure A). Regardless of concentration, MCF-7 cells did not induce the formation of lamellipodia and maintained tight intercellular attachments, further emphasizing migratory mechanisms different from those of MDA-MB-231 TNBC cells (Figure A).

3.3. Cellular Proliferation Assessment under Different FBS Concentrations

Cellular proliferation was assessed using a cell counting assay, as described in the Experimental Section (Figures A and S11A). MDA-MB-231 TNBC and MCF-7 non-TNBC cells were cultured under varying FBS concentrations (0%, 1%, or 10%) at 37 °C for 24 h under 5% CO₂, followed by cell counting using trypan blue staining. The proliferation rate was highly dependent on the FBS concentration (Figures A and S11A). These results indicate that enhanced migration and invasion of MDA-MB-231 TNBC under 1% FBS is not directly related to increased cellular proliferation.

3.

Effects of FBS concentration on cell proliferation and macropinocytosis in MDA-MB-231 TNBC cells. (A) Viable cell counts after 24 h incubation with varying FBS concentrations (0%, 1%, or 10%) at 37 °C under 5% CO₂, measured using trypan blue exclusion. (B) Cellular uptake of FITC-dextran (molecular weight: 70,000, 0.5 mg/mL) measured by flow cytometry after 24 h under the same conditions. (C) FITC-dextran uptake in MDA-MB231 cells invaded through Transwell membranes under 1% FBS (Figure C). Data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01.

3.4. Assessment of Macropinocytotic Cellular Uptake under Different FBS Concentrations

Lamellipodia formation via actin reorganization plays a key role in cell migration and macropinocytosis. − Macropinocytosis is a nonselective, clathrin-independent, actin cytoskeleton-dependent endocytic pathway, involving Rac1-GTP-mediated signal transduction, leading to lamellipodia formation (plasma membrane ruffling) and uptake of large extracellular fluid volumes (>1 μm). − Therefore, we investigated macropinocytosis activity under different FBS concentration conditions.

FITC-dextran (molecular weight: 70,000) was used as a tracer to detect macropinocytotic cellular uptake. − MDA-MD-231 TNBC cells were incubated with FITC-dextran-containing media (0.5 mg/mL) for 24 h at 37 °C under 5% CO₂, followed by flow cytometer analysis (Figure B). Our results showed that cells under 1% FBS exhibited significantly higher uptake of FITC-dextran than the cells under 0% FBS (Figure B). This finding aligns with the enhanced lamellipodia formation observed under 1% FBS (Figure ). Interestingly, cellular uptake under 1% FBS was nearly equivalent to that observed under 10% FBS (Figure B).

We also assessed the FITC-dextran in MDA-MB231 cells that had migrated through the Transwell membrane under 1% FBS (Figure C). These migrated cells exhibited FITC-dextran uptake similar to that of the original MDA-MB231 cells cultured under 1% FBS experimental conditions (Figure C). Given the increased level of lamellipodia formation under 1% FBS and stress actin fiber formation under 10% FBS (Figure ), we hypothesized that although the basic nutrient uptake under 1% FBS may be reduced, the induced lamellipodia compensate for this through macropinocytotic activity.

MCF-7 non-TNBC cells were similarly treated with the FITC-dextran (0.5 mg/mL) for 24 h at 37 °C under 5% CO₂, followed by flow cytometry (Figure S11B). In contrast to MDA-MB231 cells, 1% FBS did not increase FITC-dextran uptake relative to 0% FBS, indicating different endocytic responses (Figure S11B).

3.5. Protein Expression and Relative Quantification Detection by DIA Phosphoproteomic Analysis

To investigate signaling pathways under 1% FBS, protein expression and relative quantification using data-independent acquisition (DIA) phosphoproteomic analysis was performed (Figures S12–S15). MDA-MB-231 TNBC cells were incubated with either 1% or 10% FBS for 3 h at 37 °C under 5% CO₂, followed by DIA phosphoproteomic analysis, as described in the Experimental Section. Figure S12 lists proteins with increased phosphorylation under the 1% FBS condition, including migration/invasion-related proteins such as the tight junction protein ZO-1 and the Crk-like protein. Figure S13 lists proteins with increased phosphorylation under the 10% FBS condition, showing reduced phosphorylation under 1% FBS. Figures S14 and S15 provide additional comparisons, including proteins exclusively detected in one condition but not the other.

The results were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to predict altered signal transmission in MDA-MB-231 TNBC cells (Figure S16). From a total of 10,209 unique phosphopeptides from 3,132 proteins, 1,609 proteins (51.4%) showed at least a 2-fold change under 1% FBS (Figure S16). Interestingly, only one protein, TBCK (TBC1 domain containing kinase), was uniquely phosphorylated under 1% FBS conditions but not detected under the 10% FBS condition (Figure S16). TBCK has been shown to influence the expression of mammalian target of rapamycin (mTOR) complex components and its signaling pathway.

The top predicted upregulated pathways under 1% FBS included the ErbB signaling pathway, mTOR signaling pathway, regulation of actin cytoskeleton, and tight pathways (Figure S16). Conversely, pathways such as cell cycle regulation, ribosome biogenesis, and proteoglycans in cancer were predicted to be downregulated (Figure S16). Representative pathways such as ErbB and mTOR are shown in Figure S16, with notable activation of ERK and PI3K-related signaling pathways under 1% FBS.

3.6. Delivery of Proapoptotic Domain into MDA-MB231 Cells via Arginine-Rich Cell-Penetrating Peptides

Next, we evaluated the delivery efficiency of a proapoptotic domain into MDA-MB-231 TNBC cells using arginine-rich CPPs. ,− Arginine-rich CPPs, including the HIV-1 Tat-derived peptide [HIV-1 Tat (48–60)] and oligoarginine sequences, have been shown to facilitate efficient cellular uptake in various types of cells, including those of cancer. These peptides have proven effective as delivery vehicles for various bioactive molecules, such as peptides, proteins, nucleic acids, and synthetic macromolecules. Macropinocytosis is a critical mechanism for the uptake of arginine-rich CPPs. Cell-surface proteoglycans play crucial roles in initiating this process. ,− Specifically, syndecan-4 clustering induced by octaarginine facilitates the recruitment of PKCα to its cytosolic V-domain, leading to downstream signal transduction and enhanced cellular CPP uptake. Furthermore, multilamellar membrane structures formed at focal contact points promote direct cell membrane penetration of the peptides. , The cellular uptake efficacy of these peptides is also affected by serum concentration; high concentrations of FBS significantly reduce cellular internalization. ,

To investigate this, we assessed the cellular uptake efficacy of the flock house virus (FHV)-derived arginine-rich CPP, FHV coat (35–49), which is previously established as highly efficient intracellular delivery peptide. , We synthesized a fluorescently labeled version of the FHV peptide (FHV-Alexa488) and treated MDA-MB-231 TNBC cells at 37 °C for 30 min under 1% or 10% FBS conditions, followed by flow cytometer analysis (Figures A,B and S17). Our results showed that the 1% FBS condition yielded significantly higher cellular uptake of FHV-Alexa488 than 10% FBS. A similar trend was observed in migratory MDA-MB231 cells collected via Transwell membrane assays, also under the 1% FBS condition (Figure C), which showed significantly increased cellular uptake efficacy compared with the 10% FBS group (Figure B).

4.

Efficient cellular uptake of FHV coat (35–49) peptides in MDA-MB-231 TNBC cells under migration- and invasion-inducing FBS condition. (A) Structure of the FHV coat (35–49) peptide labeled with Alexa488 (FHV-Alexa488). (B) Flow cytometer analysis of FHV-Alexa488 peptide uptake (5 μM) in MDA-MB-231 TNBC cells after 30 min of incubation at 37 °C with varying FBS concentrations (1% or 10%). “Migrated cells”: uptake in cells migrated through the Transwell membrane under 1% FBS (Figure C). Data are expressed as mean ± SD of three experiments. (C) Structure of the FHV coat (35–49) peptide fused with a pro-apoptotic domain (FHV-PAD). (D) Cell viability and (E) microscopy (see Figure S18) of MDA-MB-231 cells treated with FHV-PAD (5 μM) in different FBS concentrations (D: 0%, 1%, or 10%, E: 1%) for 24 h at 37 °C under 5% CO₂. Viability was measured via trypan blue exclusion. (F) Confocal laser microscopy of JC-1 stained MDA-MB-231 TNBC after FHV-PAD (5 μM) treatment under 1% FBS for 24 h at 37 °C under 5% CO₂. Red: JC-1 aggregates (high mitochondrial membrane potential); green: JC-1 monomers (low mitochondrial membrane potential). The arrows point to cells showing JC-1 aggregate negativity and JC-1 monomer positivity. Scale bar: 20 μm. Data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Subsequently, we synthesized a fusion peptide combining the FHV coat (35–49) sequence with a proapoptotic domain (PAD), designated as FHV-PAD (Figure C). Once internalized, this peptide can target the mitochondrial membrane, resulting in its disruption and induction of apoptotic cell death. ,−

MDA-MB231 TNBC cells were treated with 5 μM FHV-PAD at 37 °C for 24 h under varying FBS concentrations (0%, 1%, or 10%) before microscopic observation and viability assessment (Figures D,E and S18). The highest cytotoxic efficacy was observed under the 1% FBS condition. Subsequently, JC-1 staining revealed low mitochondrial membrane potential (increased green and decreased red fluorescence) in FHV-PAD-treated cells, confirming mitochondrial dysfunction (Figures F and S19). Conversely, control cells (untreated) showed predominantly red JC-1 fluorescence, indicative of intact mitochondria (Figures F and S19). Additionally, annexin V-FITC staining revealed phosphatidylserine exposure on the plasma membrane, further confirming apoptosis induction under 1% FBS (Figure S20). Overall, these findings support that under optimal serum conditions (1% FBS), arginine-rich CPPs can achieve highly efficient intracellular delivery of functional peptide sequences to induce apoptotic death in MDA-MB231 cells during migration and invasion.

4. Discussion

In this study, we observed that FBS concentration significantly influenced cellular migration and invasion, with 1% FBS showing the highest efficacy in promoting MDA-MB231 TNBC cell migration and invasion compared with other FBS concentrations in in vitro assays. Figure summarizes our hypothesis regarding FBS-dependent proliferation, migration, invasion, and lamellipodia formation. Although 10% FBS markedly accelerated MDA-MB231 TNBC cell proliferation, it resulted in low migration and invasion efficacy (Figure , Movie S3, and Figures S3, S5, and S7). Under 0% FBS conditions, although migration and invasion of the MDA-MB231 TNBC cells were enhanced in the 3D-tumorsphere assay using Matrigel, the Transwell membrane assay showed very low efficacy (Figure , Movie S1, and Figure S5). Under 1% FBS, a distinctive cell morphology with lamellipodia formation was observed (Figure ). Additionally, tight junction-like attachments were evident in shrunken structures under 0% FBS and extended cellular structures under 10% FBS (Figures and S10).

5.

Schematic summary of serum-dependent migration and invasion in MDA-MB-231 TNBC cells and therapeutic peptide delivery via arginine-rich CPPs (FHV peptide).

Among all tested conditions, 1% FBS induced the highest migration and invasion efficacies in both the Transwell membrane assay and the 3D-tumorsphere growth assay using Matrigel. This was accompanied by enhanced lamellipodia formations (Figures and , Movie S2, and Figures S2, S4, S5, and S7). Interestingly, cells under 1% FBS condition exhibited an expanded intercellular distance compared with other FBS concentrations (Figures , S1, and S10). As described in the Introduction section, these results suggest the existence of an optimal serum concentration that promotes TNBC migration and invasion efficacy, implying that variations in serum compositions in vivo may influence tumor progression and metastasis.

Occludin, a transmembrane protein at tight junctions (TJs), is crucial for paracellular sealing and cell–cell interactions. Occludin knockdown in breast cancer cells disrupts tight junction integrity and increases invasiveness, which is associated with metastasis. Notably, the expanded intercellular spacing of MDA-MB-231 TNBC cells observed under the 1% FBS concentration condition compared with that under other FBS conditions (Figures , S1, and S10) may indicate altered tight junction function. Although we conducted DIA phosphoproteomic analysis, we did not identify any clear mechanisms that could explain serum concentration effects on TJ-related cell-to-cell adhesion either in our data or in previously published reports. Consequently, further studies are needed to elucidate these mechanisms.

Lamellipodia are involved in cell migration as well as macropinocytosis. − We studied the possible involvement of macropinocytosis under different FBS conditions. Our results showed that the 1% FBS experimental condition significantly increased FITC-dextran uptake (a macropinocytosis tracer) compared with 0% FBS (Figure B), which is consistent with the enhanced lamellipodia formation shown in Figure . Meanwhile, uptake levels under 1% and 10% FBS were nearly identical (Figure B), despite intense lamellipodia formation under 1% FBS and increased actin stress fiber formation, but no lamellipodia, under 10% FBS (Figure ). These results indicate that the 1% FBS experimental condition could induce prominent lamellipodia formation, whereas the 10% FBS experimental condition results in reduced lamellipodia and increased stress actin fiber formation. Consequently, we hypothesize that lamellipodia may compensate for lower basal uptake levels. However, the precise mechanisms of nutrition uptake under different FBS conditions remain to be clarified.

Figures S12–S16 show DIA phosphoproteomic protein expression data and KEGG pathway analysis for predicting signal transmission in MDA-MB-231 TNBC cells. Proteins with increased phosphorylation under 1% FBS include migration- and invasion-related proteins such as the tight junction protein ZO-1 and the Crk-like protein (Figure S12). Of the 10,209 unique phosphopeptides identified from 3,132 proteins, 1,609 proteins (51.4%) showed at least a 2-fold change in phosphorylation under 1% FBS (Figure S16). Only one protein, TBCK (TBC1 domain containing kinase), was exclusively upregulated under 1% FBS (Figure S16). TBCK has been reported to regulate components of the mTOR complex and actin organization.

The mTOR critically regulates cell proliferation and growth, whereas TBCK is involved in the regulation of mTOR signaling pathway, even including actin organization. KEGG pathway analysis revealed that pathways, such as ErbB signaling, mTOR signaling, regulation of actin cytoskeleton, and TJs, were among the most highly regulated, with at least a 2-fold increase, under 1% FBS (Figure S16). ErbB and mTOR signaling pathways, which include the ERK- and PI3K-related signaling, were representative of the pathways activated under 1% FBS. These findings suggest that 1% FBS induces complex signaling networks involving migration and invasion, offering valuable insights into the mechanisms underlying TNBC malignant and potential therapeutic targets. Based on these results, further detailed investigation into the differences between MDA-MB-231 and MCF-7 cells is necessary for the future. Consequently, research progress linking phosphoproteome analysis to an understanding of the molecular biological mechanisms is essential.

Arginine-rich CPPs, known to induce macropinocytosis and facilitate membrane penetration, have been used as delivery vehicles for bioactive molecules. ,− Their cationic guanidinium functional groups interact with negatively charged cell-surface molecules, including glycosaminoglycans, enhancing cellular uptake, ,− binding serum components, and reducing cellular uptake efficacy. , In our study, FHV-Alexa488 peptide uptake by MDA-MB-231 TNBC cells was significantly increased under the 1% FBS condition compared with that under 10% FBS (due to low serum concentration, interactions between the FHV peptides and serum components, which inhibit peptide internalization, might be possibly reduced) (Figure B), suggesting enhanced CPP-based delivery in migration- and invasion-promoting microenvironments. Furthermore, the FHV coat (35–49) peptide successfully delivered the apoptosis-inducible PAD peptide, resulting in efficient TNBC-killing activity under 1% FBS (Figure D–F). Although we used the FHV coat (35–49) peptide, there are diverse CPPs with different properties, including different hydrophobicities, ratios of cationic/anionic residues in sequences, serum stability, linear/cyclic structures, and helical/sheet structures. Future studies should screen diverse CPPs to identify the most effective ones under TNBC migration and invasion conditions.

Finally, our findings suggest that serum concentration has a profound effect on TNBC migration and invasion. As discussed in the Introduction, TNBC progression involves complex and interrelated signaling mechanisms. Although developing targeted therapeutics for each TNBC-related molecule poses technical challenges, our results offer a new therapeutic approach, potentially involving serum modulation, for treating breast cancer. In this study, the experiments and analyses were conducted from these perspectives (cell adhesion, phosphoproteome, analysis, cytoskeleton, and endocytosis); however, further identification of serum components that influence TNBC migration/invasion behavior and understanding their signaling pathways are crucial. Investigating how serum concentration affects cell-to-cell adhesion will also be especially crucial to developing invasion-inhibiting strategies. Regarding these points, we strongly believe that further research is necessary to contribute to the development of technologies that will advance our understanding and treatment of the TNBC.

5. Conclusion

We demonstrated that TNBC migration and invasion are highly dependent on the serum concentration. TNBC migration and invasion vary according to FBS levels, with 1% FBS emerging as an optimal concentration. In addition, the FHV coat (35–49) peptide showed enhanced cellular uptake under these conditions, enabling the efficient delivery of the apoptosis-inducing PAD peptide and resulting in robust TNBC cell death. Our findings provide a foundation for understanding how serum concentration influences breast cancer malignancy and offer novel perspectives for therapeutic development.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.5c00916.

• Effect of FBS concentration on cellular condensation, wound-healing assay, transwell migration assay, apoptosis and cell death assessments, assessments for cell proliferation and macropinocytosis, DIA phosphoproteomic analysis, KEGG pathway analysis, JC-1 assessments for mitochondrial membrane potential, annexin V staining (PDF)

• Movie S1: migration and invasion of MDA-MB-231 TNBC cells under 0% FBS in the 3D-tumorsphere growth assay (MP4)

• Movie S2: enhanced migration and invasion of MDA-MB-231 TNBC cells under 1% FBS in the 3D-tumorsphere growth assay (MP4)

• Movie S3: low migration and invasion of MDA-MB-231 TNBC cells under 10% FBS in the 3D-tumorsphere growth assay (MP4)

• Tables S1 and S2 (XLSX)

I.N. and T.T.-N. designed this study. Y.A., S.N., M.Ma., H.H., Y.K., D.F., M.Mi., M.H., T.T.-N., and I.N. performed the experiments (Y.A., H.H., Y.K., D.F., M.Mi., I.N.: peptide synthesis; Y.A., M.H.: 3D-tumorsphere growth assay, Y.A., I.N.: scratch wound migration assay, Y.A., S.N., T.T.-N., I.N.: migration assay using Transwell membrane, Y.A., S.N., I.N.: confocal laser microscopy (actin, FITC-Annexin V, and propidium iodide staining); Y.A.: cell counting assay (cell viability and FHV-PAD treatment); Y.A.: flow cytometry; M.Ma.: analysis for predicting signal transmission). T.T.-N. performed statistical analysis. S.F., H.K., T.W., and I.F. provided technical assistance with peptide synthesis, molecular analysis, and cellular experimental analysis. Y.A., M.Ma., H.K., T.W., S.F., I.F., M.H., T.T.-N., and I.N. wrote the manuscript. All of the authors discussed and analyzed the obtained results.

The authors declare no competing financial interest.