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. 2026 Mar 6;24:223. doi: 10.1186/s12964-026-02786-3

Mitochondrial preservation through the PKA–DRP1 axis empowers natural killer cells to resist acidic stress and retain cytotoxicity

Eunchong Maeng 1, Ki Seo Ryu 1, A Reum Je 2, Minwook Lee 3, Hail Park 1, Seung Hee Choi 1,✉, Kyung-Soon Park 1,✉
PMCID: PMC13077993  PMID: 41792757

Abstract

Background

Natural killer (NK) cells are critical components with potent cytotoxic capabilities in immunosurveillance against cancer. However, their function is often impaired in the acidic tumor microenvironment due to mitochondrial dysfunction.

Methods

Chemically primed NK (Chem_NK) cells were generated with 25 kDa branched polyethylenimine. Cytotoxicity and motility assays were performed at physiological (pH 7.5) and acidic (pH 6.0) conditions. Building upon our previous proteomic profiling, which identified metabolic reprogramming in Chem_NK cells, we here investigated the functional consequences of these changes under environmental stress, and pharmacological inhibition studies were used to validate the role of oxidative phosphorylation (OXPHOS).

Results

Chem_NK cells treated with 25 kDa branched polyethylenimine maintain superior cytotoxicity and motility under acidic conditions. Proteomic and metabolic analyses revealed that OXPHOS was elevated in Chem_NK cells, which supported sustained mitochondrial ATP production and respiratory capacity at pH 6.0. Inhibition of OXPHOS abolished these advantages, confirming that mitochondrial respiration is essential for acid resistance. Mechanistically, Chem_NK cells preserve mitochondrial integrity through the protein kinase A (PKA)–dynamin-related protein 1 (DRP1) axis. Under acidic stress, DRP1 phosphorylation at Ser616 (S616) was reduced, while increased PKA activity elevated phosphorylation at Ser637 (S637), suppressing mitochondrial fragmentation.

Conclusions

These findings highlight mitochondrial preservation via the PKA–DRP1 axis as a key mechanism underlying NK cell function under acidic stress and suggest a potential strategy to enhance NK cell-based immunotherapy in solid tumors.

Graphical abstract

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Under acidic stress, the PKA–DRP1 axis regulates mitochondrial dynamics in NK cells. Compared with C_NK cells, Chem_NK cells display reduced DRP1 S616 phosphorylation and increased PKA-mediated DRP1 S637 phosphorylation. This shift preserves mitochondrial integrity, thereby sustaining OXPHOS. As a result, Chem_NK cells maintain higher migration and cytotoxic activity under acidic conditions.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12964-026-02786-3.

Keywords: Natural killer cell, Acidic stress, Mitochondria, PKA–DRP1 axis

Background

Natural killer (NK) cells are innate lymphocytes that play a central role in antitumor immunity by identifying and eliminating malignant cells through a sophisticated array of effector mechanisms [1, 2]. Their primary cytotoxic activity is mediated by the release of pre-formed granules containing perforin and granzymes, as well as by the engagement of death receptor ligands such as FasL and TRAIL [3]. In addition, NK cells function as key immunomodulators by secreting pro-inflammatory cytokines, including interferon-γ (IFN-γ) and TNF-α, thereby shaping adaptive immune response [2].

However, the activity of tumor-infiltrating NK cells is frequently compromised within the hostile tumor microenvironment (TME) [4–6]. Multiple suppressive mechanisms contribute to this dysfunction, including metabolic competition for essential nutrients, accumulation of immunosuppressive metabolites such as adenosine, prostaglandin E2, and excessive lactate [5, 7]. These factors, combined with chronic exposure to hypoxia and acidosis, lead to functional exhaustion and impaired immune surveillance.

Tumor cells commonly perform aerobic glycolysis, a process known as the “Warburg effect”, resulting in subsequent acidification of the TME [8, 9], which impairs NK cell viability, metabolism, and effector function [3]. Mitochondria act as central integrators of cellular metabolism, signaling, and cell fate decisions [10]. Acidic stress in the TME disrupts mitochondrial integrity in NK cells, promoting excessive mitochondrial fragmentation, loss of membrane potential, and metabolic collapse [11]. Consistently, tumor-infiltrating NK cells often display fragmented, punctate mitochondrial network, whereas NK cells in peripheral tissues exhibit elongated mitochondrial structures, a feature associated with superior cytotoxic capacity and improved clinical outcomes [12, 13]. Strategies that preserve mitochondrial integrity by limiting excessive fission have been shown to reinvigorate NK cell effector function within tumors [12].

At the metabolic level, resting NK cells primarily depend on oxidative phosphorylation (OXPHOS), while activated NK cells increase glycolytic flux to meet heightened energy demands [14]. Disruption of either metabolic pathway under TME-associated stress conditions limits cytokine production and cytotoxic capacity [15, 16]. Thus, strategies that enhance NK cell mitochondrial fitness and metabolic resilience represent promising avenues to improve NK cell-based adoptive immunotherapies.

In the present study, we demonstrate that Chem_NK cells retain robust cytotoxicity and migratory capacity under acidic conditions. Mechanistically, this phenotype is driven by preservation of mitochondrial integrity and suppression of mitochondrial fragmentation. We further identify activation of the protein kinase A (PKA)–dynamin-related protein 1 (DRP1) axis as a key mechanism underlying mitochondrial homeostasis and functional resilience in Chem_NK cells exposed to acidic stress.

Materials and methods

Cell culture

The human NK cell line NK-92MI, the human breast cancer cell line MDA-MB-231, and the mouse breast cancer cell line 4T1 were purchased from the American Type Culture Collection (Manassas, VA, USA). NK-92MI cells were cultured in Minimum Essential Medium Alpha (12561056, Gibco/Life Technologies, Grand Island, NY, USA) supplemented with 2 mM L-glutamine (25030081, Gibco/Life Technologies), 12.5% fetal bovine serum (12483020, Gibco/Life Technologies), 0.1 mM 2-mercaptoethanol (21985023, Gibco/Life Technologies), 1% penicillin/streptomycin (15140122, Gibco/Life Technologies), 0.02 mM folic acid (F8758, Sigma-Aldrich, St. Louis, MO, USA), and 0.2 mM inositol (I5125, Sigma-Aldrich). MDA-MB-231 cells were cultured in Dulbecco’s Modified Eagle Medium (11995073, Gibco/Life Technologies) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. 4T1 cells were cultured in RPMI 1640 medium (A1049101, Gibco/Life Technologies) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin.

Human primary NK (pNK) cells (purity > 95%) were isolated from peripheral blood mononuclear cells (PBMCs) obtained from healthy donors. PBMCs were separated using Ficoll-Paque PLUS (17-1440-02, Cytiva, MA, USA) and SepMate-50 tubes (STEMCELL Technologies, Vancouver, Canada), followed by magnetic separation with an NK Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). pNK cells were cultured in RPMI 1640 medium supplemented with GlutaMAX-I (35050061, Gibco/Life Technologies), 10% fetal bovine serum, and 1% penicillin/streptomycin, and maintained in the presence of recombinant human IL-2 (10 ng/mL) and IL-15 (5 ng/mL) (Peprotech, Rocky Hill, NJ, USA). All cells were incubated at 37 °C in a 5% CO2 incubator.

Chemical reagents

Twenty-five kDa branched polyethylenimine (25KbPEI; 408727, Sigma-Aldrich) was diluted to a concentration of 5 mg/mL in ultrapure distilled water (10977023, Thermo Fisher Scientific, Rochester, NY, USA). 25KbPEI was added to NK-92MI cells at a final concentration of 5 µg/mL, and to pNK cells at a final concentration of 1.25 µg/mL.

Lactic acid (LA; L6661, Sigma-Aldrich) and hydrochloric acid (HCl; 1128, DUKSAN PURE CHEMICALS, Ansan-si, Republic of Korea) were added to adjust the medium to pH 6.0. Sodium lactate (NaL; L7022, Sigma-Aldrich) was used to establish a lactate environment at the same molar concentration as LA.

The mitochondrial complex V inhibitor oligomycin (Oligo; 75351, Sigma-Aldrich), the mitochondrial complex I/III inhibitor rotenone/antimycin A (Rot/AA; R8875 and A8674, Sigma-Aldrich), the PKA inhibitor H-89 (B1427, Sigma-Aldrich), the PKA activator Forskolin (344270, Sigma-Aldrich), and the DRP1–FIS1 inhibitor P110 (S9887, Selleckchem, Houston, TX, USA) were administered to NK cells at the indicated concentrations and for the indicated incubation times.

Mitochondrial respiration assay

The oxygen consumption rate (OCR) of NK-92MI cells was assessed using a Seahorse XFe96 extracellular flux analyzer (Agilent, Santa Clara, CA, USA) with a Seahorse XF Cell Mito Stress Test Kit (103015-100, Agilent). NK-92MI cells were incubated at pH 7.5 or 6.0 for 1 h and washed twice with Dulbecco’s Phosphate-Buffered Saline (DPBS; Welgene, Gyeongsan-si, Republic of Korea). NK cells were resuspended in Seahorse XF RPMI medium (103576-100, Agilent) and plated at a density of 1 × 105 cells per well in a 96-well Seahorse plate (Agilent). The plate was pre-coated with 22.4 µg/mL Cell-Tak (354240; Corning, Newark, NJ, USA) dissolved in 0.1 M sodium bicarbonate (S5761, Sigma-Aldrich). To assess the OCR, 1.5 µM Oligo, 0.5 µM FCCP, and 0.5 µM Rot/AA were sequentially injected during the assay. Data were analyzed using Wave software version 2.6.3 (Agilent).

Measurement of the Mitochondrial Membrane Potential (MMP, ΔΨm)

NK-92MI cells were exposed to pH 7.5 or 6.0 for 1 h and washed twice with DPBS. To measure the ΔΨm, 2 × 105 NK cells were stained for 20 min with 100 nM Tetramethylrhodamine ethyl ester perchlorate (TMRE; T669, Invitrogen, Thermo Fisher Scientific). After staining, cells were washed twice with DPBS. Fluorescence was analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Indianapolis, IN, USA).

Cytotoxicity assay

Either NK cells or target cells were initially labeled with CellTrace CFSE (C34554, Thermo Fisher Scientific) to distinguish between one and other in coculture and subsequently co-incubated with target cells for 4 h at an effector-to-target ratio of 10:1 in medium adjusted to pH 7.5 or 6.0. Then, all cells were stained with 7-aminoactinomycin D (7-AAD; A1310, Thermo Fisher Scientific) to distinguish between live and dead cells, followed by fixation with 2% paraformaldehyde. Cytotoxicity was assessed using a CytoFLEX flow cytometer and FlowJo software (Tree Star, Ashland, OR, USA).

Live-cell imaging

For time-lapse live-cell imaging, GFP-expressing MDA-MB-231 cells (5 × 103) were seeded into a confocal plate 24 h prior to the experiment. NK-92MI cells were stained with CellTrace Far Red (C34572, Thermo Fisher Scientific) and washed twice with DPBS. The two cell types were co-cultured at a ratio of 1:1 in medium adjusted to pH 7.5 or 6.0. Imaging was performed using an Olympus FV3000 incubation system (Olympus, Hachioji-shi, Tokyo, Japan). Image data were analyzed using FV31S-SW Viewer software (Olympus).

Analysis of NK cell motility

NK-92MI cells were tracked using the Manual Tracking plugin (ibidi, Martinsried, Germany) for ImageJ (NIH, Bethesda, MD, USA), and the resulting tracks were analyzed with the Chemotaxis and Migration Tool (ibidi). Tracking was conducted at 3.5 min intervals per frame within 2 h of co-culture initiation. Tracking was terminated if an NK-92MI cell moved out of the field of view or formed a stable conjugate with a target cell. Migration trajectories were visualized using the Chemotaxis and Migration Tool, and cell velocity was calculated by dividing the accumulated migration distance by the total tracking time, as implemented in the software.

In vivo animal experiments

To evaluate the migratory capacity of NK-92MI cells within the TME, orthotopic 4T1 tumor models were established by injecting 2 × 105 4T1 cells into the fourth mammary fat pad of 5-week-old female nude/SCID mice (JA BIO, Suwon-si, Republic of Korea). To assess the migratory capacity of adoptively transferred NK-92MI cells within the acidic TME, tumors were allowed to grow to a volume of 300–400 mm3. At this stage, each mouse received two intravenous injections of 1 × 107 CFSE-labeled NK cells, administered 24 h apart. The day after the final injection, tumors were harvested, and infiltrated NK-92MI cells were visualized by immunofluorescence staining, while the proportion of migrated NK-92MI cells was analyzed by flow cytometry.

To evaluate phospho-dynamin-related protein 1 (DRP1) expression in tumor-infiltrating NK cells using the same orthotopic 4T1 tumor model, tumors were allowed to grow to a volume of 500–600 mm3, followed by a single intratumoral injection of 1 × 107 NK-92MI cells. Tumors were collected the following day, and infiltrating NK-92MI cells were analyzed using PE-conjugated anti-human CD45 (304058, BioLegend, San Diego, CA, USA) and anti-phospho-DRP1 (S637; PA5-37534, Thermo Fisher Scientific) antibodies.

All mice were maintained under specific pathogen-free conditions at CHA University’s animal facility (Seongnam, Republic of Korea). Experimental protocols were reviewed and approved by both the CHA University Institutional Animal Care and Use Committee (IACUC 240127, 250123) and the institutional ethics review board.

Immunofluorescence staining

Orthotopic 4T1 tumors were embedded in OCT compound (4583, Sakura, CA, USA) and sectioned at a thickness of 9 μm using a cryotome (Leica, Hesse, Germany). To visualize NK-92MI cell infiltration within tumor tissues, NK-92MI cells were labeled with CFSE prior to intravenous injection. Nuclei were counterstained with DAPI (F6057, Sigma-Aldrich). Frozen tumor sections were examined using a Zeiss LSM510 microscope (Carl Zeiss, Oberkochen, Germany) to detect CFSE-labeled NK-92MI cells within the tumor.

Flow cytometric analysis

Cells were fixed with 2% paraformaldehyde for 15 min and then washed with Stain Buffer (554656, BD Biosciences, Franklin Lakes, NJ, USA). Permeabilization was performed using FOXP3 Perm Buffer (353097, BioLegend) for 20 min at room temperature, and cells were subsequently washed with Stain Buffer. Cells were stained with antibodies for 30 min and then washed twice with Stain Buffer. Data were acquired using a CytoFLEX flow cytometer and analyzed with CytExpert (Beckman Coulter) and FlowJo software.

Migration assay

The migratory capacity of NK cells was evaluated using a 24-well Transwell insert chamber (353097, Corning). To evaluate NK cell motility, 3 × 103 NK cells stained with 1 µM CellTrace CFSE were loaded into the upper chamber containing serum-free medium at pH 6.0 with or without the indicated inhibitor. The pH of the complete medium in the bottom chamber was adjusted to match that of the upper chamber (pH 6.0). After 18 h, NK cells in the bottom chamber were harvested and counted using a Luna cell counter (Logos, Anyang-si, Republic of Korea). Imaging was performed with the EVOS M5000 imaging system (Thermo Fisher Scientific).

Immunoblot assay

A total of 2 × 106 NK cells were incubated in medium at pH 7.5 or 6.0 for 1 h and washed twice with DPBS. NK cells were lysed with Cell Lysis Buffer (#9803, Cell Signaling Technology, Danvers, MA, USA) containing 1% Triton X-100, supplemented with a protease and phosphatase inhibitor cocktail (P3300-001, GenDepot, Houston, TX, USA). Total proteins were extracted and heated at 95 °C for 10 min. Protein concentrations of cell lysates were measured by a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 3% bovine serum albumin (A0100-010, GenDEPOT) for 1 h and incubated overnight at 4 °C with the following primary antibodies: Phospho-PKA Substrate (RRXS*/T*) (#9624, Cell Signaling), DRP1 (#5391, Cell Signaling), phospho-Ser637 (S637) DRP1 (PA5-37534, Thermo Fisher Scientific), phospho-Ser616 (S616) DRP1 (#3455, Cell Signaling), MFN2 (#9482, Cell Signaling), FIS1 (sc-376447, Santa Cruz, Santa Cruz, CA, USA), ERK1/2 (#4695, Cell signaling), phospho-ERK1/2 (T202/Y204) (#4695, Cell signaling) and anti-GAPDH (sc-166574, Santa Cruz), After two washes with 1% Tween-20 (P1379, Sigma-Aldrich) prepared in Tris-buffered saline (1706435, Bio-Rad), the membranes were incubated with a secondary antibody for 1 h at room temperature. Immunoreactivity was detected using enhanced chemiluminescence solution (Thermo Fisher Scientific), and immunoreactive bands were visualized using LAS 4000 (GE HealthCare, Barrington, IL, USA). Immunoblots were then quantified using ImageJ software (NIH).

Transmission Electron Microscopy (TEM)

NK cells were incubated in medium at pH 7.5 or 6.0 for 1 h and then fixed with 2.5% glutaraldehyde prepared in 0.1 M cacodylate buffer (pH 7.3) at room temperature. After this fixation, the cells were treated with 2% OsO4 plus 3% potassium ferrocyanide prepared in 0.1 M cacodylate buffer (pH 7.3) for 1 h at 4 °C in the dark and embedded in Epon 812 after dehydration in an ethanol and propylene oxide series. Polymerization was conducted using pure resin at 70 °C for 2 days. Ultrathin Sect.  (70 nm) were obtained with an ultramicrotome (EM UC7; Leica, Vienna, Austria), which were then collected on 150-mesh copper grids. After staining with UranyLess (5 min) and lead citrate (3 min), the sections were examined by JEM-1400Plus TEM (JEOL Ltd., Tokyo, Japan) at 120 kV.

Statistical analysis

All data were statistically analyzed using GraphPad Prism v.9.3.0 software (GraphPad, La Jolla, CA, USA). The details of the statistical tests performed are indicated in the figure legends. Graphical data are presented as the mean ± standard deviation (SD) or as box-and-whisker or violin plots, as indicated in the figure legends. Statistical significance is indicated as follows: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001.

Results

Chem_NK cells with enhanced mitochondrial metabolism retain cytotoxicity under acidic stress

To investigate the mechanisms underlying the enhanced effector functions of Chem_NK cells, we first re-examined the proteomics-based metabolic landscape established in our previous study [17, 18]. Among 535 differentially expressed proteins (fold change > 1.25, p < 0.05), 332 proteins were associated with metabolic processes (Fig. 1A). KEGG pathway enrichment analysis further revealed significant enrichment of the oxidative phosphorylation (OXPHOS) pathway in Chem_NK cells (Fig. 1B). Based on this metabolic pre-adaptation, we hypothesized that Chem_NK cells may be better equipped to maintain functional integrity under the acidic constraints of the tumor microenvironment.

Fig. 1.

Fig. 1

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Chem_NK cells exhibit enhanced mitochondrial metabolism and acid resistance. A Pie graph showing expression of protein products of significantly regulated genes. Heatmap showing metabolic genes identified by proteomics analysis. B Dot plot showing the top enriched GO biological processes related to metabolism in Chem_NK cells based on DEGs, as determined by DAVID analysis. The number of genes enriched within each GO term is represented by dot size. Metabolic changes were confirmed in KEGG analysis. C OCR of C_NK and Chem_NK cells. D–F Parameters extracted from the OCR. (D) Basal and maximal respiration, (E) ATP production, and (F) SRC. G TMRE staining indicating the ΔΨm in C_NK and Chem_NK cells. Quantification of Mean Fluorescence intensity (MFI) is shown on the right. H Cytotoxicity of C_NK and Chem_NK cells against MDA-MB-231 cells was assessed by the CFSE/7-AAD assay at an E: T ratio of 10:1 in the presence of LA (lactate 20 mM, pH 6.0), NaL (lactate 20 mM, pH 7.5), and HCl (non-lactate, pH 6.0) for 4 h. All experiments were performed at least in triplicate. Statistical analyses were performed using an unpaired Student’s t-test for panels C–G and a two-way ANOVA with Tukey’s multiple comparisons test for panel H. Values represent the mean ± SD. ns, non-significant. **p< 0.01, ***p< 0.001, ****p< 0.0001.

To validate these proteomic findings, we assessed mitochondrial function using Seahorse XF analysis. Chem_NK cells exhibited a markedly higher OCR than C_NK cells (Fig. 1C), which was reflected by elevated basal and maximal respiration levels (Fig. 1D). Furthermore, Chem_NK cells demonstrated increased ATP production (Fig. 1E) and significantly greater spare respiratory capacity (SRC), indicative of enhanced metabolic flexibility under stress (Fig. 1F). TMRE staining further confirmed that Chem_NK cells maintained a higher ΔΨm than C_NK cells, indicative of higher electron transport chain activity (Fig. 1G).

Given that prior reports link mitochondrial fitness to resistance against acidic stress in immune cells [11, 13], we investigated whether Chem_NK cells could better withstand LA-induced functional suppression under a TME-mimicking environment. To first characterize the impact of extracellular acidity on NK-92MI cell function, cytotoxicity was assessed across a range of LA-induced pH conditions. We observed a progressive, pH-dependent reduction in NK-92MI cytotoxicity, confirming lactate-driven acidosis as a key determinant of functional impairment (Supplementary Fig. 1). Specifically, to differentiate the effects of low pH from those of lactate itself, NK-92MI cells were exposed for 4 h to one of the following: LA (20 mM, pH 6.0), NaL (20 mM, pH 7.5), or HCl (pH 6.0). Cytotoxic activity was assessed using the CFSE/7-AAD-based assay. LA exposure significantly reduced NK-92MI cytotoxicity, whereas NaL had minimal impact. Notably, HCl-induced cytotoxic impairment was comparable to that observed with LA, suggesting that acidic pH, rather than lactate per se, is the primary driver of NK-92MI cell dysfunction (Fig. 1H). In addition, Chem_NK cells maintained superior cytotoxic activity compared with C_NK cells across all acidic stress conditions (Supplementary Fig. 2), exhibiting significantly greater resilience upon both LA and HCl exposure. Acidic conditions did not compromise NK-92MI viability, indicating that the observed functional impairment was not attributable to cell death (Supplementary Fig. 3). Moreover, elevated expression of activating receptors in Chem_NK cells, as previously reported [17], was preserved under pH 6.0 conditions (Supplementary Fig. 4).

Taken together, these findings show that Chem_NK cells display both enhanced OXPHOS and preserved cytotoxic function under acidic conditions.

Chem_NK cells rely on enhanced mitochondrial functions to sustain cytotoxicity under acidic stress

Mitochondrial and metabolic fitness is essential for NK cell function in the TME [19], where acidic stress presents a major immunosuppressive barrier. Building upon our earlier observations, we hypothesized that the superior mitochondrial respiration observed in Chem_NK cells may underpin their preserved cytotoxicity under acidosis.

To test this, we evaluated mitochondrial function in C_NK and Chem_NK cells under acidic conditions (pH 6.0). As seen at neutral pH (pH 7.5), Chem_NK cells maintained a significantly higher OCR than C_NK cells, even at pH 6.0 (Fig. 2A). Specifically, Chem_NK cells exhibited elevated basal and maximal respiration, ATP production, and SRC, all of which remained significantly higher than those in C_NK cells at pH 6.0 (Fig. 2B–D). TMRE staining further confirmed that Chem_NK cells preserved a higher ΔΨm under acidic conditions, indicative of sustained mitochondrial activity (Fig. 2E). Collectively, these results demonstrate that Chem_NK cells retain robust mitochondrial function even in the presence of acidosis.

Fig. 2.

Fig. 2

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Chem_NK cells maintain high mitochondrial metabolism under acidosis, which confers acid resistance. A OCR of C_NK and Chem_NK cells after exposure to pH 6.0 for 1 h. B–D Parameters extracted from the OCR. (B) Basal and maximal respiration, (C) ATP production, and (D) SRC. E TMRE staining indicating the ΔΨm after exposure to pH 6.0 for 1 h in C_NK and Chem_NK cells. MFI is shown on the right. F Cytotoxicity of C_NK and Chem_NK cells was evaluated at pH 6.0 at an E: T ratio of 10:1 in the presence or absence of Oligo (1 µM) or Rot/AA (0.5 µM) for 4 h, with a 30 min pre-treatment with Oligo and Rot/AA. All experiments were performed at least in triplicate. Statistical analyses were performed using an unpaired Student’s t-test for panels B–E and a two-way ANOVA with Tukey’s multiple comparisons test for panel F. Values represent the mean ± SD. ns, non-significant. **p<0.01, ***p<0.001, ****p<0.0001.

To determine whether this mitochondrial advantage directly contributes to cytotoxic activity under acidic stress, we performed cytotoxicity assays following pharmacological inhibition of OXPHOS (Fig. 2F). Both C_NK and Chem_NK cells were treated with Oligo (a complex V inhibitor) or Rot/AA (complex I/III inhibitors), and then exposed to pH 6.0. In C_NK cells, cytotoxic activity was already suppressed by the acidic conditions and was only marginally affected by OXPHOS inhibition. By contrast, OXPHOS inhibition significantly diminished the cytotoxic function of Chem_NK cells, which was otherwise preserved under acidic conditions .

These findings indicate that the sustained antitumor function of Chem_NK cells under acidosis is critically dependent on their enhanced mitochondrial respiration. This underscores the pivotal role of metabolic reprogramming in conferring resistance to TME-associated immunosuppression.

Enhanced mitochondrial activity of Chem_NK cells promotes migration and tumor engagement under acidic conditions

Immune cell migration is closely linked to mitochondrial oxidative metabolism and ATP production [20–23]. Based on these insights, we assessed the migratory capacity of NK-92MI cells under pH 6.0 conditions using a transwell migration assay (Supplementary Fig. 5). Chem_NK cells exhibited significantly enhanced migratory capacity compared with C_NK cells and, notably, maintained migration at levels comparable to those observed under control pH conditions despite acidic stress. Importantly, a similar preservation of migratory capacity under acidic conditions was also observed in chemically primed pNK cells, indicating that this phenotype is not restricted to an engineered NK cell line (Supplementary Fig. 6). We therefore hypothesized that the OXPHOS-dependent resistance of Chem_NK cells to acidosis may be associated with enhanced migratory capacity, which in turn facilitates effective tumor engagement. To test this, we conducted time-lapse imaging of NK-92MI cells co-cultured with MDA-MB-231 tumor cells under pH 6.0 conditions. Cell migration was monitored for the first 1.5 h to assess motility and tumor cell contact (Fig. 3A) [24].

Fig. 3.

Fig. 3

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Mitochondrial metabolism in Chem_NK cells supports their migration and tumor infiltration in acidosis, which contributes to antitumor activity. A Schematic illustration showing the sequential process of NK cell migration, target recognition, and killing. B Representative time-lapse images of C_NK or Chem_NK cells (outlined with white dotted lines) exhibiting motility and interacting with MDA-MB-231 cells (green) at pH 6.0. Nuclei were stained with DAPI (blue). Red arrows indicate points of contact between NK cells and target cells. Scale bar, 20 μm. Quantitative killing rates (%) of target cells are shown in the accompanying graph. (C_NK: n=64; Chem_NK: n=62) C Cell trajectory plots of C_NK and Chem_NK cells co-cultured with MDA-MB-231 cells at pH 6.0. Corresponding quantification of migration velocity is shown (C_NK: n=55; Chem_NK: n=46). Each dot represents an individual cell. D Immunofluorescence staining of NK-92MI cells (green, CFSE) and nuclei (blue, DAPI) in the indicated tumor sections. Scale bar, 100 μm. E The percentage of C_NK and Chem_NK cells that migrated into tumor tissue determined by flow cytometry (n=5). F Migration assay of C_NK and Chem_NK cells incubated in medium at pH 6.0 for 12 h, with or without Oligo (1 µM) or Rot/AA (0.5 µM). NK cells were pre-treated with inhibitors for 30 min prior to the assay. Representative fluorescence images (left) and quantification of relative migration ratios (right) are shown. Scale bar, 100 μm. All experiments were performed at least in triplicate. Statistical analyses were performed using an unpaired Student’s t-test for panels B, C, and E and a two-way ANOVA with Tukey’s multiple comparisons test for panel F. Values represent the mean ± SD. ns, non-significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Chem_NK cells demonstrated more rapid target recognition and efficient killing of tumor cells, whereas C_NK cells exhibited delayed contact and reduced cytolytic activity (Fig. 3B). Tracking analysis revealed that Chem_NK cells exhibited significantly greater motility, which was characterized by higher migration velocities and more dynamic movement patterns, in contrast to the limited, linear trajectories of C_NK cells (Fig. 3C).

To assess whether the superior migratory capacity of Chem_NK cells translates into improved tumor infiltration in vivo, we used the 4T1 mouse model, which generates an acidic tumor microenvironment [25–27]. We adoptively transferred CFSE-labeled NK-92MI cells into 4T1 tumor-bearing mice and tumor tissues were harvested and analyzed to assess NK-92MI cell infiltration (Supplementary Fig. 7). Fluorescence imaging of harvested tumor sections revealed that Chem_NK cells achieved significantly greater infiltration depth and density compared with C_NK cells (Fig. 3D). To quantify this, tumor tissue was dissociated and analyzed using flow cytometry. Chem_NK cells infiltrated tumors at significantly higher levels than C_NK cells, indicative of a superior ability to infiltrate the acidic TME (Fig. 3E). These findings confirm that chemical priming effectively promotes NK cell infiltration within the acidic TME in vivo.

To determine whether this enhanced migratory phenotype is dependent on mitochondrial function, NK-92MI cells were treated with Oligo or Rot/AA and then transwell migration assays were performed. OXPHOS inhibition markedly reduced the enhanced motility of Chem_NK cells, indicating that respiratory activity of mitochondria is essential for sustaining migration under acidic conditions (Fig. 3F). Taken together, these results suggest that the superior mitochondrial fitness of Chem_NK cells enables enhanced motility, which facilitates rapid tumor cell engagement and contributes to their improved infiltration within the acidic TME.

Chem_NK cells resist acidic stress by preserving mitochondrial integrity via the PKA–DRP1 axis

Mitochondrial dynamics are critical regulators of NK cell function because they directly influence metabolic activity and immune surveillance. It has been reported that exposure to the TME induces mitochondrial fragmentation and dysfunction in NK cells, which are largely mediated by activation of DRP1 [12]. Phosphorylation of DRP1 at S616 promotes mitochondrial fission, while its phosphorylation at S637 by PKA inhibits DRP1 activation and prevents fragmentation (Fig. 4A) [28].

Fig. 4.

Fig. 4

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Chem_NK cells resist acidosis-induced mitochondrial fragmentation via the PKA–DRP1 axis. A Schematic of the PKA–DRP1 regulatory axis. PKA-mediated phosphorylation of DRP1 at S637 inhibits mitochondrial fission, while phosphorylation at S616 promotes fission. B–C Western blot analysis of NK cells cultured at pH 7.5 and 6.0. B Detection of phosphorylated PKA substrates. C DRP1 phosphorylation status showing levels of p-DRP1S637 and p-DRP1S616. Quantification graphs are shown on the right. D TEM images of mitochondria in NK cells cultured at pH 7.5 and 6.0. Red boxes indicate regions shown in the magnified insets, and red outlines highlight individual mitochondria. Scale bars, 1 μm. Quantification of mitochondrial length from TEM images is shown on the right. E Schematic of the 4T1 orthotopic breast cancer mouse model. C_NK or Chem_NK cells were intratumorally injected to evaluate p-DRP1S637 expression in tumor-infiltrating NK cells. F Flow cytometric analysis of dissociated tumor tissues comparing p-DRP1S637 expression between C_NK cells and Chem_NK cells (n = 5). MFI is shown on the right. All experiments were performed at least in triplicate. Statistical analyses were performed using a two-way ANOVA with Tukey’s multiple comparisons test for panel B–D and an unpaired Student’s t-test for panel F. Values represent the mean ± SD. ns, non-significant. *p<0.05, **p<0.01.

To investigate whether the acid resistance of Chem_NK cells involves protection against mitochondrial fragmentation, we assessed PKA activity and DRP1 phosphorylation following exposure to pH 6.0. Immunoblot analysis revealed that phosphorylation of PKA substrates was higher in Chem_NK cells than in C_NK cells, indicative of elevated PKA activity under acidosis (Fig. 4B). Correspondingly, p-DRP1S637 levels were significantly increased, while p-DRP1S616 levels were decreased in Chem_NK cells relative to C_NK cells (Fig. 4C), indicative of inhibition of DRP1-mediated fission. Importantly, a similar phosphorylation pattern of DRP1 was observed in pNK cells subjected to the same acidic conditions, confirming that PKA–DRP1 axis–mediated regulation of mitochondrial dynamics is conserved in pNK cells (Supplementary Fig. 8). To determine whether chemical priming induces mitochondrial remodeling beyond the PKA–DRP1 axis, we performed additional immunoblot analyses of mitochondrial dynamics–related proteins under acidic stress (Supplementary Fig. 9). MFN2 levels showed a modest reduction at pH 6.0 in both C_NK and Chem_NK cells, irrespective of 25KbPEI treatment, and FIS1 expression remained unchanged, indicating that alterations in these fusion/fission regulators are not the dominant contributors to mitochondrial preservation in our system. In parallel, we examined ERK1/2 signaling as a potential upstream regulator of DRP1 S616 phosphorylation [29–31]. Consistent with the observed changes in DRP1 S616 phosphorylation, phospho-ERK1/2 levels were markedly increased in C_NK cells under acidic conditions but were only weakly induced in Chem_NK cells. These molecular findings were corroborated by ultrastructural analysis using TEM. At pH 6.0, C_NK cells exhibited shortened, fragmented mitochondria, consistent with DRP1 activation. By contrast, Chem_NK cells retained elongated mitochondrial structures, indicative of preserved mitochondrial integrity (Fig. 4D). These results suggest that, under acidic stress, mitochondrial integrity in Chem_NK cells is preserved through suppression of the ERK–DRP1 pro-fission pathway rather than through broad remodeling of the mitochondrial dynamics machinery, further supporting the PKA–DRP1 axis as a key mechanism.

To assess whether these molecular changes in DRP1 also occur in vivo, we injected NK-92MI cells intratumorally into an orthotopic 4T1 tumor model in nude mice and examined p-DRP1S637 levels (Fig. 4E). Consistent with the in vitro findings, flow cytometric analysis of tumor-infiltrating NK-92MI cells showed that, under the acidic TME, Chem_NK cells maintained significantly higher p-DRP1S637 level than C_NK cells, further supporting the physiological relevance of this pathway (Fig. 4F). Furthermore, temporal analysis showed that the favorable DRP1 S637/S616 phosphorylation ratio in Chem_NK cells is most robustly maintained during the early phase of acidic stress, particularly within the first 1 h, before gradually declining by 12 h (Supplementary Fig. 10). This early window of mitochondrial protection is likely sufficient to support initial NK cell effector functions, including tumor infiltration and target cell lysis under acidic conditions. From a therapeutic perspective, this transient but potent metabolic resilience suggests that chemical priming can empower NK cells to overcome the initial inhibitory barriers of the acidic tumor microenvironment.

Collectively, these results indicate that the enhanced resistance of Chem_NK cells to acid-induced mitochondrial dysfunction is mediated through increased PKA activity and suppression of DRP1-dependent mitochondrial fragmentation.

The PKA–DRP1 axis governs acid resistance and antitumor function of Chem_NK cells

H-89 is a well-characterized pharmacological inhibitor of PKA that suppresses phosphorylation of DRP1 at S637 (Fig. 5A) [32, 33]. To determine whether the PKA–DRP1 signaling axis underlies the acid resistance of Chem_NK cells, we treated cells with H-89 and evaluated functional changes under pH 6.0 conditions. Immunoblot analysis confirmed that the elevated levels of p-DRP1S637 observed in Chem_NK cells under acidic stress were markedly reduced following H-89 treatment (Fig. 5B), indicating effective blockade of this pathway.

Fig. 5.

Fig. 5

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Chem_NK cells preserve functional activity in acidosis through the PKA–DRP1 axis. A Schematic of the PKA–DRP1 regulatory axis and the effect of H-89 treatment. H-89, a pharmacological inhibitor of PKA, suppresses PKA activity and thereby reduces DRP1S637 phosphorylation, resulting in increased mitochondrial fission. B Western blot analysis of p-DRP1S637 in NK cells cultured at pH 6.0 with or without H-89. Quantification is shown on the right. C OCR of Chem_NK cells after exposure to pH 6.0 for 1 h with or without H-89. D–F Parameters extracted from the OCR. (D) Basal and maximal respiration, (E) ATP production, and (F) SRC. G Migration assay of C_NK and Chem_NK cells incubated at pH 6.0 for 12 h in the presence or absence of H-89. Representative fluorescence images (left) and quantification of relative migration ratios (right) are shown. Scale bar, 100 μm. H Cytotoxicity of C_NK and Chem_NK cells was evaluated at pH 6.0 at an E: T ratio of 10:1 in the presence or absence of H-89 for 4 h. H-89 (20 µM, 30 min) was applied before all experiments. All experiments were performed at least in triplicate. Statistical analyses were performed using a two-way ANOVA with Tukey’s multiple comparisons test for panel B, G, and H and an unpaired Student’s t-test for panel D–F. Values represent the mean ± SD. ns, non-significant. *p<0.05, **p<0.01, ***p<0.001.

Given the close relationship between mitochondrial dynamics and oxidative metabolism, we next examined whether PKA signaling directly contributes to the maintenance of mitochondrial respiration in Chem_NK cells. PKA inhibition significantly suppressed basal and maximal respiration, ATP production, as well as SRC (Fig. 5C–F), indicating that the metabolic advantage of Chem_NK cells is intrinsically linked to the activation of the PKA signaling pathway. Functionally, PKA inhibition had little impact on the already low motility and cytotoxic ability of C_NK cells (Fig. 5G, H). However, the enhanced motility and cytotoxic activity of Chem_NK cells were significantly diminished by H-89, which reduced them to levels comparable with those of C_NK cells.

Altogether, these findings indicate that PKA-mediated regulation of DRP1 is a critical determinant linking mitochondrial preservation to sustained migratory and cytotoxic capacities of Chem_NK cells under acidic stress.

The PKA–DRP1 axis preserves NK cell metabolic fitness and effector functions under acidic stress

Based on these results, we hypothesized that pharmacological activation of PKA or inhibition of DRP1-mediated mitochondrial fission might enhance the resistance of NK-92MI cells to acidic stress. To first examine the contribution of PKA signaling, we employed forskolin, a well-established small-molecule activator of adenylyl cyclase that elevates intracellular cAMP levels and thereby activates PKA [33–35]. Forskolin at 50 µM did not affect NK-92MI cells viability and was sufficient to induce PKA phosphorylation (Supplementary Fig. 11, 12). Under acidic conditions, forskolin treatment restored the impaired mitochondrial respiratory capacity of C_NK cells, as evidenced by significant recovery of basal respiration, maximal respiration, ATP production, and SRC (Fig. 6A–D). Consistent with the restoration of mitochondrial bioenergetics, forskolin-treated C_NK cells also exhibited a marked recovery of migratory capacity under acidic stress, reaching levels comparable to those observed in Chem_NK cells (Fig. 6E). However, despite these improvements in mitochondrial function and migration, forskolin treatment failed to restore cytotoxic activity (Supplementary Fig. 13A). Our previous findings indicated that the enhanced cytotoxicity of Chem_NK cells is partially attributable to increased perforin accumulation [17]. This lack of cytotoxic rescue was associated with the absence of intracellular perforin accumulation, indicating that activation of the PKA axis alone is insufficient to prime cytolytic effector machinery under acidic conditions (Supplementary Fig. 13B). These observations suggest that while activation of the PKA–DRP1 axis contributes to maintaining NK cell motility under acidic stress, restoration of full cytotoxic function requires additional mechanisms.

Fig. 6.

Fig. 6

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The PKA–DRP1 axis preserves effector function of NK cells in acidosis. A OCR of C_NK cells after exposure to pH 6.0 for 1 h with or without Forskolin. B–D Parameters extracted from the OCR. (B) Basal and maximal respiration, (C) ATP production, and (D) SRC. E Migration assay of C_NK and Chem_NK cells incubated at pH 6.0 for 12 h in the presence or absence of Forskolin. Representative fluorescence images (left) and quantification of relative migration ratios (right) are shown. Scale bar, 100 μm. F OCR of C_NK cells after exposure to pH 6.0 for 1 h with or without P110. G–I Parameters extracted from the OCR. (G) Basal and maximal respiration, (H) ATP production, and (I) SRC. J Migration assay of C_NK and Chem_NK cells incubated at pH 6.0 for 12 h in the presence or absence of P110. Representative fluorescence images (left) and quantification of relative migration ratios (right) are shown. Scale bar, 100 μm. K Cytotoxicity of C_NK and Chem_NK cells was evaluated at pH 6.0 at an E: T ratio of 10:1 in the presence or absence of P110 for 4 h. Forskolin (50 µM, 30 min) or P110 (0.5 µM, 24 h) was applied before the indicated experiments. All experiments were performed at least in triplicate. Statistical analyses were performed using an unpaired Student’s t-test for panel B, C, D, F, H, and I and a two-way ANOVA with Tukey’s multiple comparisons test for panel E, J, and K. Values represent the mean ± SD. ns, non-significant. *p<0.05, **p<0.01, ****p<0.0001.

To further dissect the contribution of mitochondrial dynamics, we next specifically targeted DRP1 using P110, a selective peptide inhibitor that disrupts pathological DRP1–FIS1 interactions [36–38]. Inhibition of DRP1 with P110 significantly enhanced mitochondrial oxidative metabolism in NK-92MI cells under acidic conditions, as demonstrated by increased basal respiration, maximal respiration, ATP production, and SRC (Fig. 6F–I). Also, P110 treatment restored migratory capacity under acidic stress to levels comparable to those of Chem_NK cells (Fig. 6J). Notably, DRP1 inhibition also led to a partial recovery of cytotoxic activity (Fig. 6K), suggesting that suppression of mitochondrial fragmentation directly contributes not only to metabolic fitness and migration but also, to a limited extent, to effector function under acidic stress. In summary, these data highlight that the PKA–DRP1 axis plays a central role in regulating NK cell migration under acidic stress, while robust antitumor function under such conditions appears to require coordinated enhancement of both mitochondrial dynamics and cytotoxic effector machinery.

Discussion

In our previous studies, we demonstrated that Chem_NK cells not only infiltrate solid tumors more effectively but also exhibit superior antitumor efficacy compared with C_NK cells [17]. Although it is well-known that the antitumor functions of NK cells are significantly compromised within the TME [4–6], our findings suggested that Chem_NK cells possess an enhanced ability to overcome these inhibitory conditions. To further elucidate the mechanisms underlying this resistance, we focused on one of the key stressors within the TME, namely, acidic stress, and investigated the specific advantages of Chem_NK cells under such conditions.

To address this limitation, our study identifies a previously unappreciated mitochondrial adaptation mechanism that enables Chem_NK cells to retain functionality under acidic stress. Our findings clearly demonstrate that NK cells experience a significant reduction in antitumor activity when exposed to acidic conditions. This is consistent with previous reports showing that acidic pH in the TME can inhibit effector functions of various immune cells, including NK cells [39, 40]. We identified that diminished cytotoxicity in NK cells under acidic stress is largely attributed to impaired migration toward cancer cells, a critical function for tumor surveillance and elimination. Furthermore, mitochondrial integrity is essential for NK cell antitumor responses, and the preservation of mitochondrial dynamics under hostile TME conditions emerges as a key determinant of therapeutic efficacy. Accordingly, our data identify the PKA–DRP1 signaling axis as a central regulatory mechanism through which Chem_NK cells preserve mitochondrial stability under acidic stress, conferring robust resistance to acid-induced mitochondrial fragmentation and a fundamental metabolic advantage via chemical priming. Collectively, these findings underscore a major obstacle for NK cell–based therapies in solid tumors with acidic TMEs, while simultaneously revealing a mechanism by which Chem_NK cells overcome this limitation.

Mitochondria are dynamic organelles whose morphology is regulated by balanced fusion and fission processes, which are critical for cellular metabolism and survival [41, 42]. Disruption of this balance, particularly excessive DRP1-mediated mitochondrial fission, has been associated with impaired NK cell antitumor function, as exemplified by hypoxia-driven mTOR–DRP1 activation in liver cancer–infiltrating NK cells [12]. Consistent with this paradigm, we found that acidic stress similarly induces pronounced mitochondrial fragmentation in NK cells, leading to defective energy production and impaired migration. To counteract this stress-induced mitochondrial dysfunction, Chem_NK cells selectively engage a protective regulatory mechanism centered on DRP1 phosphorylation. As PKA-mediated DRP1 phosphorylation at S637 is a known stress-responsive event [43, 44], our data indicate that acidic stress acts as a trigger for this adaptive response. Under physiological pH, mitochondrial networks in NK cells are already stable, so DRP1 phosphorylation at S637 is not required to maintain their dynamics. This selective activation of the PKA–DRP1 axis explains how Chem_NK cells maintain mitochondrial integrity and bioenergetic capacity in hostile TMEs, thereby sustaining their antitumor activity.

The potent priming effect observed in our study is likely initiated by the high cationic charge density of 25KbPEI. Polycationic molecules are known to interact with mitochondrial membranes, where they can modulate electrochemical gradients and trigger the production of reactive oxygen species (ROS). Indeed, we have observed that 25KbPEI treatment induced a rapid rise in MMP and mtROS of NK-92MI cells (data not shown). It suggests that 25KbPEI-mediated priming utilizes these transient stress signals as a form of mitohormesis, activating downstream pathways such as the PKA–DRP1 axis to enhance mitochondrial fitness. While redox signaling is a plausible driver of this metabolic rewiring, the exact molecular sensors governing this adaptation warrant further investigation to fully map the signaling landscape of chemically primed NK cells.

Chemical priming with 25KbPEI activates an additional Ca²⁺-dependent signaling axis involving TRPM2, which facilitates intracellular perforin accumulation [17]. Because NK cell cytotoxicity critically depends on the accumulation and availability of cytolytic granules, particularly perforin [45], this pathway is essential for restoring effective target cell killing under acidic stress. In contrast, PKA–DRP1 signaling primarily contributes to mitochondrial fitness and oxidative metabolism and does not directly promote perforin accumulation. Consistent with this distinction, pharmacological activation of the PKA axis using forskolin or inhibition of DRP1-mediated mitochondrial fission using P110 effectively restored mitochondrial integrity and oxidative metabolism, which was sufficient to recover migratory capacity. However, these interventions failed to fully restore cytotoxic function, indicating that mitochondrial preservation and bioenergetic support alone are insufficient to re-establish robust NK cell-mediated cytotoxicity under acidic conditions. Together, these findings demonstrate that restoration of NK cell migration and cytotoxicity is governed by partially overlapping but mechanistically distinct pathways, and that the dual engagement of mitochondrial protection and Ca2+-dependent perforin accumulation enables Chem_NK cells to simultaneously preserve migration and potent cytotoxicity in acidic conditions.

Taken together, our findings identify the PKA–DRP1 axis as a critical regulator of mitochondrial integrity in NK cells under acidic stress. Through chemical priming–induced modulation of this pathway in Chem_NK cells, we demonstrate how stress-adaptive control of mitochondrial dynamics preserves migratory capacity in the TME. More broadly, these results highlight mitochondrial regulation via the PKA–DRP1 axis as a central determinant of NK cell resilience within the TME and suggest a rational strategy for enhancing NK cell–based immunotherapies against solid tumors.

Conclusions

In conclusion, our findings highlight the detrimental effects of acidic stress on NK cell function and underscore the therapeutic potential of Chem_NK cells as a viable strategy to overcome hostile TME. Further studies dissecting the mechanisms by which Chem_NK cells preserve mitochondrial integrity and activity under acidic stress will be essential for advancing their clinical applications.

Supplementary Information

Supplementary Material 1. (514.8KB, docx)
Supplementary Material 2. (213.3KB, pdf)

Acknowledgements

Not applicable.

Abbreviations

NK cells

Natural killer cells

25KbPEI

25kDa branched polyethylenimine

C_NK cells

Control NK-92MI cells

Chem_NK cells

NK-92MI cells primed by 25KbPEI

TME

Tumor microenvironment

OXPHOS

Oxidative phosphorylation

MMP

Mitochondrial membrane potential

PKA

Protein kinase A

DRP1

Dynamin-related protein 1

pNK

Human primary NK

C_pNK

Control primary NK

Chem_pNK

Primary NK cells primed by 25KbPEI

Authors’ contributions

SHC and K-S P designed the research; EM, SHC, KSR, ARJ, ML and HP performed experiments; SHC and EM analyzed data; K-S P and SHC wrote the manuscript; K-S P and SHC supervised the project and reviewed and edited the manuscript.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (Ministry of Science and ICT) (No. RS-2025-00557411, RS-2023-00271041 to SHC.; RS-2025-20182971 and RS-2019-NR40073 to K-S P.).

Data availability

All relevant data are available from the corresponding author.

Declarations

Competing interests

The authors declare no competing interests.

Conflict of interest

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Seung Hee Choi, Email: choicat14@gmail.com.

Kyung-Soon Park, Email: kspark@cha.ac.kr.

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Associated Data

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Supplementary Materials

Supplementary Material 1. (514.8KB, docx)
Supplementary Material 2. (213.3KB, pdf)

Data Availability Statement

All relevant data are available from the corresponding author.

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