PrP^C Glycoprotein Modulates Atmospherically Relevant Artificial Particulate Matter-Induced Development of Lung Cancer in Mice

Abstract

Fine particulate matter with a diameter less than 2.5 μm (PM_2.5) is an environmental risk factor for lung cancer. However, the molecular mechanisms linking PM_2.5 exposure to tumorigenesis remain unclear. We identified the cellular prion protein (PrP^C) as a critical regulator of susceptibility to PM_2.5-induced lung pathologies. PrP^C and Sirt1 expression levels were lower, whereas HIF-1α expression was higher, in aged compared to younger C57BL/6 mice, which correlated with increased mortality and lung cancer susceptibility following PM_2.5 exposure. Prnp mice (PrP^C wild-type (WT) and knockout (KO) mice) were exposed to PM_2.5 at 50 μg/m³ for 2 h per day over 5 days. Two PM_2.5 sources were used: a synthetic ion–organic acid mixture and an urban standard (NIST 1648a), which are rich in heavy metals and polycyclic aromatic hydrocarbons. Lung pathology was evaluated by using imaging, histology, immunohistochemistry, and Western blotting. PrP^C deficiency recapitulated and exacerbated age-associated pathology, promoting emphysema, hypoxia, angiogenesis, and tumorigenesis via dysregulating the Sirt1-p53-HIF1α axis. NIST triggered more aggressive tumorigenesis than the synthetic mixture, underscoring the role of particle composition. PM_2.5 has environmental and public health impacts, particularly in older adults, and PrP^C is a mechanistic regulator and potential biomarker of pollution-associated lung cancer.

Introduction

The cellular prion PrP^C, encoded by the Prnp gene on chromosome 20 in humans and chromosome 2 in mice, is widely expressed across various organs and tissues. The highest levels of PrP^C expression occur in the central nervous system, where it plays a crucial role in neuronal function and protects against neurodegenerative diseases. Research has shown that PrP^C exerts neuroprotective effects by preventing neuronal apoptosis, combating oxidative stress, , and facilitating synaptic function. Conversion of PrP^C into its misfolded and pathogenic isoform, PrP^Sc, is a well-known hallmark of prion diseases. Extensive research has investigated PrP^C’s physiological roles beyond its association with these disorders. While the nervous system has been the primary focus of PrP^C research, recent studies have revealed that PrP^C is also expressed in a variety of other organs, including the heart, liver, kidneys, and lungs. In the lungs, PrP^C is found in the alveolar walls and is present in lung epithelial cells, including alveolar types I and II cells, as well as Clara cells. This suggests that they may play a role in respiratory health. PrP^C has been shown to protect mice from lethal infection with the influenza A virus and the human bronchial epithelial barrier against oxidative stress. However, the functional role of PrP^C in the lungs, particularly in lung cancer, remains largely unelucidated, and further research is needed to fully understand its role in lung physiology. Given the lungs’ direct exposure to environmental pollutants, exploring the function of PrP^C in this context is particularly interesting.

The escalating concentration of fine particulate matter (PM) in the atmosphere is causing detrimental effects on multiple fronts, including in the heart, lungs, brain, immune system, and metabolism. , The presence of PM with a diameter of 2.5 μm or smaller (PM_2.5) is linked to an increased incidence of respiratory ailments such as asthma, chronic obstructive pulmonary disease (COPD), and adult-onset lung cancer, resulting in a decline in pulmonary function. , Among these adverse health outcomes, lung cancer is the most prevalent disease associated with PM_2.5 exposure. Numerous studies have established a compelling connection between PM_2.5 and the heightened risk of lung cancer and subsequent mortality. The American Cancer Society has affirmed the existence of a causal relationship between PM_2.5 exposure and elevated risk of death from lung cancer. Nevertheless, the precise mechanistic underpinnings of the influence of PM_2.5 on the development of lung cancer remain unclear.

In our recent study, we found that exposure to PM2.5 during pregnancy has a detrimental effect on the fetal lungs, inducing oxidative stress. These harmful effects persist into adulthood and contribute to the senescence of hematopoietic stem cells, ultimately leading to the development of myeloproliferative disease. We demonstrated that exposure of newborn mice to PM_2.5 results in initial lung damage that persists throughout their lifespan. Specifically, exposure to PM_2.5 induced early and extensive lung inflammation in infant mice, which persisted up to 6 and 12 months postexposure, indicating that the effects of PM_2.5 did not diminish over time. This prompted us to investigate the effects of PM_2.5 in other mouse models, including aged and transgenic mice. In this study, we found that PM_2.5 exposure resulted in lung cancer development and a high related mortality rate in older mice, accompanied by decreases in Prnp gene expression and PrP^C protein levels. These findings led us to focus on the impact of PM_2.5 in PrP^C-deficient mice.

Recent research has highlighted the destructive impact of PM_2.5 on lung tissue, including its ability to induce oxidative stress and inflammation, both of which are key drivers of carcinogenesis. We also considered the role of emphysema, a chronic lung condition characterized by the destruction of alveoli that eventually leads to reduced respiratory function and chronic hypoxia. Chronic inflammation, oxidative stress, and the resultant tissue remodeling associated with emphysema create an environment conducive to tumor growth. Several studies have reported that patients with emphysema are at increased risk of lung cancer, with the chronic inflammatory state being a key factor in this association. To explore this further, we utilized Prnp KO mice, which lack PrP^C, to investigate whether the absence of PrP^C would exacerbate the harmful effects of PM_2.5 exposure, leading to increased lung cancer incidence. Our findings revealed that Prnp KO mice exposed to atmospherically relevant artificial PM_2.5 exhibited significantly higher rates of lung damage, emphysema, and lung cancer than wild-type mice. This suggests that PrP^C plays a critical role in mitigating the adverse effects of PM_2.5 on lung tissue by modulating oxidative stress and inflammatory pathways. The exacerbated lung pathology observed in Prnp KO mice underscores the crucial role of PrP^C in lung defense mechanisms.

To assess the carcinogenic potential of our synthetic PM_2.5, we utilized PM_2.5 NIST (Sigma-Aldrich), a certified reference material containing polycyclic aromatic hydrocarbons (PAHs) and heavy metals, as a positive control. Exposure to PM_2.5 NIST rapidly induced visible lung tumors and high mortality in both WT and KO mice. These results confirm the potent carcinogenicity and systemic toxicity of PM_2.5 NIST, with more severe outcomes observed in PrP^C-deficient mice. Collectively, our findings highlight the pronounced toxic effects of PM_2.5 and underscore a critical protective role of PrP^C in mitigating PM_2.5-induced lung injury, inflammation, and tumorigenesis.

Materials and Methods

Animals

C57BL/6 mice were acquired from Damul Science (Daejeon, Korea) and maintained until 18–24 months old. Prnp (encoding PrP^C) knockout mice on the FVB background were purchased from The Jackson Laboratory (Bar Harbor, ME). Detailed information regarding mice has been previously published; these mice develop and behave normally for at least 7 months, with no apparent immunological defects. To reduce genetic variability, these Prnp ^0/0 mice were backcrossed to C57BL/6J mice and subsequently to FVB/NCrl mice for 10 generations (JAX stock #018122).

Fine Particulate Matter (PM_2.5) Samples

Two types of PM_2.5 samples were used for the mouse exposure studies.

• 1.Synthetic PM_2.5 Mixture (PM_2.5): A synthetic particulate matter mixture was prepared to mimic common atmospheric components. The mixture contained oxalic acid, malonic acid, glutaric acid, sucrose, 2,5-dihydroxybenzoic acid, glycine, ammonium sulfate, ammonium nitrate, acetate, glycerol, and water in defined proportions (see Table S4). This synthetic mixture primarily consists of inorganic ions and small organic acids, lacking polycyclic aromatic hydrocarbons (PAHs) or heavy metals, representing a controlled chemical environment.
• 2.Urban Particulate Matter Standard Reference Material (PM_2.5 NIST): As a positive control, reference urban particulate matter obtained from the National Institute of Standards and Technology (NIST), SRM 1648a (Sigma-Aldrich), collected from the urban environment of St. Louis, MO. This reference contains a complex mixture of inorganic elements (including heavy metals such as Pb, Cd, Zn, Cu), carbonaceous compounds, and hazardous organic pollutants, including PAHs, polychlorinated biphenyls (PCBs), and chlorinated pesticides. Due to its composition, NIST SRM 1648a is widely used in environmental toxicology studies as a model of urban air pollution. The chosen PM_2.5 NIST suspensions were prepared by sonicating the particles in sterile phosphate-buffered saline (PBS) for 15 min in a cooling water bath. Particles larger than PM_2.5 were filtered out using a commercial polypropylene melt-blown filter with a microscale diameter.

Experimental Design

For the experiment, female young C57BL/6 mice (6 weeks old), old C57BL/6 mice (18–24 months old), young Prnp mice (6 weeks old), and old Prnp mice (15–18 months old) were randomized into two groups: control (CTL) and PM_2.5-exposed. The randomization process was stratified by age, sex, and genotype to ensure a balanced distribution across experimental groups. Throughout the experiment, all mice were housed under a 12 h light/dark cycle at a temperature of 22 ± 2 °C, with free access to water and a standard diet. All experimental procedures were approved by the Animal Welfare Committee of Jeonbuk National University (JBNU 2021-0165).

C57BL/6 and Prnp mice in the PM_2.5 group were exposed to nebulized particles for 2 h daily over 5 days. A control group was exposed to distilled water under the same conditions. Each cage contained 4–5 mice. The health of the mice was assessed daily following exposure, and their general condition was observed. Lung imaging was conducted using DEXA X-ray every month. Mice were euthanized at 1 month, 6 months, or 9 months postexposure, or sooner if they exhibited signs of poor health, such as shortness of breath, or if lung imaging abnormalities were detected using DEXA X-ray. The lung tissue was collected for analysis.

To validate the findings, a separate cohort of young Prnp KO mice was exposed to NIST 1648a PM_2.5 (Sigma-Aldrich), a reference material containing polycyclic aromatic hydrocarbons (PAHs), heavy metals, and urban particulates. Exposure conditions were the same as those for the PM_2.5 synthetic. Prnp mice in the PM_2.5 group were exposed to nebulized particles for 2 h daily over 5 days. A control group was exposed to sterile PBS under the same conditions. In this cohort, microcomputed tomography (micro-CT) was performed when mice exhibited clinical signs of distress, such as labored breathing, weight loss, or general physical deterioration, to assess early structural lung changes and potential tumor formation. Lung tissues were subsequently harvested for comparative pathological and molecular analyses.

Genotyping of Prnp Knockout Mice

Genomic DNA was extracted from the tail of each mouse using the QIAamp DNA Blood and Tissue Kit (Qiagen, Hilden, Germany), following the manufacturer’s protocol. The polymerase chain reaction (PCR) mixture comprised 2.5 μL of 10× H-star Taq reaction buffer, 5 μL of 5× band helper, 1 μL of 10 mM dNTP mix, 1 μL of each primer (10 μM), 0.2 μL of H-star Taq DNA polymerase (BIOFACT, Daejeon, Korea), and nuclease-free water to a total volume of 25 μL. The PCR conditions were as follows: denaturation at 95 °C for 15 min; 34 cycles of 95 °C for 20 s, 56 °C for 40 s, and 72 °C for 1 min for annealing and extension; and final extension at 72 °C for 5 min. PCR was performed using a C1000 Touch Thermal Cycler instrument (Bio-Rad, Hercules, CA). The PCR products were separated via electrophoresis in a 1% agarose gel, followed by staining with ethidium bromide, and analyzed for genotyping (wild-type allele: 388 bp; mutant allele: 341 bp). The PCR products were sequenced on an ABI 3730 sequencer (Applied Biosystems, Foster City, CA), and the DNA sequences of the amplicons were confirmed by using Finch TV software (Geospiza Inc., Seattle, WA). The wild-type and mutant alleles of the Prnp gene were amplified from genomic DNA using the specific primer sets listed in Table S2.

Atmospheric Simulation Chamber (ASC) and Exposure Setup

The ASC and exposure setup have been reported previously. A chemical solution containing 10 components dissolved in purified water was atomized to generate PM using a nebulizer (TQ-50-C0.5; Meinhard) with optimized timing. The aerosol particles produced were diluted in a dilution chamber with purified air and then passed through a diffusion dryer to remove moisture. This dried PM was then delivered to mice in a whole-body exposure chamber. Control mice were exposed to distilled water without the 10-component mixture via an ASC system. Real-time monitoring of PM in the chamber was conducted using an OPC (OPC-N2; α Sense, U.K.) for size distribution and number concentration measurements. Additionally, the mass concentration of PM was determined by collecting aerosols on a 47 mm PTFE filter (PALL). Temperature, relative humidity, and oxygen level in the chamber were maintained at 19.3 ± 1.9 °C, 47.0 ± 11.6%, and 21.4 ± 1.4%, respectively.

The composition of the particulate matter (PM) used was as previously reported. Particles were collected at ∼10% relative humidity on a Teflon filter for 2 h in the absence of mice and immediately extracted with 5 mL of ultrapure water (18.2 MΩ cm at 25 °C). The concentrations of inorganic salts were determined by ion chromatography (ICS-90; Dionex). The mean mass ion composition from three filters was: NO₃ ^– = 11.7 μg/m³, SO₄ ^2– = 11.9 μg/m³, and NH₄ ⁺ = 0.96 μg/m³. Assuming full neutralization by ammonium, the mass of ammonium sulfate was calculated as sulfate mass × 1.38, and ammonium nitrate as nitrate mass × 1.29, resulting in ∼16.4 μg/m³ for ammonium sulfate and ∼15.0 μg/m³ for ammonium nitrate. Inorganic salts accounted for ∼50% of the PM_2.5 mass.

C57BL/6 and Prnp mice in the PM_2.5 group were subjected to nebulized particles for 2 h per day over 5 days. A control group of mice was exposed to distilled water in the ASC chamber without PM_2.5 under identical conditions. Each cage housed 4–5 mice, all of which were exposed to PM_2.5. Real-time monitoring of particle levels in the chamber was conducted by using a particle counter (BT-610; Met One). Particles larger than PM_2.5 were filtered out using a commercial polypropylene melt-blown filter with a microscale diameter. The exposure dose and duration were selected based on a previous study, , where a concentration of 50 μg/m³ was used to expose conscious infant mice or pregnant mice over 5 days, resulting in induced lung inflammation in the offspring.

PM_2.5 Concentration

The mean PM_2.5 concentration in the exposure chamber for 2 h exposure was 51.8 ± 11.3 μg/m³. PM concentrations used in the mouse exposure tests are listed in Table S1. The measurement of PM concentrations and the assessment of air quality in the absence of mice have been previously reported. This concentration of 51.8 ± 11.3 μg/m³ was selected based on our previous studies, , which demonstrated that this concentration produced biologically relevant effects, including hematopoietic stem cell senescence and the development of myeloproliferative diseases in the offspring.

Dual-Energy X-ray Absorptiometry (DEXA)

Mice received anesthesia throughout the experimental procedure (5 min) via an intraperitoneal injection of 100 mg/kg of ketamine. Each mouse was positioned in the prone orientation on the scanning bed with the limbs and tail extended away from the body. Pulmonary images were obtained by using the imaging system. Mice were allowed to breathe freely during the imaging process.

Micro-CT Imaging and 3D Lung Segmentation

Micro-CT imaging was performed using a SkyScan 1276 system (Bruker, Belgium) with a source voltage of 60 kV, a current of 200 μA, and a 0.5 mm aluminum filter. Mice exhibiting signs of labored breathing or weight loss were anesthetized via intraperitoneal injection of a Zoletil/Rompun mixture (1:1 ratio) before scanning. Images were acquired over a 360° rotation with 0.6° angular steps, using 2 × 2 binning and a final voxel size of 20.2 μm. Each scan included frame averaging and a flat-field correction to improve image quality. Following the acquisition, images were reconstructed using NRecon software (Bruker, version 1.7.4.6) and exported in the BMP format. The reconstructed data sets were then processed with 3D Slicer (version 5.2.2) for lung segmentation and 3D visualization. Semiautomated thresholding and manual editing tools were used to isolate lung tissues and generate 3D models.

Lung Primary Cell Isolation

Lung primary cells were isolated from the lung tissue of mice as previously described. The mice were euthanized, and their lungs were collected. The lung tissue was rinsed in phosphate-buffered saline (PBS) and minced into small pieces by using scissors. The tissue fragments were transferred to a 10 cm culture dish, covered with 10% fetal bovine serum (FBS) in α-MEM media, and incubated at 37 °C for 3 h. An additional 10–12 mL of media was added to facilitate the migration of lung fibroblasts from the tissue pieces. Media changes were performed every 3 days.

Hematoxylin and Eosin Staining (H&E)

The lung tissue was fixed in 4% paraformaldehyde and subsequently embedded in paraffin. The paraffin block was then sectioned into 5-μm-thick sections that were stained with H&E solution (Gill No. 3 hematoxylin, Sigma-Aldrich LLC), followed by counterstaining with 0.25% Eosin Y (Sigma-Aldrich LLC). For morphometric analysis, all slides were evaluated by two independent observers who were unaware of the sample origins.

Immunohistochemistry (IHC) Analysis

Deparaffinization of the paraffin-embedded, 5-μm-thick lung tissue sections was performed using xylene, followed by rehydration with graded ethanol washes in water. Endogenous peroxidase activity was quenched by incubating sections in 0.3% H₂O₂ in methanol for 30 min at room temperature. The sections were washed in buffer for 5 min and then blocked for 30 min with diluted normal blocking serum. Tissue slides were incubated overnight at 4 °C with specific antibodies, including c-Myc (sc-42-Santa Cruz Biotechnology), Sox2 (sc-365823-Santa Cruz Biotechnology), TTF1 (sc-53136-Santa Cruz Biotechnology), K-ras (TA801672-Origene), Ki67 (ab16667-abcam), CK7 (ET1609-63 Huabio), PrP^C (A03202-SPI bio), HIF-1α (NB-100-449-Novus Biologicals), COX2 (BS1076-BioWord), IL-1β (sc-52012-Santa Cruz Biotechnology), TNFα (AB1793-Abcam), 8-OHdG (Ab62623-Abcam), CD4 (25229-Cell Signaling), CD8 (sc-7970-Santa Cruz Biotechnology) and VEGF-A (BS2853-Bio World). After washing, the slides were incubated with a diluted biotinylated secondary antibody solution for 30 min. Slides were treated with the VACTASTAIN ABC reagent to visualize immune complexes. Finally, the sections were counterstained with hematoxylin (Sigma-Aldrich). Images of the entire cross-section were acquired using an EasyScan slide scanner (Motic), and the analysis of these images was performed by using Motic ImagePlus software (Motic). IHC scoring was conducted in 10 randomly chosen, nonoverlapping fields using ImageJ software (http://rsb.info.nih.gov/ij, accessed April 12, 2022).

Mean Line Intercept (MLI) Analysis

After fixation, 5-μm sections were subjected to hematoxylin and eosin staining. The mean linear intercept (MLI), serving as an indicator of the interalveolar wall distance, was assessed through light microscopy at a magnification of 100×. To determine the MLI, the total length of a line drawn across the lung section was divided by the total number of intercepts encountered, with at least 72 lines per lung, following a previously reported methodology.

Western Blotting

Protein extracts from lung tissue and cell lysates were separated using 7–12% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk (Bio-Rad) and then incubated with specific primary antibodies against K-ras (TA801672-Origene), CK7 (ET1609-63-Huabio), Sox2 (sc-365823-Santa Cruz Biotechnology), Ki67 (ab16667-Abcam), TTF1 (sc-53136-Santa Cruz Biotechnology), c-Myc (sc-42 Santa Cruz Biotechnology), PrP^C (A03202- SPI bio), HIF-1α (NB-100-449-Novus Biologicals), COX2 (BS1076-BioWord), IL-1β (sc-52012-Santa Cruz Biotechnology), TNF-α (AB1793-Abcam), Sirt1 (Millipore, Billerica, MA),p-p53 (sc-71786 Santa Cruz Biotechnology), p53 (sc-55476 Santa Cruz Biotechnology), Caspase-1 (2225 Cell Signaling), and γH2AX (AB26350 Abcam). β-actin served as an internal control. Membranes were washed and incubated with horseradish peroxidase-conjugated goat antirabbit IgG or goat antimouse IgG. Signals were visualized by a chemiluminescent detection kit (Amersham Pharmacia Biotech) and analyzed by a densitometric scanner (FUSION SOLO 4S. WL, Vilber).

Total RNA Extraction and Real-Time PCR

Total mRNA was isolated from lung tissue using Trizol lysis reagent according to the manufacturer’s protocol (Invitrogen Corp., Carlsbad, CA). The first-strand cDNA was synthesized from 0.5 to 1 μg of total RNA using an AmpiGene cDNA synthesis kit (Enzo Life Sciences, Inc., Farmingdale, NY). Quantitative real-time polymerase chain reaction (qRT-PCR) was then performed using Power SYBER^@ Green PCR Master Mix (Life Technologies, CA) and the Applied Biosystem platform (Thermo Fisher Scientific). The S18 ribosomal subunit served as the endogenous control for normalizing the raw qPCR data. The primer sequences used in the assays are detailed in Table S3. Expression levels of target genes are presented as delta-C _t (ΔC _t) values relative to S18 to enable the precise quantification of individual gene expression.

Statistical Analysis

Statistical analysis was performed with GraphPad Prism software (version 8.0, La Jolla, CA), and the results are presented as the mean ± standard error of the mean (SEMs) across the number of experiments conducted. Statistical analyses included adjustments for Student’s t-test and one-way analysis of variance (ANOVA), with statistical significance defined at the 95% confidence level (p < 0.05).

Results

PM_2.5 Causes a High Rate of Fatality and Lung Cancer Only in Old Mice, but Not in Young Mice

To investigate the age-dependent effect of PM_2.5 exposure on mouse lung physiology, we exposed C57BL/6 young (6 weeks old) and old C57BL/6 (18–24 months old) mice to approximately 50 μg/m³ PM_2.5 mass concentration in an atmospheric simulation chamber (ASC) (Table S1). PM_2.5 exposure did not impact the survival rate of young mice (Figure A, unfilled gray circles). In contrast, old mice exposed to PM_2.5 began to die starting at 1-month postexposure, with approximately 80% of mice dying within 3 months (Figure A, unfilled red diamonds). These findings suggest that older mice are more susceptible to exposure to PM_2.5.

1.

Age-dependent effect of PM_2.5 on lung cancer development. (A) Analysis of the animal survival rate in PM_2.5-exposed young and old C56BL/6 mice (n = 20). (B) Representative lung photo and sections from control and PM_2.5-exposed old mice stained with H&E (scale bar = 200 μm); a representative result is shown (n = 6). (C) Molecular markers of adenocarcinoma (c-Myc, Sox2, TTF1, K-ras) in the lungs of PM_2.5-exposed young and old mice using immunohistochemistry (10× scale bar = 200 μm; 20× scale bar = 120 μm); (n = 6). (D) Age-dependent expression levels of PrP^C protein and mRNA in the lungs using immunohistochemistry (scale bar = 200 μm), Western blotting analyses, and RT-PCR; a representative result is shown (n > 6). (E) Age-dependent expression levels of HIF1α and Sirt1 in the lungs using immunohistochemistry (scale bar = 200 μm) and Western blotting analyses; a representative result is shown (n = 6). Data are shown as mean ± SEM *p < 0.05, **p < 0.01, and ***p < 0.001 in young vs old or control vs PM_2.5 by Student’s t-test.

Different from the lung tissues from PM_2.5-exposed young mice, lung tissues from exposed old mice exhibited phenotypes associated with lung cancer (Figure B). Moreover, the lungs from PM_2.5-exposed old mice, but not those from exposed young mice, expressed detectable levels of c-Myc, Sox2, thyroid transcription factor-1 (TTF-1), and K-ras proteins (Figure C), which are well-known markers of lung adenocarcinoma, , compared with levels in the corresponding control mice (Figures C and S1). Conversely, the tumors were negative for cytokeratin 7 (CK7), which is diagnostically valuable in distinguishing certain epithelial carcinomas. The tumors exhibited sporadic positivity (approximately 30–40%) for K _i-67, a biomarker linked to cancer growth (Figure S2). PM_2.5 induced levels of oxidative stress (8-OHdG) and inflammation (COX2 and TNF-α), and caused the infiltration of immune cells (CD4⁺ and CD8⁺ T cells) to increase in the lungs of young and old mice. These phenomena induced by PM_2.5 exposure were more severe in old mice than in young mice (Figure S3). These findings indicate an age-dependent effect of PM_2.5 on the development of lung cancer.

PrP^C Deficiency Amplifies Mortality and Lung Cancer Incidence in Response to PM_2.5

PrP^C expression was detected in the lung, albeit at a lower level compared to the nervous system. In line with previous findings, PrP^C protein and mRNA were expressed at high levels in the lungs of young mice, but transcript and protein expression levels of PrP^C were decreased in the lungs of old mice (Figure D). During aging, the lung environment becomes more hypoxic with an elevated inflammatory status, which triggers emphysema and lung cancer. , The expression of HIF1α was higher in the lungs of old mice compared with those of young mice (Figure E, top panel). Sirt1 is involved in modulating lung cancer progression and migration. Similar to PrP^C, Sirt1 was hardly expressed in the lungs of old mice (Figure E, bottom panel).

To investigate the role of PrP^C as a molecular determinant for the heightened mortality and increased incidence of lung cancer following PM_2.5 exposure, we used wild-type (WT) mice and Prnp knockout (KO) mice with deleted PrP^C, which showed a similar phenotype (Figure S4). As shown in Figure A, the survival rates of young WT (6 weeks old) unexposed mice and those exposed to PM_2.5 were comparable. Young KO mice (6 weeks old) also exhibited similar survival rates. In contrast, young KO mice were notably vulnerable to PM_2.5 exposure, with mortality rates reaching up to 60% within 6 months postexposure (Figure A, unfilled red diamonds). Lung tissues of young KO PM_2.5-exposed mice, but not young WT PM_2.5-exposed mice, exhibited solid adenocarcinoma foci and significantly elevated levels of tumor-associated factors, including c-Myc, Sox2, TTF-1, and K-ras, compared with those of young KO and WT control mice, respectively (Figures B–D and S8A). The level of CK7 showed no significant changes, and Ki67 was infrequently observed in young control and PM_2.5-exposed mice (Figure S5). In instances where it was not a common occurrence, tumor metastasis occurred around the brains of PM_2.5-exposed KO young mice that exhibited noticeable expression of c-Myc, Sox2, TTF-1, and K-ras proteins but not those with Ki67 and CK7 expression (Figure E).

2.

Protective role of PrP^C in modulating survival and lung cancer incidence in young mice exposed to PM_2.5. (A) Analysis of survival rates in young WT and Prnp KO mice exposed to PM_2.5 (n = 20). (B) Dual-energy X-ray absorptiometry (DEXA) analysis. The arrow indicates the tumor; a representative result is shown (n = 6). (C) (D) Evaluation of c-Myc, Sox2, TTF1, and K-ras expression in the lungs of young WT and KO mice with PM_2.5 exposure using immunohistochemistry (scale bar = 200 μm); higher-magnification images (20×; scale bar = 120 μm) are provided in Figure S13, and Western blotting analyses; a representative result is shown (n = 6). (E) DEXA analysis of young WT and KO mice exposed to PM_2.5; tumor metastasis around the brain was observed in PM_2.5-exposed young KO mice. A representative photo of Prnp mice is shown. Assessment of c-Myc, Sox2, TTF1, and K-ras, Ki67, and CK7 expression levels in the tumor metastasis of PM_2.5-exposed young KO mice using immunohistochemistry (scale bar = 200 μm). Arrows indicate the tumors. (F, G) Levels of Sirt1, p-p53, p53, HIF1α, oxidative stress and inflammation markers, and immune cell infiltration were analyzed in the lungs of PM_2.5-exposed young KO mice using Western blotting and immunohistochemistry (scale bar = 200 μm); higher-magnification images (20×; scale bar = 120 μm) are provided in Figure S13. Data are shown as mean ± SEM **p < 0.01 and ***p < 0.001 in WT control vs WT PM_2.5 or KO control vs KO PM_2.5 by Student’s t-test. ns indicates no significance.

Compared to young WT mice, old WT mice (15–18 months old) exposed to PM_2.5 exhibited rapid death within 4 weeks postexposure, with a survival rate of 50% (Figure S6A, unfilled blue circles). Old KO mice (15–18 months old) displayed reactions to PM_2.5 that were even more severe than those of their WT counterparts. Old KO PM_2.5-exposed mice all died within 4 weeks postexposure (Figure S6A, unfilled red diamond). Tumor-associated factors such as c-Myc, Sox2, TTF-1, and K-ras, but not Ki67 and CK7, were exclusively expressed in old KO PM_2.5-exposed mice (Figure S6B,C). These findings suggest that PrP^C plays a pivotal protective role in modulating survival rates and lung cancer incidence in response to exposure to PM_2.5.

We next investigated the protective role of PrP^C in various organs from young and old KO mice exposed to PM_2.5. No significant morphological changes were observed in the kidneys of both young and old KO PM_2.5-exposed mice compared to corresponding WT PM_2.5-exposed mice (Figure S7A). The livers of young KO PM_2.5-exposed mice showed an observable increase in adipocytes and hepatocyte damage and further, those of old KO PM_2.5-exposed mice exhibited interfacial hepatitis (Figure S7B). WT mice did not exhibit any histological changes in their livers following PM_2.5 exposure. The spleens of old KO PM_2.5-exposed mice but not young KO PM_2.5-exposed mice displayed an incomplete shape, a decreased density and size, and unclear borders in their white pulps compared to those of corresponding WT PM_2.5-exposed mice (Figure S7C).

PrP^C Deficiency Accelerates Lung Emphysema and Subsequent Hypoxia, Inflammation, and Angiogenesis as Risk Factors for Lung Cancer Development

We next examined how a PrP^C deficiency exacerbates mortality and lung cancer incidence in response to PM_2.5. We observed marked morphological changes in the lungs of KO mice, characterized by emphysema-like changes such as enlarged distal airspaces, universal expansion of alveolar airspaces merging into larger cavities, expanded alveolar pores, and the narrowing and fragmentation of numerous alveolar septa (Figure A). Among the 15 KO mice, 11 exhibited lung emphysema compared to 5 of 15 WT mice. Emphysema was present in both young and old KO mice; the incidence of emphysema was significantly higher in older KO mice than in younger KO mice. In contrast, emphysema was observed only in old WT mice and not in young WT mice (Figure A,D, bottom panels). The mean linear intercept (MLI) significantly increased in KO mice compared to WT mice, regardless of age (Figure A). Emphysema mainly occurs in older patients and is closely associated with hypoxia and the induction of lung cancer. , If it occurred infrequently, lung cancers were generated in KO-old mice not exposed to PM_2.5 (Figure B).

3.

PrP^C deficiency causes lung emphysema, hypoxia, inflammation, and angiogenesis, and generates lung cancers. (A) Comparison of lung morphology in Prnp WT and KO mice by H&E staining (2× scale bar = 1200 μm; 10× scale bar = 200 μm) and mean line intercept (MLI) analysis; a representative result is shown (n = 6). Data are shown as mean ± SEM *p < 0.05, **p < 0.01, and ***p < 0.001 by one-way ANOVA. (B) Expression levels of c-Myc, Sox2, TTF1, K-ras, Ki67, and CK7 in the lungs of old Prnp KO mice with emphysema that were not exposed to PM_2.5 were visualized by H&E staining (scale bar = 200 μm) and immunohistochemistry (scale bar = 200 μm); a representative result is shown (n = 6). (C) Expression levels of Sirt1 and HIF1α were evaluated in primary lung cells isolated from Prnp WT and KO mice by Western blotting; a representative result is shown (n = 6). (D) Expression levels of Sirt1 in normal and emphysema lung tissues of Prnp WT and KO mice. Left panel: The expression level of Sirt1 was detected in Prnp KO mice using Western blotting and H&E staining (scale bar = 200 μm). Right panel: the expression level of Sirt1 in emphysema of Prnp mice; a representative result is shown (n = 6). (E, F) Levels of Sirt1, p-p53, p53, HIF1α, oxidative stress, inflammation markers, and immune cell infiltration were analyzed in the lungs from PM_2.5-exposed old Prnp mice by Western blotting and immunohistochemistry (10× scale bar = 200 μm; 20× scale bar = 120 μm); a representative result is shown (n = 6). (G) Expression level of VEGF-A in the lungs of PM_2.5-exposed young and old Prnp mice was assessed by analysis of immunohistochemistry (10× scale bar = 200 μm; 20× scale bar = 120 μm). Data are shown as mean ± SEM *p < 0.05, **p < 0.01, and ***p < 0.001 in WT control vs WT PM_2.5 or KO control vs KO PM_2.5 by Student’s t-test. ns indicates no significance.

Emphysema in the lungs of KO mice became more evident with age. It caused hypoxia by triggering hypoxia-related signaling, as evidenced by the elevated levels of HIF1α and Sirt1 in primary cells of old KO mice (Figures C and S9A). No changes in Sirt1 levels were detected in the pulmonary tissue of WT and KO unexposed mice (Figure D, left panel; Figure S9B, left graphs). In KO mice exhibiting an elevated incidence of emphysema, Sirt1 levels were upregulated (Figure D, right panel; Figure S9B, right graphs). Exposure to PM_2.5 elicited a discernible augmentation in Sirt1, phosphorylated p53 (p-p53), and p53 levels in both young and old KO mice (Figure F; Figure E, left panels; Figures S8B and S9C). Additionally, Western blot analysis revealed elevated levels of HIF1α and inflammatory markers such as COX2, IL-1β, and TNF-α in both young and old KO mice following exposure to PM_2.5 (Figure F; Figure E, right panels; Figures S8B and S9C). Immunohistochemistry results revealed heightened levels of HIF1α, oxidative stress (8-OHdG), and inflammation (COX2 and TNF-α), along with increased levels of infiltration of immune cells (CD4⁺ and CD8⁺ T cells) in the lungs of both young and old KO mice exposed to PM_2.5 (Figures G and F). Hypoxia and inflammation share an interdependent relationship. A significantly increased level of VEGF-A, a key mediator of angiogenesis in cancer, was observed in the lungs of the exposed KO mice (Figure G). These findings strongly suggest that PrP^C deficiency induces emphysema-like changes, leading to the activation of angiogenesis and the promotion of lung cancer development under hypoxic conditions.

PM_2.5 NIST, as a Positive Control, Demonstrates Broader Tumorigenic Potential Than Synthetic PM_2.5

To validate and compare the carcinogenic potential of our synthetic PM_2.5, we used Sigma-Aldrich-certified PM_2.5 NIST, which contained PAHs and heavy metals, as a positive control. Both WT and KO young mice exposed to PM_2.5NIST developed visible lung tumors within 1–2 months. As shown in Figure A, death occurred as early as 3–5 weeks, with 50% of WT mice and 80% of KO mice dying by week 11. In contrast, our synthetic PM_2.5-lacking carcinogenic PAHs and heavy metals induced lung tumors only in KO mice, reaching 60% mortality after 6 months of exposure (Figure A). Micro-CT revealed multiple ill-defined, ground-glass opacities of varying sizes, with poorly demarcated margins, suggestive of early tumor formation. Subsequent 3D reconstruction using 3D Slicer segmentation highlighted solid nodular masses with irregular shapes and heterogeneous density, consistent with neoplastic lesions or tumor foci. H&E staining of lung sections confirmed the presence of multiple foci with abnormal cellular growth and a disrupted alveolar architecture. These foci exhibited increased cellular density, nuclear pleomorphism, and localized infiltration into adjacent alveolar spaces. The spatial distribution of lesions matched the nodules identified in the micro-CT analysis (Figure B). IHC staining and Western blot analysis demonstrated marked upregulation of lung adenocarcinoma markers including c-Myc, TTF-1, Sox2, and K-Ras (Figure C,D). The expression level of PrP^C in WT mice remained unchanged following PM_2.5 NIST exposure (Figure D), supporting the presence of active oncogenic signaling pathways. These results suggest that PM_2.5 NIST drives a rapid and aggressive tumor phenotype independent of PrP^C, with clear evidence of early malignant transformation at both the structural and molecular levels. This carcinogenic process is likely accelerated by the chemical complexity of NIST particles, which can bypass normal inflammatory barriers and directly activate oncogenic cascades.

4.

PM_2.5 NIST exposure induces early and widespread lung tumorigenesis in both Prnp WT and KO mice. (A) Survival analysis of young Prnp WT and KO mice following PM_2.5 NIST exposure (n = 10 per group). (B) Representative gross lung images, micro-CT scans (coronal, transverse, and sagittal views), 3D-reconstructed lung images showing tumor nodules (brown) within lung tissue (green), and H&E-stained (2× scale bar = 1200 μm; 10× scale bar = 200 μm) lung tissue sections (n = 5). Red arrows indicate tumor foci. (C) Immunohistochemical analysis of Ki67, CK7, c-Myc, TTF1, Sox2, and K-ras in lung tissues from each group (scale bar = 200 μm); higher-magnification images (20×; scale bar = 120 μm) are provided in Figure S14. Quantification of positive staining areas is presented (n = 4 per group). (D) Western blot analysis of CK7, c-Myc, TTF1, Sox2, K-ras, and PrP^C in lung tissues from control and PM_2.5 NIST-exposed WT and KO mice. Quantified band intensities are shown (n = 4 per group). (E) Protein levels of SIRT1, p53, γ-H2AX, HIF-1α, TNF-α, IL-1β, COX-2, and Caspase-1 were analyzed by Western blotting in lung tissues from PM_2.5- and PM_2.5 NIST-induced lung tumors. Quantified protein expression levels are presented (n = 4 per group). Data are shown as mean ± SEM *p < 0.05, **p < 0.01 in WT control vs WT PM_2.5 NIST or KO control vs KO PM_2.5 NIST or PM_2.5 vs PM_2.5 NIST by Student’s t-test. ns indicates no significance.

To investigate potential differences in the underlying mechanisms, we compared tumors induced by synthetic PM_2.5 to those induced by PM_2.5 NIST. Western blot analysis revealed significantly higher expression of COX-2, HIF-1α, and Caspase-1, a component of inflammasomes, in tumors from only the NIST-exposed group (Figure E), suggesting a more aggressive inflammatory and hypoxic tumor microenvironment driven by the complex chemical composition of the PM_2.5 NIST. In contrast, expression levels of SIRT1, p53, γ-H2AX (a marker for DNA double-strand breaks), TNF-α, and IL-1β were comparable between the two groups (Figure E), indicating overlapping stress response and DNA damage pathways. Together, these findings indicate that both types of PM_2.5 induced tumor formation; however, PM_2.5 NIST triggers a more severe inflammatory and hypoxic response, indicating a higher carcinogenic potential.

Discussion

The protein is expressed ubiquitously throughout the body and exists in two isoforms, PrP^C and PrP^Sc2. While PrP^Sc is notorious for its role in a spectrum of neurodegenerative diseases in both humans and other mammals, PrP^C has been recognized for its functions in stress protection, cellular differentiation, and copper homeostasis. PrP^C has emerged as a multifaceted player with functions in various aspects of cancer biology, including cell proliferation, metastasis, cell death, drug resistance, and the regulation of cancer stem cells. In lung cancer, PrP^C is involved in promoting tumor progression by upregulating the development of Tregs through TGF-β- and PD-1-mediated pathways. , We investigated how PrP^C modulates susceptibility to environmental stressors, using a PM_2.5 concentration of 50 μg/m³, which reflects the PM_2.5 levels commonly observed in highly polluted urban environments, such as major cities in East Asia. , This biologically relevant PM_2.5 exposure, which we used in our previous studies, allows for modeling PM_2.5-induced lung pathology and tumorigenesis in mice while maintaining ethical standards for animal welfare. Our findings in Prnp WT and KO mice can be interpreted in terms of environmental relevance in this context, providing mechanistic insight into how the age-related loss of PrP^C increases susceptibility to air-pollutant-associated lung disease. This framework strengthens the translational value of our results by linking experimental observations to potential human health outcomes under ambient PM_2.5 exposure.

In our study, we demonstrated for the first time that PrP^C expression in the lungs noticeably declined during aging (Figure D). Furthermore, exposure to PM2.5 in old mice resulted in increased mortality rates and lung cancer (Figure A–C). PM_2.5-induced lung cancer was positive for c-Myc, Sox2, TFF1, and K-ras, and solid tumor foci were diagnosed as adenocarcinoma. , The protective role of PrP^C in lung physiology was demonstrated by experiments using Prnp WT and KO mice exposed to PM_2.5 (Figure ). In our comparative analysis of lung morphology between Prnp WT and KO mice, a significantly higher percentage of emphysema was observed in the lungs of KO mice (Figure A). Emphysema was present in both young and old KO mice, whereas it was detected only in old WT mice, not in young WT mice. The mean linear intercept (MLI) was higher in KO mice than in the control mice (Figure A). The expression levels of inflammatory mediators, including COX2, TNF-α, and IL-1β, as well as of the hypoxia marker HIF1α were also higher in the KO mice (Figures F and S8B). These findings indicate that the absence of PrP^C upregulates inflammatory and hypoxic responses following exposure. Structural changes in the lung tissue due to emphysema, such as reduced alveolar surface area and impaired gas exchange, may indirectly contribute to lung cancer by creating a hypoxic environment. We observed elevated VEGF-A expression levels in both young and old PrP^C-deficient mice (Figure G), consistent with the results of a previous study. Elevated VEGF-A expression may not directly cause cancer but generate a microenvironment that favors carcinogenesis via activating hypoxia-related signaling pathways, particularly the HIF1α pathway. This interpretation aligns with the view that emphysema and lung cancer share common risk factors, including cigarette smoking and air pollution, without implying a direct causal relationship. However, chronic tissue hypoxia and inflammation promote genetic mutations and cellular alterations that facilitate the malignant transformation of cells.

PrP^C deficiency affects organs in addition to the lungs under exposure to PM_2.5. Hepatic adipocytes accumulated, and hepatocytes were damaged in young KO mice (Figure S7B), consistent with previous reports on lipid metabolism in the absence of PrP^C. PM_2.5 exposure also caused the loss of splenic white pulp in aged KO mice (Figure S7C), consistent with previous findings of disrupted T- and B-cell segregation. These systemic pathologies correlated with the high mortality in KO mice, which was 60% within six months in the young animals and 100% within 4 weeks in the aged animals (Figures A–D and S6A). In contrast, young C57BL/6 and WT mice exposed to the same conditions did not develop lung cancer, and their mortality rate was high. These results together support the role of PrP^C in pulmonary and systemic resilience to environmental stress. We also emphasize the need for further research into the role of PrP^C in metabolism as well as in immune and hematopoietic organs.

At the molecular level, we observed that the Sirt1 expression level, which normally declines with age (Figure D, left panel; Figure E, bottom panel) markedly increased under hypoxic conditions in Prnp KO mice (Figure D, right panel), in agreement with the findings of previous studies. , The levels of Sirt1, p-p53, and p53 were substantially upregulated in the lung tissue of KO mice upon exposure to PM_2.5 (Figure F; Figure E; Figures 8B and S9C), which promotes lung cancer development. Sirt1 is an NAD⁺-dependent deacetylase that plays two roles in stress responses: upregulation protects against oxidative stress, and deacetylates p53, reducing its proapoptotic activity. , The PM_2.5-induced oxidative stress and DNA damage , in KO mice triggered the activation of p53 and Sirt1; however, the absence of PrP^C appeared to strengthen this response. Loss of PrP^C likely halts a regulatory mechanism that normally balances Sirt1-p53 signaling, thereby allowing the survival and proliferation of cells with DNA damage and increasing the risk of cancer. This dysregulation was not observed in WT mice, where PrP^C helped to preserve homeostasis. Consistent with our findings, Sirt1 is overexpressed in several human cancers, , and Sirt1 levels positively correlate with p53 in patients with lung cancer. Inflammation and p53 mutations are key drivers of tumor progression, , where these factors also positively correlate with Sirt1 levels. The upregulation of Sirt1 and p53 in response to PM_2.5 in KO mice in this study underscores the potential for similar molecular alterations in humans exposed to high levels of air pollution, reinforcing the need for preventive measures and targeted therapies. Understanding how Sirt1 modulates p53 activity in this mouse model provides insights into potential therapeutic strategies targeting this pathway in human lung cancer.

Our study highlights the critical influence of both PM_2.5 composition and host genetic background on lung tumorigenesis. We observed that Sigma-certified PM_2.5 NIST, containing known carcinogens such as PAHs and heavy metals, rapidly induced tumor formation and high mortality in both WT and KO mice (Figure A,B). In contrast, our synthetic PM_2.5, which lacks such carcinogens, led to tumor formation only in KO mice after long-term exposure, suggesting that chemical complexity can markedly accelerate carcinogenesis, while genetic vulnerability, such as the loss of PrP^C, can predispose hosts to tumor development even from less potent particles. Histological and imaging analyses confirmed lung adenocarcinoma features in both PM_2.5 exposure models; however, tumors from the NIST group were more aggressive, with larger, ill-defined lesions (Figure C). Molecular analyses revealed that both particle types activated core oncogenic pathways (c-Myc, TTF-1, Sox2, and K-Ras), while NIST-induced tumors showed higher levels of acute inflammatory and hypoxia-related markers such as COX-2, HIF1α, and Caspase-1, indicating a more hostile tumor microenvironment. COX-2 and Caspase-1 are associated with inflammasome activation and pyroptosis, whereas HIF1α promotes angiogenesis and metabolic reprogramming under hypoxic stress, a common feature of PAH-induced carcinogenesis. , Stress-related proteins (SIRT1, p53, γ-H2AX, TNF-α, and IL-1β) were similarly elevated in both groups, indicating a shared cellular stress and DNA damage response following PM_2.5 exposure. Synthetic PM triggered tumorigenesis only in KO mice with compromised antioxidant defense, relying on cumulative DNA damage and cellular stress over time. Meanwhile, PM_2.5 NIST exerts potent carcinogenic effects via inflammation and hypoxia, irrespective of PrP^C status due to its toxic components. Figure E shows variability in biomarker expression among individuals within the same group, likely due to interindividual differences in PM_2.5 susceptibility, tumor microenvironment, and disease stage.

Our findings demonstrate that even brief exposure to PM_2.5 can induce long-term pathological changes, particularly in genetically vulnerable individuals. Similarly, the epidemiological evidence indicates that short-term PM_2.5 exposure increases the risk of acute lung injury and long-term lung cancer susceptibility. − The KO mouse model does not fully mimic human lung cancer; however, the conservation of key pathways such as the Sirt1 and p53 pathways between mice and humans, together with the well-established carcinogenicity of PM_2.5, supports the translational relevance of our findings. Our results suggest a protective role of PrP^C against lung disease, with the loss of PrP^C predisposing individuals to the aberrant activation of oncogenic pathways. However, this study has some limitations. Short-term exposure to PM_2.5 caused high mortality in aged C57BL/6 and PrP^C-deficient mice but did not induce lung cancer or mortality in young WT mice, indicating age- and genotype-dependent vulnerability to lung cancer. Future studies should extend the duration of exposure to PM_2.5, conduct postexposure monitoring, and validate our proposed mechanisms of PrP^C in human tissues or in vitro models. Furthermore, whether reduced PrP^C expression or function due to aging, genetic variations, or comorbidities could serve as a biomarker of susceptibility to pollution-related lung disease and cancer remains to be determined.

Environmental and Ecological Implications

Our findings highlight the significant public health and ecological risks associated with exposure to PM_2.5, particularly among vulnerable populations, such as the elderly. The exacerbation of lung cancer and mortality in aged mice lacking PrP^C highlights the synergistic effects of environmental stressors and the age-related decline in protective molecular mechanisms. This study not only reveals the potential role of PrP^C as a biological safeguard against air pollution-induced lung damage but also suggests its value as a biomarker for susceptibility in polluted environments. From a public health perspective, these results reinforce the need for more rigorous air quality monitoring and policies aimed at reducing fine particulate emissions, especially in densely populated and industrial areas. PM_2.5 exposure poses a threat to respiratory health across species, potentially disrupting ecosystem balance through increased disease burden in wildlife. These insights support integrative approaches combining environmental protection, aging research, and respiratory health strategies to mitigate the far-reaching effects of air pollution.

Glossary

Abbreviations

ASC

Atmospheric simulation chamber

CK7

Cytokeratin 7

DEXA

Dual-energy X-ray absorptiometry

HE

Hematoxylin and eosin

IHC

Immunohistochemistry

KO

Knockout

MLI

The mean linear intercept

PrP^C

The prion protein

PrP^Sc

The prion scrapie

PM_2.5

Particulate matter with a diameter of less than 2.5 μm.

TTF-1

Thyroid transcription factor-1

WT

Wild-type

WP

White pulp

PBS

Phosphate-Buffered Saline

Data will be made available upon request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.est.5c08365.

• Histopathological analyses; IHC; Western blotting; quantitative protein analyses; PCR-based genotyping; primer sequences; particulate matter concentration measurements; and lists of chemicals used in the study (PDF)

§.

T.T.T.K. and H.-J.S. contributed equally to this work. J.-C.L. and S.-H.K. conceived and designed the experiments. T.T.T.K., H.-J.S., B.G., S.-H.S., and S.-H.K. performed the experiments and analyzed the data. J.-C.L. and S.-H.K. contributed to reagents, materials, and analytical tools. T.T.T.K., H.-J.S., J.-C.L., and S.-H.K. wrote and/or edited the manuscript. All authors have reviewed the manuscript.

The Animal Welfare Committee of Jeonbuk National University (JBNU 2023-114) approved all experimental procedures. The study was carried out in accordance with relevant guidelines and regulations. All efforts were made to minimize the numbers and suffering of animals used in this study.

The authors declare no competing financial interest.