Cold atmospheric plasma sensitizes head and neck cancer to chemotherapy and immune checkpoint blockade therapy.

Introduction

Head and neck cancer (HNC) is the seventh most prevalent cancer worldwide, with approximately 890,000 new cases reported each year. HNC encompasses various types of tumors, including those affecting the upper aerodigestive tract (nasal, sinuses, pharynx, throat, and oral), head and neck adenocarcinomas (thyroid and salivary glands), and head and neck skin cancers [ 1 ]. Among these, squamous cell carcinoma of the head and neck (HNSCC) constitutes over 90 % of cases [ 2 ]. Current treatment modalities for HNC primarily include surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy [ 3 ]. However, HNC remains a devastating disease, posing a significant burden on the global healthcare system [ 3 ]. Therefore, the search for more effective drugs to combat HNC is of utmost importance.

Cold atmospheric plasma (CAP) has gained increasing attention as a potential treatment modality for cancer. Plasma is an ionized gas produced by adding energy to a neutral gas in the form of heat or electromagnetic fields. This excited gas contains charged particles, free radicals, ultraviolet radiation, and usually high temperature [ [4] , [5] , [6] ]. According to previous literature, there have been copious preclinical and clinical reports supporting the central role of reactive oxygen and nitrogen species (RONS) in CAP treatment of various types of cancer, such as breast, melanoma, ovarian, metastatic bone, pancreatic, oral, and thyroid cancers [ [7] , [8] , [9] , [10] , [11] , [12] , [13] ]. Because the free radicals induced by cold atmospheric plasma have hemolytic activity on red blood cells, the mild conditions of ROS/RNS induced by CAP are necessary for the safe treatment of human diseases. CAP demonstrates great potential in cancer therapy by selectively inducing apoptosis in tumor cells and inhibiting their migration and invasion [ 14 ]. However, the precise underlying CAP's therapeutic advantages in oncology is not completely elucidated.

Cancer cells employ diverse survival mechanisms, including glycolytic metabolism, disrupted redox balance, and proliferative signaling, to evade therapy [ 15 ]. These pathways often interact and reinforce each other, posing a significant challenge in treating malignant tumors. Interestingly, malignant cells exhibit abnormal elevation of reactive oxygen species (ROS), which plays a crucial role in regulating glycolysis, redox balance, and proliferation signals [ [16] , [17] , [18] , [19] ]. Therefore, targeting ROS may offer a potential therapeutic strategy to exploit this common vulnerability.

In this study, we utilize the Piezobrush compact handheld plasma device, based on piezoelectric direct discharge (PDD) technology, to investigate its impact on HNC treatment. The main objectives are to assess the influence of this plasma platform, CAP PZ2 , on HNC cancer cells and explore its effects on proliferation, apoptosis, migration/invasion, and autophagy in Fadu, Cal27, A375, and Hth83 cells. Additionally, we aim to investigate whether CAP PZ2 can simultaneously target the redox balance, glycolysis, and proliferation signals in a head and neck cancer model, providing valuable insights into its potential as a therapeutic approach for HNC.

Cisplatin, a metal-based anticancer drug, is commonly used in clinical practice for treating various cancers, including head and neck cancer [ 20 ]. Despite it has been used in cancer patients for decades, cisplatin is associated with significant side effects such as bone marrow suppression, hearing problems, electrolyte disorders, nephrotoxicity, and cardiac toxicity, which hinder its clinical application and adversely affect patients' quality of life [ [20] , [21] , [22] ]. Since cisplatin induces oxidative damage in both normal and cancer cells, combining low doses of cisplatin with CAP could maximize oxidative stress in cancer cells especially, while potentially minimizing adverse reactions in normal cells [ 21 , 23 , 24 ]. Immune checkpoint blockade (ICB) therapy enhances anti-tumor immunity by inhibiting intrinsic regulators of the immune system. Although it has revolutionized cancer treatment, challenges such as low objective response rates and severe systemic side effects persist, limiting its widespread clinical use. For example, programmed cell death protein 1 (PD-1) is an immune inhibitory receptor expressed by T cells, and is involved in various physiological responses, such as infection, cancer, and immune homeostasis. PD-L1 is a ligand of PD1, and is highly expressed in many cancers, including HNC [ 25 ]. Therefore, blocking the interaction between PD-1/PD-L1 using antibodies has been shown to inhibit tumor progression [ 26 , 27 ]. The anti-PD-1 agent, nivolumab, was recently approved by the FDA as a standard care regimen for patients with platinum refractory recurrent/metastatic (R/M) HNSCC [ 28 ]. However, the response rate to ICB therapy remains unsatisfactory among patients [ 28 ]. Previous studies have shown a synergistic effect by combining CAP with immunotherapy [ 26 , 27 , 29 ]. For instance, Park et al. utilized CAP along with gold nanoparticles-conjugated PD-L1 antibody in oral SCC cells, and resulted in a significant increase in apoptotic cells compared to monotherapy [ 26 ]. Additionally, Miebach et al. validated that local application of medical gas plasma technology synergizes with mitochondria-targeted drugs and anti-PD1 checkpoint therapy for melanoma treatment [ 29 ].

Here, the last but most important objective of this study was to investigate the synergistic potential between CAP PZ2 and chemotherapy drugs (cisplatin) or immune checkpoint inhibitors (PD-1 antibody), focusing on their combined efficacy. By exploring this synergism, we seek to provide novel insights for the clinical management of head and neck cancer, offering a promising avenue for innovative oncotherapy.

Materials and methods

Cell culture and cell lines

Head and neck upper aerodigestive tract cancer cells (human nasopharyngeal carcinoma Fadu cells, human tongue squamous carcinoma Cal27 cells), head and neck skin cancer cells (human melanoma A375 cells), head and neck glandular cancer cells (human thyroid cancer Hth83 cells) and mouse-derived HNSCC cell line SCC7 were obtained from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). Cal27, A375, and Hth83 cells were cultured in Dulbecco's modified eagle medium (DMEM, Corning, USA) supplemented with 10 % fetal bovine serum (FBS, Gibco, USA) and penicillin-streptomycin (Gibco, USA), and maintained in a 37° incubator with 5 % CO 2 . Fadu cells were cultured in MEM containing 10 % FBS, while SCC7 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10 % FBS under similar conditions.

CAP PZ2 device and treatment

Throughout the research, Piezobrush® PZ2, a handheld piezoelectric cold atmospheric plasma equipment (CAP PZ2, Relyon Plasma Co., Regensburg, Germany), was utilized ( Supplemental Figs. 1A–G ) [ 30 , 31 ]. CAP PZ2, which operated at a temperature below 50 °C, was generated using the Piezo Direct Discharge (PDD®) technology [ 32 ]. The core of this handheld plasma device is the TDK CeraPlas™ piezoelectric plasma generator, a high-voltage discharge device used to produce cold atmospheric pressure plasma. The typical parameter of CAP PZ2 in Supplemental Figs. 1C–E . The frequency of 50 kHz leads to a mean discharge power of 0.9 mW and the optical emission spectroscopy (OES) was performed in the UV, visible (VIS), and near infrared (NIR) region [ 30 , 33 , 34 ]. The multigas nozzle produced direct discharges to 10 mm distance, whereas their intensity and frequency decreased with increasing distance. In phases without discharge, the nozzle created a plasma jet accompanied by stable and relatively low leakage currents [ 30 ]. The culture medium was removed before treating cancer cells with CAP PZ2 , and positioned the plasma nozzle 10 mm from the bottom of the well plates. To assess the effect of CAP PZ2 therapy on human head and neck carcinoma cells, direct plasma therapy was performed on Fadu, Cal27, A375, and Hth83 cells.

Cell viability assay

Cell viability of Fadu, Cal27, A375, and Hth83 cells following CAP PZ2 treatment was evaluated using the cell counting kit-8 (CCK-8, Dojindo Laboratories, Japan). The CAP PZ2 was adjusted to a height of plasma nozzle 10 mm from the bottom of the well plates and administered for the specified time. The cells were treated with CAP (5, 10, 20, 30, 60, and 90 s) in 96-well plates (3 × 10 3 cells/well), and the cells were allowed to regenerate for 24, 48, and 72 h. CCK-8 was diluted with cell culture medium at a 1:9 ratio, then 100 μL was added to each well and incubated at 37 °C for 1.5 h, according to manufacturer's protocol. The optical density at 450 nm was measured using a microplate reader (Molecular Devices, Sunnyvale, USA).

Colony formation assay

Fadu, Cal27, A375, and Hth83 cells were seeded in 6-well plates at a density of 1 × 10 5 cells/well and treated with CAP PZ2 for 24 h at durations of 0, 30, and 60 s. The cells were collected, resuspended in complete medium, and then inoculated into the wells at a density of 2000 cells per dish. The culture medium was changed periodically until visible colonies formed. Finally, the colonies were fixed with methanol and stained with a crystal violet solution (Beyotime Biotechnology, China). This experiment was repeated three times.

RNA-sequencing and bioinformatic analysis

To examine the mRNA expression profiles of CAP PZ2 treated head and neck cancer (HNC) cells, RNA sequencing (RNA-seq) was performed using the HiSeq3000 System (Illumina, USA). The LifeScope Genomic Analysis Software v2.5.1 was utilized to align the reads to the genome, generate raw counts for known genes, and calculate fragments per kilobase million (FPKM) values. Differentially expressed genes (DEGs) with |logFC|≧0.6 were subjected to clustering, and gene ontology (GO) analysis was performed for pathway enrichment using the Cytoscape plug-in ClueGO, considering a P -value <0.05.

Migration and invasion assay

Transwell migration and invasion assays were performed using 8-μm pore transwell compartments (Cat. No. PTEP24H48, Millipore, Billerica, USA). Fadu, Cal27, A375, and Hth83 cells were seeded into 6-well plates at a density of 2 × 10 5 cells/well and treated with CAP PZ2 (0, 30 and 60 s). For the migration assay, 200 μL of Fadu, Cal27, A375 or Hth83 cells (1 × 10 5 ) were seeded into the upper chamber containing serum-free medium, while the bottom of the chamber contained 800 μL of DMEM supplemented with 10 % FBS. For the invasion assay, Matrigel (BD Biosciences, USA) was thawed in a 4 °C refrigerator one day before the experiment. It was then removed 6 h prior to the experiment and diluted with serum-free medium at a 1:19 ratio. 100 μL of the diluted Matrigel was carefully added to each chamber to prevent the introduction of bubbles. The chambers were incubated for 6 h, and 1 × 10 5 cells were suspended in the upper compartment. The subsequent steps mirrored those of the migration assay. Following incubation at 37 °C for 24 h, the medium was discarded, and cells that had invaded the lower surface of the membrane were fixed in methanol for 30 min. Staining was performed using crystal violet (Beyotime Biotechnology, China) for 30 min at room temperature. The migrated and invaded cells were counted in 5 separate visual fields at × 20 magnification using an Olympus microscope.

Western blot assay

For the Western blot assay, Fadu, Cal27, A375, and Hth83 cells were seeded in 6-well plates at a density of 2 × 10 5 cells/well and incubated for 24 h. Subsequently, CAP PZ2 treatment was performed for 30 and 60 s, and the cells were allowed to regenerate for 24 h. The HNC cells were collected and washed with ice-cold PBS solution, then lysed in RIPA cell lysis buffer (Solaibao, Beijing, China) containing complete™ Protease Inhibitor Cocktail. Protein concentrations in the supernatant were quantified using the bicinchoninic acid (BCA) Assay (Solaibao, Beijing, China). The protein extracts were separated by 10–15 % SDS-PAGE, and the gels were transferred to PVDF membranes (Immobilon-P, CA, USA). The membranes were blocked in 5 % skimmed milk, incubated with the primary antibody overnight at 4 °C, washed three times in TBST, and then incubated with the secondary antibody at room temperature for 1 h. The signals were developed using an enhanced chemiluminescence (ECL) kit (Immobilon-P, CA, USA), and the bands were visualized using the ChemiDoc™ Touch Imaging System (Bio-Rad). Relative quantification was performed using β-actin as an internal reference. The primary antibodies used are listed in Table S1 .

RNA extraction and qRT-PCR

For the qRT-PCR analysis, Fadu, Cal27, A375, and Hth83 cells were seeded in 6-well plates and incubated for 24 h. Subsequently, CAP PZ2 treatment was performed for 30 and 60 s, and the cells were allowed to regenerate for 24 h. Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA). Complementary DNA (cDNA) was synthesized using the RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific). The PowerUp SYBR Green Master Mix (Applied Biosystems) was used for qRT-PCR on an FTC-3000p Real-Time PCR System (Funglyn Biotech, China). The 2 −ΔΔ CT method was employed for relative gene expression analysis, with β-actin serving as the internal control. The PCR primers used are listed in Table S2 .

Wound healing assay

Fadu, Cal27, A375, and Hth83 cells were seeded into 12-well plates at a density of 1 × 10 5 cells/well and treated with CAP PZ2 (0, 30 and 60 s). Subsequently, a scratch was made using a sterile 10 μL pipette tip, and unattached cells and fragments were removed by washing with PBS. The wounded areas were photographed under an Olympus microscope at 0 h and 24 h after creating the wound.

Hoechst fluorescent staining

Fadu, Cal27, A375, and Hth83 cells were seeded in 12-well plates and exposed to CAP PZ2 treatment for 0, 15, 30 and 60 s. After incubation for 24 h, the cells were stained with Hoechst 33342 (Beyotime Biotechnology, China) at 37 °C for 30 min, followed by washing with phosphate buffered saline (PBS). Nuclear morphological changes were observed using an Olympus fluorescent microscope.

Detection of apoptosis

Fadu, Cal27, A375, and Hth83 cells were seeded into 6-well plates at a density of 2 × 10 5 cells/well overnight. A375 and Hth83 cells were subsequently treated with CAP PZ2 for 0, 30, and 60 s. Fadu and Cal27 cells (1 × 10 5 cells/well) were treated with CAP PZ2 (20 s), cisplatin (DDP, 20 μg/mL), or co-treated with CAP PZ2 (20 s) and DDP (20 μg/mL). Apoptosis was assessed using the PE Annexin V apoptosis detection kit I (BD Biosciences, San Jose, CA) and flow cytometry. In brief, 24h after the CAP PZ2 treatment, the cells were collected, washed, resuspended in ice-cold PBS, and incubated with PE Annexin V and 7-AAD for 15 min in the dark at 25 °C. The cells were then analyzed using a flow cytometer (BD LSRFortessa™, BD Biosciences) within 1 h.

Acridine orange and ethidium bromide (AO/EB) cell staining

To distinguish viable cells from apoptotic and necrotic cells, AO/EB staining (BestBio, China) was employed [ 35 ]. A375 and Hth83 cells (1 × 10 5 cells/well) were seeded in 12-well plates overnight. Subsequently, CAP PZ2 treatment was performed for 30 and 60 s, and the cells were allowed to regenerate for 24 h. Fadu and Cal27 cells (1 × 10 5 cells/well) treated with CAP PZ2 (20 s), cisplatin (DDP, 20 μg/mL), or co-treated with CAP PZ2 (20 s) and DDP (20 μg/mL), and the cells were allowed to regenerate for 24 h. The cells were then collected, centrifuged, resuspended in 100 μl of culture medium, and stained with 5 μl of AO and EB for 15 min at 4 °C. After washing the cell suspension with PBS, a fluorescence microscope (Olympus) was used for analysis. The cell suspension was then washed with PBS and analyzed using a fluorescence microscope (Olympus). Viable cells displayed uniformly bright green nuclei and an organized structure, apoptotic cells exhibited orange to red nuclei with condensed or fragmented chromatin and green cytoplasm, while necrotic cells featured uniformly orange to red nuclei, an organized structure, and red cytoplasm.

Cell cycle analysis

Flow cytometry was used to assess the potential inhibitory effect of CAP PZ2 on cell cycle progression. Fadu, Cal27, A375 and Hth83 cells were seeded in 6-well plates overnight. CAP PZ2 treatment was performed for 30 and 60 s, and the cells were allowed to regenerate for 24 h. The cells were collected and fixed with 70 % ethanol at 4 °C for 2 h. Subsequently, the fixed cells were stained with propidium iodide (PI, Beyotime Biotechnology, China) and analyzed using a flow cytometer (BD Biosciences, USA) to determine the percentage of cells in different phases of the cell cycle.

Measurement of extracellular ROS in PBS

An electron spin resonance (ESR) spectrometer (Bruker, Germany) and the following spin traps: 5,5-dimethyl-1-pyrroline N-oxide (DMPO, Dojindo Laboratories) were used. Long-lived ROS produced by CAP PZ2 in PBS were measured using a microplate reader with a hydrogen peroxide assay kit (S0038, Beyotime Biotechnology, China) for H 2 O 2 . The Griess Reagent Nitrate/Nitrite kit was used for nitrite (NO 2 − ) and nitride (NO 3 − ). Short-lived ROS were measured including OH • , N-(dithiocarboxy) sarcosine (DTCS) and N-methyl-d-glucamine dithiocarbamate (MGD; Dojindo Laboratories, Japan) for NO • , 2,2,6,6-tetramethylpiperidine (TEMP; Sigma-Aldrich, St. Louis, MO, USA) for 1 O 2 , and 1-hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine (TEMPONE-H; Enzo Biochem, Farmingdale, NY, USA) for O 2 •– and ONOO − . Spin trap concentrations were chosen based on dosing experiments. For 30s or 60 s CAP PZ2 treatment, 5 mM TEMPONE-H was found to be adequate for trapping ONOO − and O 2 •– . For TEMP and DMPO, 30 mM and 50 mM were chosen, respectively. Spin traps were added to PBS before and after CAP PZ2 treatment for measurement of ROS produced during the treatment and residual ROS post-treatment, respectively.

ROS detection assay

The levels of reactive oxygen species (ROS) were measured using the Reactive Oxygen Species Assay Kit (S0033S, Beyotime Biotechnology, China) following the manufacturer's instructions. Fadu, Cal27, A375, and Hth83 cells were cultured in 6-well plates at a density of 2 × 10 5 cells per well and incubated overnight. Subsequently, they were treated with CAP PZ2 for 30 and 60 s, followed by a 24-h regeneration period. The cells were collected, washed, and resuspended in serum-free DMEM. Subsequently, they were stained with 10 μM fluorescent dye DCFH-DA at 37 °C for 20 min, washed twice with ice-cold PBS, and analyzed by flow cytometry (BD Biosciences) or observed under an Olympus microscope. A minimum of 10,000 cells were analyzed for each group in the flow cytometer. The experiments were performed in triplicate. N-Acetyl- l -cysteine (NAC, Purity≥99 %, Beyotime Biotechnology, China) was used to scavenge ROS. Additionally, we also used the following fluorescent dyes in these experiments: dihydroethidium at 1 μM for O 2 •– (S0063, Beyotime Biotechnology, China), DAF-FM diacetate at 10 μM for NO • (S0019S, Beyotime Biotechnology, China), and Cell Meter™ Fluorimetric Intracellular Peroxynitrite Assay Kit at 5 μM for ONOO − (Cat.16317, AAT Bioquest, Inc, USA).

Measurement of the antioxidant system

To measure the cellular antioxidant system, Fadu, Cal27, A375, and Hth83 cells were seeded into 6-well plates at a density of 2 × 10 5 /well and treated with CAP PZ2 for 60 s. At 24 h post-treatment, the cells were lysed in RIPA cells lysis buffer, and the supernatant was collected in 96-well plates. The glutathione to oxidized glutathione ratio (GSH/GSSG) was determined using the GSH/GSSG Assay (Promega, Madison, WI, USA), and the total antioxidant capacity was assessed using the Total Antioxidant Capacity Assay (Abcam, Cambridge, UK).

Measurement of lactate production

Lactate production was measured using Lactate Assay Kit to measured (Sigma-Aldrich). Duplicate samples for the lactate standard curve were prepared with final concentrations of 0, 25, 50, 100, 200, 400, 600, and 1000 μM. The experimental samples were incubated at room temperature for 20 min and then analyzed using a microplate reader with an excitation wavelength of 530–540 nm.

Cell autophagy assay

Cell autophagy was assessed by analyzing the protein expression of Beclin-1, P62, and LC3B, as well as through the transfection with Ad-mCherry-GFP-LC3B. The protein expression was determined using western blotting. Fadu, Cal27, A375, or Hth83 cells were seed in 6-well plates (1 × 10 5 /well) and then transfected with Ad-mCherry-RFP-LC3B (C3011, Beyotime Biotechnology, China). After 24 h of transfection, the cells were subjected to CAP PZ2 treatment for 30 and 60 s. Subsequently, the cells were cultured for another 24 h, and autophagy was observed using an Olympus fluorescence microscope.

Tumor xenografts in nude mice

The Animal Ethics Committee of Tongji University School of Medicine approved the animal experiments conducted in this study. Six-week-old female BALB/c nude mice were obtained from the Shanghai Lingchang Biotechnology Co., Ltd (Shanghai, China). The xenograft tumor model was established by subcutaneously inoculating 200 μL (6 × 10 6 ) of Fadu cells suspension per tumor into the nude mice. Once the tumor size reached 50 mm 3 , the tumor-bearing mice were randomly divided into the control group, CAP PZ2 group, DDP group, and comb group. Mice in the CAP PZ2 group were treated for 1 min each time, three times a week until the end of the study. Mice in the DDP group were administered 5 mg/kg Cisplatin every three day. In the combo group, mice received 5 mg/kg Cisplatin twice weekly along with CAP PZ2 three times weekly until the study endpoint.

Female mice, (8 weeks old, Shanghai Lingchang Biotechnology Co., Ltd, China) were subcutaneously injected with SCC7 cells (1 × 10 6 ) at the right flank. Once the tumor size reached approximately 50 mm 3 , mice with analogous types of tumors were randomly divided into four groups. Mice in the combo group were injected intraperitoneally with 200 μg of anti-PD-1 (BioXcell, Clone RMP1-14, USA) in combination with CAP PZ2 three times weekly until the study endpoint.

The tumor size was monitored using a caliper and calculated as follows: tumor volume (mm 3 ) = 0.5 × length × width 2 . At the end of the study, the mice were euthanized, and the tumors were excised, photographed, and weighed. Subsequently, the tumors were fixed with 4 % paraformaldehyde, embedded, and sectioned. All slides were stained using Hematoxylin and eosin (HE) staining and immunohistochemistry (IHC) staining.

PET/CT imaging

18 F-FDG PET/CT was performed at one week, and two weeks after CAP PZ2 treatment. All imaging acquisition was performed on a clinical PET/CT scanner (Biograph64, Siemens Healthcare, Germany). The PET scan was performed 1 h after the injection of 18 F-FDG at 7.4 MBq/20 g. The maximum standardized uptake value (SUV max ) was recorded to determine the degree of glucose metabolism by the tumor.

Histological and immunohistochemistry analysis

The paraffin-embedded tumor sections were deparaffinized using xylene followed by a series of ethanol concentrations (100 %, 90 %, 80 %, and 70 %) and then rinsed with PBS. For IHC staining, the tumor sections were immersed in methanol containing 0.1 % H 2 O 2 for 10 min to block endogenous peroxidase activity. Subsequently, antigen repair was performed by treating the sections with pH 6.0 citric acid buffer in a microwave oven. The primary antibodies, including ki67 (Abcam, 1:200), MMP2 (Proteintech, 1:200), MMP9 (Proteintech, 1:200), E-cadherin (Proteintech, 1:1000), N-cadherin (Proteintech, 1:1000), HK1 (Cell Signaling Technology, 1:400), PKM2 (Cell Signaling Technology, 1:200), PDK1 (Cell Signaling Technology, 1:400), LDHA (Cell Signaling Technology, 1:200), CD3 (Proteintech, 1:400), CD4 (Cell Signaling Technology, 1:100), and CD8 (Cell Signaling Technology, 1:100) were incubated with the slides overnight at 4 °C. Following PBS washing, the sections were then incubated with appropriate biotinylated secondary antibodies provided in the ABC kit (PV-6000D, Beijing Zhongshan Jinqiao Biotechnology Co., Ltd, Beijing, China), and visualized using the diaminobenzidine substrate. For HE staining, the sections were counterstained with hematoxylin, cleared with a graded series of ethanol and xylene, and mounted with neutral balsam. The images were captured using an Olympus microscope equipped with a digital imaging system.

Statistical analysis

All statistical calculations were performed using the GraphPad Prism 8 software package (GraphPad Software Inc., United States). The data were presented as mean ± SD. The comparison between the control group and the CAP PZ2 treatment groups was assessed using Student's t-test, with a significance level of P < 0.05.

Results

CAP PZ2 inhibits cell proliferation in head and neck cancer (HNC) cells

To investigate CAP PZ2 's potential as a therapy for head and neck cancer (HNC), we studied its anti-tumor effects on various HNC cell lines: Fadu (nasopharyngeal carcinoma), Cal27 (tongue squamous carcinoma), A375 (melanoma), and Hth83 (thyroid cancer). Under phase contrast microscopy ( Fig. 1 A–D), we observed distinct changes in cell morphology and density in the tested HNC cells. CAP PZ2 treatment for 15 s or 30 s resulted in significant cell shrinkage, reduced adherence, and detachment from the culture dish. Prolonged treatment for 60 s caused cells to become spherical, shrunken, and visually suspended in the culture medium ( Fig. 1 A–D). CCK-8 assays revealed that CAP PZ2 exhibited time- and dose-dependent inhibition of cell viability in Fadu, Cal27, A375, and Hth83 cells compared to the control group ( P < 0.05, P < 0.01, and P < 0.001) ( Fig. 1 E–H). The colony formation assay demonstrated treatment time-dependent reduction in the number of colonies formed by Fadu, Cal27, A375, and Hth83 cells ( Fig. 1 I-L). Notably, co-administration of CAP PZ2 with the chemotherapy drug Cisplatin (DDP) enhanced Fadu cell sensitivity, resulting in significant proliferation inhibition ( Supplemental Fig. 2 ). We also calculated the Drug Interaction Index (CDI) in vitro . Our results indicated that when Fadu cells were treated with 20 s of CAP and co-administered with DDP (20 μg/mL), the CDI was found to be 0.235. This suggests a highly significant synergistic effect in vitro 24 h after the co-treatment.

These findings suggest a potential synergistic effect between CAP PZ2 and Cisplatin against head and neck cancer. Overall, CAP PZ2 exhibits promising anti-tumor properties in HNC cells and holds potential as a therapeutic approach. Further research is needed to understand its mechanism of action and optimize clinical application.

Functional enrichment analysis of CAP PZ2 treatment HNC cells obtained from RNA-seq

To further investigate the effect of CAP PZ2 on HNC cells, we separately treated Fadu, Cal27, A375 and Hth83 cells with CAP PZ2 . Transcriptome sequencing was performed, followed by differential gene analysis. Heatmaps and volcano map of the results are showed in Fig. 2 A-H. In the CAP PZ2 treated group, we identified 1810 differentially expressed genes in Fadu cells, 708 in Cal27 cells, 1260 in A375 cells, and 1753 in Hth83 cells. KEGG enrichment analysis revealed the involvement of differentially expressed genes in various pathways, such as metabolic pathways, PI3K-Akt signaling pathway, mTOR signaling pathway, Glycolysis/Gluconeogenesis, apoptosis, Cell cycle, Glutathione metabolism, ferroptosis, p53 signaling pathway, and MAPK signaling pathway ( Fig. 2 I-L). GO enrichment analysis results, shown in Supplementary Figs. 3A–D , indicated that these genes are primarily associated with cell migration, apoptotic process, response to glucose, gluconeogenesis, cell cycle, and immune response. To some extent, these RNA sequencing findings provide valuable insights for our subsequent experiments.

CAP PZ2 attenuates the epithelial-mesenchymal transition (EMT) process in HNC cells

Based on the aforementioned findings, we further investigated the impact of CAP PZ2 on the migration and invasion capabilities of HNC cells. Transwell assays demonstrated that CAP PZ2 significantly reduced the migration and invasion of Fadu, Cal27, A375, and Hth83 cells in a treatment time-dependent manner ( Fig. 3 A–D). Similarly, the wound-healing assay revealed that CAP PZ2 effectively attenuated the migration of HNC cells ( Supplemental Figs. 4A–D ). qRT-PCR and Western blot analyses revealed significant downregulation of VEGF , MTDH , MMP9 , and MMP2 mRNA levels ( Fig. 3 E–H), which correlated with decreased protein expression in Fadu, Cal27, A375, and Hth83 cells ( Fig. 3 I and G). Moreover, CAP PZ2 treatment resulted in decreased expression of N-cadherin and vimentin, known stromal marker molecules, and increased expression of the epithelial adhesion molecule E-cadherin, suggesting a potential reversal of the EMT process in these cell lines ( Fig. 3 I and G).

CAP PZ2 produces ROS in PBS and elevates intracellular ROS in A375 cell lines

The generation of reactive oxygen species (ROS) is considered to be a crucial mediator in the apoptotic signaling transduction induced by various apoptosis inducers. The ROS produced by CAP PZ2 disrupted critical cancer cell survival pathways, particularly redox deregulation [ 19 ]. CAP inhibits cancer development through various mechanisms, including the generation of ROS and reactive nitrogen species (RNS) that increase oxidative stress in tumor cells. These reactive species can be categorized as short-lived, including hydroxyl radicals (OH • ), singlet oxygen ( 1 O 2 ), superoxide anions (O 2 − ), and peroxynitrite (ONOO − ). Short-lived species like 1 O 2 play a key role in apoptosis and the production of cytotoxic compounds. O 2 − is highly reactive and further elevates oxidative stress in tumor cells. Hydroxyl radicals, known for their extreme reactivity, oxidize lipids, nucleic acids, and proteins, thereby leading to cell death. On the other hand, long-lived species like hydrogen peroxide (H 2 O 2 ), nitrite (HNO 2 /NO 2 − ), and nitrate (HNO 3 /NO 3 − ) also contribute to CAP's anti-tumor effects. H 2 O 2 acts as a secondary messenger, triggering signaling cascades within tumor cells. Nitrite and nitrate enhance CAP's anti-tumor effects by generating short-lived ONOO − . The 15-s treatment produces 17.55 ± 0.74 μM of H 2 O 2 in PBS, and this amount significantly increases as the CAP PZ2 treatment time extending from 15 s to 90 s, and reaching 154.31 ± 1.67 μM ( Fig. 4 A and B). It is also consistent with the H 2 O 2 levels of most CAP regimens for cancer cell treatment. Furthermore, the levels of nitrite (NO 2 − ) and nitride (NO 3 − ) in the PBS are largely increased during and after CAP PZ2 treatment for 15–90 s ( Fig. 4 C–F), similar to current plasma jets that increase NO 2 − and NO 3 − in cell medium [ 36 ]. Finally, it is worth noting that H 2 O 2 , NO 2 − , and NO 3 − underwent little change 120 min or 24 h post-CAP PZ2 treatment in PBS ( Fig. 4 C–F).

In addition, CAP PZ2 produces diverse, short-lived ROS, including singlet oxygen ( 1 O 2 ), superoxide (O 2 •– ), hydroxyl rad-ical (OH • ), nitric oxide (NO • ), and peroxynitrite (ONOO − ), in the PBS, detected with spin traps to form spin trap adducts and quantified with electron spin resonance (ESR) spectrometry, as shown in Supplemental Figs. 5A–D . We find that CAP PZ2 notably increased spin trap adducts of OH • , O 2 •– , NO • and ONOO − , respectively. For A375 cells treated with 30s or 60 s CAP PZ2 increases intracellular NO • , O 2 •– , and ONOO − ( Fig. 4 G-L).

CAP PZ2 treatment raised intracellular reactive oxygen species accumulation

Next, we assessed whether ROS generated by CAP PZ2 is associated with the disruption of critical cancer cell survival pathways, such as redox deregulation. To assess the effect of CAP PZ2 treatment on HNC cells, we examined intracellular ROS levels and cellular antioxidant activity in Fadu, Cal27, A375, and Hth83 cells. As depicted in Fig. 5 , exposure to CAP PZ2 for 24 h at durations of 30 and 60 s resulted in a significant increase in intracellular ROS accumulation in Fadu, Cal27, A375, and Hth83 cells ( Fig. 5 A–H). A notable increase in fluorescence emitted by ROS-induced oxidation of DCFH-DA was observed in CAP PZ2 -treated cells compared to untreated cells ( Fig. 5 I). Pretreatment with N-acetyl- l -cysteine (NAC), an ROS scavenger, largely rescues in in Fadu, Cal27, A375, and Hth83 cells, indicating that CAP PZ2 acts largely through ROS ( Fig. 5 J-M). Furthermore, we assessed antioxidant activity by determining the ratio of reduced glutathione (GSH) to glutathione disulfide (GSSG). GSH is a major cellular ROS-scavenging system that replenishes antioxidant enzymes, while GSSG represents oxidized glutathione [ 16 , 17 ]. The results indicated that both the GSH:GSSG ratio and total antioxidant capability were attenuated following CAP PZ2 treatment in Fadu, Cal27, A375, and Hth83 cells ( Fig. 5 N). These findings collectively demonstrate that CAP PZ2 disrupts redox deregulation in HNC cell lines through two mechanisms: excessive ROS production and attenuated antioxidant defense.

CAP PZ2 treatment induced autophagy in HNC cells

Autophagy and apoptosis are vital processes involved in tumorigenesis and exhibit an interactive relationship in anti-tumor therapy. Therefore, investigating the mechanism underlying the interplay between autophagy and apoptosis in tumors is crucial for understanding the therapeutic effects of anticancer drugs. Our study revealed that the expression levels of Beclin 1 and LC3B showed the most significant increase after 24 h of CAP PZ2 treatment in Fadu, Cal27, A375, and Hth83 cells. Conversely, the expression of the P62 protein was notably reduced ( Fig. 6 A–H). Furthermore, immunofluorescence analysis demonstrated a significant increase in the number of LC3B dots in HNC cells treated with CAP PZ2 30 and 60s for 24 h ( Fig. 6 I-L). These findings suggest that CAP PZ2 treatment promotes the formation of autophagosomes upstream of the lysosomes.

CAP PZ2 treatment targets multiple key components involved in cancer metabolism and reduces lactate production

Glycolysis is a critical metabolic process in tumor cells, and targeting over-activated enzymes in HNC cells is a promising therapeutic approach. Glucose is converted into pyruvate through glucose transporters (e.g., GLUT1) and glycolytic enzymes, generating ATP and lactate. Hexokinases 1 (HK1) and HK2 initiate glucose metabolism, while pyruvate dehydrogenase (PDH) kinases (e.g., PDK1 and PDK3) enhance pyruvate-lactate conversion by regulating PDH [ 37 ]. Lactate dehydrogenase A (LDHA) facilitates pyruvate-to-lactate conversion, and pyruvate kinase isozyme M2 (PKM2) diverts glucose intermediates towards the pentose phosphate pathway (PPP) for macromolecule synthesis [ 16 , 17 , 38 ]. Lactate, the primary glycolysis product, serves as a nutrient and is associated with antioxidant stress and lipid biosynthesis in cancer cells. To investigate the underlying mechanism, we evaluated the impact of CAP PZ2 treatment on glucose transporters and glycolytic enzymes at the protein and mRNA levels using Western blot and qRT-PCR analysis. After 60 s of CAP PZ2 treatment, the protein expression levels of PKM2, pyruvate kinase M1/2, HK1, HK2, and PDK1 decreased progressively at 24 h ( Fig. 7 A). Similarly, Glut1 , HK1 , HK2 , LDHA , PKM2 , and PDK1 mRNA levels in Fadu, Cal27, A375, and Hth83 cells showed reduction at 24 h ( Fig. 7 B). This decrease in LDHA mRNA levels correlated with reduced lactate production in HNC cells ( Fig. 7 C). Overall, CAP PZ2 disrupts glycolysis at multiple enzymatic points and significantly decreases lactate production in HNC cells ( Fig. 7 C).

Attenuates oncogenic proliferative signaling in cancer cells

The PI3K/Akt/mammalian target of rapamycin (mTOR) pathway is crucial for cell proliferation, apoptosis, migration, invasion, and autophagy [ 39 , 40 ]. It has been associated with human cancers and plays a role in growth control and glucose metabolism [ [41] , [42] , [43] , [44] ]. Inactivating the PI3K/Akt/mTOR pathway has been shown to inhibit HNC [ [45] , [46] , [47] ]. This pathway regulates glucose transporters and glycolytic enzymes through AKT and mTOR, respectively [ 19 , 48 ]. Reciprocal activation between glycolysis and the PI3K/Akt/mTOR/HIF-1α signaling pathway promotes cancer cell survival [ 43 ]. We investigated the impact of CAP PZ2 on HIF-1α and PI3K/Akt/mTOR signaling in HNC cell lines. CAP PZ2 treatment significantly reduced the protein expression of PI3K, p-AKT, HIF-1α, and p-mTOR in Fadu, Cal27, A375, and Hth83 cells ( Fig. 7 E and F).

CAP PZ2 induced apoptosis in HNC cells

Based on enrichment analysis results, we investigated the mechanism of cell death induction by CAP PZ2 . CAP PZ2 treatment induced chromatin condensation and apoptotic bodies in Fadu, Cal27, A375, and Hth83 cells, as observed with Hoechst fluorescent staining ( Fig. 8 A, Supplemental Fig. 6A ). Additionally, AO/EB staining demonstrated reduced cell viability and increased apoptotic cells in Fadu, Cal27, A375, and Hth83 cells treated with CAP PZ2 ( Fig. 8 B and C and Supplemental Figs. 6B and C ). Combining CAP PZ2 with DDP further enhanced chromatin condensation and apoptotic bodies in Fadu and Cal27 cells ( Fig. 8 A–C). Furthermore, apoptosis was confirmed by using the PE annexin-V/7-AAD apoptosis kit and flow cytometry. The results exhibited a treatment time-dependent increase in apoptotic cell population in A375 and Hth83 cells with CAP PZ2 treatment ( Supplemental Figs. 6D–G ). Compared to the control sample (2.19 % in Fadu or 0.25 % in Cal27), treatment with DDP alone resulted in only 8.63 % (Fadu) or 23.17 % (Cal27) apoptotic cells ( Fig. 8 D–G). However, CAP PZ2 alone increased apoptosis to 16.94 % or 45.58 %, and the co-treated group showed a significant increase in apoptotic cells (53.4 % in Fadu or 70.1 % in Cal27) compared to the control group ( Fig. 8 D–G). Western blot analysis showed increased levels of cleaved caspase-3, cleaved caspase-9, and Bax, and decreased expression of Bcl2 in CAP PZ2 -treated cells ( Supplemental Figs. 6H and I ). The combination treatment of CAP PZ2 and DDP further enhanced these effects ( Fig. 8 H and I). Additionally, CAP PZ2 induced cell cycle arrest at the S phase and modulated the expression of cyclin D1 and p21 in HNC cells ( Supplemental Figs. 7A–H ). Overall, CAP PZ2 induces apoptosis and cell cycle arrest in HNC cells, and the combination with DDP enhances these effects.

CAP PZ2 treatment inhibited in vivo tumor growth in a xenograft mouse model

To investigate CAP PZ2 's anti-neoplastic effect on HNC in vivo , we conducted a proof-of-principle experiment using cell-derived xenograft (CDX) models. Mice were subcutaneously injected with Fadu cells and treated with isotype control PBS, CAP PZ2 , DDP, or the combination of CAP PZ2 with DDP (combo) ( Fig. 9 A and B). Tumor growth inhibition was observed in the CAP PZ2 or DDP alone groups, but the combo groups showed the most significant inhibition ( Fig. 9 C–E). In addition, the average tumor weight was significantly lower in the CAP PZ2 -, DDP-, or combination-treated mice compared to the control group. We calculated a Drug Interaction Index (CDI) of 0.172, indicating a highly significant synergistic effect between CAP PZ2 and DDP in cell-derived xenograft (CDX) models. The combination treatment demonstrated remarkable effects on both tumor growth inhibition and prolonged survival ( Fig. 9 F).

SUVmax was used as a parameter to calculate the 18F-FDG uptake in each group. The SUVmax of the tumor were 1.01 ± 0.14, 0.74 ± 0.12, 0.73 ± 0.14 in the control, CAP PZ2 and combination treatment groups on day one week after treatment and 1.15 ± 0.29, 0.68 ± 0.03, 0.48 ± 0.17 on day two weeks after treatment, respectively. ( Fig. 9 G and H). The SUVmax of tumors was reduced in both the CAP PZ2 group ( P < 0.05) and the combination treatment group ( P < 0.05) compared with the control group. ( Fig. 9 G and H). The tumor of control group exhibited the highest uptake of 18F-FDG, whereas the tumor of combined treatment group demonstrated the lowest 18F-FDG uptake, indicating a suppressed tumor glucose metabolism. These findings suggest that CAP PZ2 treatment methods and combined treatment methods are effective in reducing tumor SUVmax, with the combined treatment showing the greatest effect.

To explore the molecular mechanisms involved in CAP PZ2 's in vivo treatment of head and neck cancer cells, we analyzed the activation of Akt and the proliferative index Ki67 in the developed tumors using IHC staining. The results showed that Akt phosphorylation and the Ki67 proliferative index were significantly decreased in the CAP PZ2 and DDP co-treatment groups compared to the other groups ( Fig. 9 I). Additionally, we assessed the expression of migration and invasion-related proteins MMP2, MMP9, N-cadherin, and E-cadherin. CAP PZ2 treatment markedly downregulated MMP2, MMP9, and N-cadherin expression and significantly upregulated E-cadherin expression. These effects were more pronounced in the combo treatment group ( Fig. 9 J). Moreover, we examined the expression of glucose transporters and glycolytic enzymes-related proteins using IHC. Control group tumors exhibited moderate to strong staining intensity of HK1, PKM2, LDHA, and PDK1, whereas CAP PZ2 -treated tumors showed weak or undetectable expression of these proteins ( Fig. 9 K). IHC staining also revealed that the combo groups had significantly decreased levels compared to other groups ( Fig. 9 K). Collectively, these results indicate that the inhibition of HNC tumor growth by CAP PZ2 may involve the blockade of glycolysis at multiple enzymatic points and the AKT pathway.

CAP PZ2 enhances the anti-tumor effect of PD-1 immunotherapy in HNC

Immune checkpoint blockade therapies targeting the PD-L1/PD-1 axis have demonstrated clinical benefits. However, many patients exhibit poor responses to these therapies due to the immunosuppressive nature of the tumor microenvironment [ 49 , 50 ]. Here, we explore the potential of combines CAP PZ2 and immune checkpoint blockade (ICB) in HNC cells. We observed that CAP PZ2 treatment upregulated the expression of PD-L1, indicating that CAP can regulate PD-L1 in these cells ( Fig. 10 A–C). In immune-component mice, CAP PZ2 treatment led to a significant reduction in tumor weight and size ( Fig. 10 E–G). The IHC staining of the xenograft tumors revealed an increase in tumor infiltration of CD3, CD4, and CD8 T cells upon CAP PZ2 treatment ( Fig. 10 H), suggesting that CAP PZ2 might ameliorate the immunosuppressive microenvironment of head and neck cancer. In mice injected with SCC7 cells, both CAP PZ2 and anti-PD-1 treatments inhibited tumor growth and prolonged survival ( Fig. 10 J-L), with the combo group showing the most significant effects ( Fig. 10 M). Compared to the control group, the average tumor weight in mice treated with CAP PZ2, anti-PD-1 or a combination therapy was significantly lower. We calculated a CDI of 0.188, indicating a significant synergistic effect between CAP PZ2 and anti-PD-1 in SCC7 CDX models. The combo treatment also resulted in a more pronounced increase in CD8 and CD4 T cells compared to individual treatments ( Fig. 10 N, L).

Discussion

In this study, we used the handheld plasma device, CAP PZ2 , to investigate its efficacy in treating head and neck cancer (HNC) and understand its mechanisms of action. We employed human nasopharyngeal carcinoma Fadu cells, human tongue squamous carcinoma Cal27 cells, human melanoma A375 cells, and human thyroid cancer Hth83 cells as our in vitro experimental models. Our findings revealed that CAP PZ2 affects multiple molecular pathways involved in HNC, including E-cadherin/N-cadherin switch, GSH oxidation-reduction pathway, metabolic pathway, and PI3K/Akt/mTOR/HIF pathway. These pathways influence cancer-related processes like proliferation, migration, invasion, epithelial-to-mesenchymal transition, oxidative defense, programmed cell death, and metabolic reprogramming. Additionally, We have unveiled that combining CAP PZ2 with chemotherapy drugs (cisplatin) or immune checkpoint blockade (ICB) therapy effectively inhibits tumor growth and prolongs survival in mice.

The application of CAP in oncology has witnessed rapid development since its therapeutic effect on melanoma was initially reported. Numerous studies have demonstrated the inhibitory impact of CAP on the growth and induction of tumor cell death in various malignant tumors, including hepatocellular carcinoma, prostate cancer, cervical cancer, cervical squamous cell carcinoma, oral squamous cell carcinoma, osteosarcoma, multiple myeloma, and lymphoma [ 51 , 52 ]. Furthermore, a series of clinical trials have revealed that CAP has tremendous therapeutic potential for HNCs [ 53 ]. This is partially because of CAP's distinct technical advantages. Some of the most common cancers, such as those arising from solid organs (lung and liver) and gastrointestinal tracts (colorectum and stomach), are anatomically deep [ 54 ]. This poses real challenges for direct CAP treatment. In comparison, most cancers in the head and neck should be accessible by CAP, as they are either superficial (skin cancers), subcutaneous (thyroid and parotid glands), or reachable through upper ADT (oral cavity, pharynx, and larynx) [ 1 , 53 ].

The latest preclinical and clinical trials of CAP oncology primarily focused on the following aspects. Firstly, it enhances preoperative sensitivity in HNC surgery. Schuster et al. tested CAP on nine advanced cases pre-surgery, showing promise [ 55 ]. Another study displayed CAP's effect on skin melanoma, inducing cell death and less inflammation [ 56 ]. Secondly, CAP aids intraoperative procedures for surgeons due to its non-thermal nature [ 57 ]. Plasma jets function akin to handheld tools, assisting surgeries or linked with existing instruments. Thirdly, CAP acts post-surgery, eliminating pathogens, boosting clotting, and aiding tissue recovery [ 58 ]. Notably, it kills residual cancer cells, curbing metastasis risk [ 55 , [58] , [59] , [60] , [61] ]. Mettelman et al. highlighted CAP's benefits for HNSCC patients, including reduced pain, improved healing, and social well-being [ 53 ]. Considering the global prevalence and mortality rate of head and neck tumors, the current body of CAP-related studies addressing HNC treatment remains relatively limited [ 62 ]. Notably, existing qualified research is predominantly confined to cell experiments, focusing on fundamental aspects such as selective cell death. Moreover, the mechanisms explored in these studies primarily center around CAP-induced DNA damage or genetic alterations in cancer cells [ 1 , 58 , 63 ]. In contrast, our study takes a comprehensive approach by utilizing four types of human HNC cell lines and incorporating both human and mouse CDX models. We undertook a thorough analysis of the treatment effects, employing classical toxicity assessments, as well as in-depth gene and protein expression analyses. This multi-faceted approach allows us to elucidate the underlying mechanisms of CAP PZ2 's tumor treatment effects. Additionally, our study provided evidence of a synergistic effect when combined with chemotherapy or immune checkpoint therapy, further contributing to the potential clinical applications of our findings. On the other hand, Previous plasma technologies used in cancer treatment, such as dielectric barrier discharge (DBD), atmospheric pressure plasma jet (APPJ), and pulsed atmospheric arc (PAA), each exhibit inherent limitations [ 64 ]. For instance, while certain CAP machines feature a handheld electrode head, their base units significantly increase the size compared to the commonly found bipolar diathermy machines in operating theaters [ 65 ]. In this study, we are the first to introduce piezoelectric direct discharge (PDD)-based CAP to HNC treatment, attempting to surmount technical obstacles that prevent clinical application of CAP in HNC surgery.

Our results showed that CAP PZ2 treatment exhibited a significant and plasma treatment time-dependent inhibition of cell viability in Fadu, Cal27, A375, and Hth83 cells, accompanied by observable morphological changes such as cellular contraction, chromatin condensation, and nuclear fragmentation. Cisplatin (DDP) induces oxidative damage in both normal and cancer cells. Combining CAP PZ2 with DDP can maximize oxidative stress on cancer cells while potentially minimizing harm to normal cells through localized cancer cell treatment. Lee et al. reported that the combination of CAP PZ2 and DDP has a synergistic effect on the treatment of oral squamous cell cancer [ 21 ]. In our study, we investigated the synergistic effect of combining CAP PZ2 with cisplatin-based chemotherapy using Fadu cells in vitro . The co-administration of CAP PZ2 with cisplatin (DDP) rendered Fadu cells more sensitive to the therapeutic agent, resulting in notable proliferation inhibition. Notably, a lower concentration of DDP (20 μg/mL) enhanced the effects of CAP PZ2 on Fadu cells and induced cellular toxicity and apoptosis at non-toxic levels. When Fadu cells were co-treated with CAP PZ2 (20 s) and DDP (20 μg/mL), the CDI was 0.235 at 24 h. This indicates a highly significant synergistic effect in vitro . This combinatorial therapy not only enhances anti-tumor effectiveness but also reduces the required chemotherapeutic drug dosage.

CAP PZ2 treatment effectively inhibits invasion and metastasis of HNC cells, providing a novel therapeutic strategy. MMPs, including MMP9 and MMP2, degrade the basement membrane and facilitate tumor metastasis [ 66 ]. VEGF promotes angiogenesis and tumor cell proliferation [ 66 ]. MTDH contributes to cancer progression, chemoresistance, and metastasis [ 67 ]. CAP PZ2 down-regulates these markers in Fadu, Cal27, A375, and Hth83 cells. EMT affects tumor cell invasion and metastasis [ 68 ]. E-Cadherin and N-Cadherin are markers of EMT and tumor progression [ 69 ]. Vimentin is involved in cell integrity and resistance to stress [ 70 , 71 ]. CAP PZ2 treatment increases E-Cadherin expression and decreases Vimentin and N-Cadherin expression in Fadu, Cal27, A375, and Hth83 cells. These findings suggest that CAP PZ2 modulates the migration and invasion abilities of these cells by influencing the EMT process.

Autophagy is a conservative pathway dependent on lysosomes, utilized for the degradation of cytoplasmic proteins, macromolecules, and organelles through a dynamic and multi-step process [ 72 , 73 ]. LC3 is a key molecule involved in autophagy membrane formation and is present throughout the process. Beclin 1 contributes to autophagy formation, and P62 plays a role in metabolic crosstalk in tumor stroma. Autophagy and apoptosis are important in tumorigenesis and anti-tumor therapy. Autophagy induces tumor cell death, inhibits proliferation, and degrades cancer proteins, suppressing tumor development, metastasis, and enhancing chemotherapy sensitivity [ 72 , [74] , [75] , [76] , [77] , [78] , [79] , [80] ]. In this study, CAP PZ2 treatment substantially increased the levels of LC3B and Beclin 1 in HNC cells, while significantly reducing the level of P62. These findings suggest the activation of the autophagy-lysosomal pathway in tumor suppression. Furthermore, these results highlight the crucial role of lethal autophagy in the process of CAP PZ2 inhibiting head and neck cancer cells.

An ideal cancer treatment would selectively induce cancer cell death and inhibit cancer cell survival pathways. Cold atmospheric plasma (CAP) generates reactive oxygen species (ROS) and charged particles that can interact with the surrounding medium, inhibiting cancer survival pathways, including redox deregulation [ 19 ]. Cancer therapies based on ROS-generating strategies, including radiotherapy, are not new and usually rely on accumulation of ROS to induce direct cancer cell death [ 17 , 81 ]. Significantly, CAP PZ2 used in our study could produce high-level, steady, and diverse populations of ROS, thereby resulting in high-rate of apoptosis in HNC cell lines. Our results demonstrated a significant increase in intracellular ROS levels following CAP PZ2 treatment. Glutathione (GSH) serves as the primary cellular system for clearing ROS by supplementing antioxidant enzymes, with glutathione disulfide (GSSG) being the oxidized form of glutathione [ 17 ]. Furthermore, we evaluated antioxidant activity based on the ratio of reduced GSH to GSSG. Interestingly, we observed a reduction in both the GSH:GSSG ratio and total antioxidant capability after CAP PZ2 treatment. These findings indicate that CAP PZ2 induces a dual disruption of redox deregulation in Fadu, Cal27, A375, and Hth83 cells, characterized by excessive ROS production and weakened antioxidant defense.

Metabolic reprogramming is a crucial characteristic of malignant tumors, supporting cell survival, bioenergy demand, and macromolecule synthesis. In aerobic glycolysis, cancer cells rely on glucose fermentation instead of aerobic mitochondrial respiration, generating lactic acid for ATP and biomolecule production [ 82 ]. Key enzymes in glycolysis, such as HK1, HK2, PKM2, and LDHA, play essential roles in tumor cell metabolism [ 16 , 17 , 38 ]. Overexpression of GLUT1 and SGLT2 contributes to tumorigenicity in HNC. Dysregulation of glucose metabolism is a recognized feature in HNC initiation and progression. CAP PZ2 treatment enhances energy metabolic processes and cancer growth macromolecule synthesis. Glycolysis is particularly important in HNC metabolism, making it a potential target for selective therapy. In this process, glucose is converted to pyruvate through GLUT1 and glycolytic enzymes, producing ATP and lactate [ [82] , [83] , [84] , [85] ]. Inhibition of glycolytic enzymes by CAP PZ2 reduces lactate production, disrupting cancer survival pathways. PKM2 inhibition diverts glucose intermediates to the PPP, impairing macromolecule synthesis and redox balance. LDHA inhibition affects cancer stem and progenitor cells, while lactate amplifies the PI3K/AKT signaling pathway [ 43 ].

The findings suggest that CAP PZ2 -induced inhibition of lactate production and LHDA expression effectively blocks the glycolytic activation of PI3K/AKT signaling. The PI3K/AKT/mTOR pathway is a commonly activated signaling pathway in human cancers, playing a critical role in cell proliferation, apoptosis, autophagy, protein synthesis, and glucose metabolism [ [41] , [42] , [43] , [44] ]. On one hand, the PI3K signal regulates glucose transporter expression through AKT, enhancing glucose uptake by hexokinase and stimulating phosphofructokinase activity. On the other hand, mTOR activates transcription factors, including HIF-1α, which promotes the expression of glycolytic enzymes involved in cancer progression [ 19 , 48 ]. Conversely, lactate production resulting from glycolysis amplifies PI3K/AKT signaling [ 43 ]. This reciprocal activation between glycolysis and PI3K/AKT/mTOR/HIF-1α signaling perpetuates cancer cell survival. In our in vitro experiments, the PI3K/AKT/mTOR/HIF-1α signaling pathway was inhibited. CAP PZ2 treatment significantly reduced the protein levels of PI3K, p-AKT, HIF-1α, and p-mTOR in Fadu, Cal27, A375, and Hth83 cells, respectively.

Apoptosis, a programmed cell death process, plays a crucial role in tumor destruction by inducing marked morphological changes and biochemical events. In our study, CAP PZ2 treatment inhibited the proliferation of Fadu, Cal27, A375, and Hth83 cells, increasing early and late apoptotic rates and displaying apoptotic morphological features. CAP-induced ROS generation disrupts mitochondrial function and promotes cell apoptosis [ 86 ]. CAP PZ2 treatment arrested the cell cycle at the S phase by regulating cyclin D1 and p21. The increased Bax/Bcl-2 ratio activated cleaved caspase-3 and cleaved caspase-9, leading to apoptosis. Co-treatment of CAP PZ2 and cisplatin (DDP) enhanced apoptotic markers and apoptotic cell numbers, with a decrease in Bcl2 levels. These findings confirm that CAP PZ2 induces apoptosis through caspase modulation in HNC cells. Intracellular RONS increase caspase activity, promoting apoptosis [ 87 ]. The upregulation of cleaved caspase-3 and caspase-9 reduces HNC progression. The synergistic effect of CAP PZ2 and DDP explains the observed apoptosis in Fadu cells.

Following the successful application of CAP PZ2 treatment in vitro for HNC cells, we proceeded to investigate its therapeutic potential in cell-derived xenograft (CDX) models in animals. Our findings revealed that tumor growth was partially inhibited in the single administration CAP PZ2 or DDP groups compared to the control group. However, the most pronounced inhibition of tumor growth was observed in the combination therapy groups. Furthermore, we confirmed that the anticancer effect on Fadu cells xenografted mice was attributed to the induction of apoptosis, promotion of autophagy, and inhibition of glycolytic enzymes by CAP PZ2 . These effects were particularly significant in the combination therapy group.

While immune checkpoint blockade (ICB) holds promise for cancer treatment, its clinical application is limited due to challenges such as low objective response rates and severe systemic side effects. Combining immunocheckpoint blockade with other types of anti-tumor drugs presents an opportunity to improve response rates, prolong response duration, and activate anti-tumor immune memory. Such combination therapy strategies can reduce adverse reactions and enhance drug safety by adjusting dosages without compromising treatment efficacy. Our research demonstrates that the combination of CAP PZ2 and ICB therapy effectively inhibits tumor growth, leading to prolonged survival in tumor-bearing mice. CAP PZ2 treatment rapidly induces the accumulation of CD8 and CD4 T cells among cancer cells, synergizing with anti-PD-L1 therapy. The damaging effect of CAP PZ2 on cancer cells exposes tumor-specific antigens, which may render them visible during immune surveillance, thereby promoting the initiation and activation of cytotoxic T cells. Furthermore, CAP PZ2 -induced regulation of the tumor microenvironment may be facilitate the recruitment and infiltration of immune cells. This unique relationship provides a theoretical basis for combining CAP PZ2 with immune checkpoint blockade.

Conclusion

In this preclinical study, we are the first to systematically investigate the potent anti-tumor effect of CAP PZ2 on head and neck cancer cells and the underlying molecular mechanism. Using various in vitro models (Fadu, Cal27, A375, and Hth83 HNC cells) and in vivo model (CDX mice), we first established that CAP PZ2 exerts anti-tumor functions through inhibiting various cancer hallmarks, such as cell proliferation, migration and invasion, EMT, and antioxidant defense in HNC cells. Further phenotype and mechanism studies unveiled the dual anti-cancer strategy of CAP PZ2 : inducing cancer cell death and disrupting its survival mechanisms. The former is evidenced by CAP PZ2 -induced diverse forms of programmed cell death, such as apoptosis and autophagy, while the latter is demonstrated through CAP PZ2 weakened survival mechanisms, such as tumor glycolysis and carcinogenic proliferation. Assisted by bioinformatic analysis, we discovered and verified numerous signaling pathways that are sensitive to CAP PZ2 treatment of HNC, such as, E-cadherin/N-cadherin switch of EMT, apoptotic pathway, oxidation-reduction pathway of GSH, glycolytic pathway, and PI3K/Akt/mTOR/HIF pathway. Most importantly, CAP PZ2 achieved aforementioned anti-HNC functions with exceptional power efficiency, compactness and versatility, and without additional gas requirement other than just ambient air. Additionally, our study provided evidence of a synergistic effect when combined with chemotherapy or immune checkpoint therapy, further contributing to the potential clinical applications of our findings. In conclusion, CAP PZ2 emerges as a promising adjunctive physicochemical therapy for HNC. Based on the transcriptomic, protein, cellular and animal data from this study, the ideal next stage would be preparation for the clinical trial of human HNC.

Ethics approval and consent to participate

The animal study was reviewed and approved by The Laboratory Animal Care and Use Committees of the School of Medicine, Tongji University (Ethics number: TJAA11923102).

Consent for publication

Not applicable.

Author contributions

Conceptualization, F.T. and Y.W.; methodology, Y.W., X.M. D.L. and Z.W.; writing-original draft preparation, Y.W.; writing-review and editing, Y.W. and F.T.; supervision, Z.C. and F.T.; funding acquisition, Y.W. and F.T. All authors have read and agreed to the published version of the manuscript. The authors declare no conflict of interest.

Funding

This work is sponsored by the Fundamental Research Funds for the Central Universities. It was also supported by the National Natural Science Foundation of China (No.82271192) and the Natural Science Foundation of Gansu Province (No. 21JR1RA015).

Declaration of competing interest

The authors declare no competing interests

Footnotes

Supplementary data to this article can be found online at https://doi.org/10.1016/j.redox.2023.102991 .

Abbreviations

head and neck cancer

Cold atmospheric plasma

Cell Counting Kit-8

RNA sequencing

gene ontology

reactive oxygen species

transmission electron microscopy

glutathione

oxidized

glutathione

Quantitative real time polymerase chain reaction

vascular endothelial growth factor

Metadherin

phosphate buffered saline

sequestosome 1

microtubule-associated protein 1 light chain 3 beta

matrix metallopeptidase 2

matrix metallopeptidase 9

Hexokinase I

Hexokinase II

Pyruvate Dehydrogenasemuscle

pyruvate kinase isozyme 2

Lactate dehydrogenase

facilitated glucose transporter 1

Phosphoinositide 3-kinase

hreonine protein kinase

mammalian target of rapamycin

hypoxia inducible factor-1

Acridine orange and ethidium bromide

cyclin-dependent kinase inhibitor 1A

programmed cell death protein 1

Programmed cell death 1 ligand 1

Supplementary data

The following is the Supplementary data to this article:

Multimedia component 1

Data availability

Data will be made available on request.

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Data Availability Statement

Data will be made available on request.