Ⅰ. INTRODUCTION

Oral cancer is one of among the malignancies with a globally increasing incidence, the majority of which are oral squamous cell carcinomas (OSCC).(1) OSCC arises from the abnormal proliferation of squamous epithelial cells in the oral mucosa. Major risk factors include smoking, alcohol consumption, human papillomavirus (HPV) infection, and chronic mechanical irritation.(2) Without early diagnosis and appropriate treatment, OSCC typically demonstrates a poor prognosis due to its high rates of recurrence and metastasis, highlighting the need for continued research into novel therapeutic strategies.

Current standard treatment for OSCC involves multimodal approaches that combine surgical resection, radiation therapy, and chemotherapy.(3) However, these therapies are often accompanied by severe side effects and are limited in efficacy due to the development of resistance mechanisms by tumor cells. (4) Conventional chemotherapy is particularly problematic owing to its non-specific cytotoxicity, which harms normal tissues and significantly reduce patient’s quality of life. Consequently, immunotherapy has recently emerged as a promising alternative, particularly due to its ability to elicit tumor-specific immune responses. (4)

Immunotherapy enhances antitumor activity by either activating the host immune system or suppressing tumor-driven immune evasion mechanisms. Among these approaches, immune checkpoint inhibitors have garnered significant attention in recent years. (5, 6) Pembrolizumab (Keytruda), a monoclonal antibody targeting programmed death-1 (PD-1), has demonstrated considerable therapeutic benefits across various cancer types and is widely used in clinical practice.(6) PD-1, expressed on the surface of activated T-cells, binds to its ligand PD-L1 on tumor cells, leading to T-cell inactivation and immune evasion.(7) Keytruda blocks this interaction between PD-1 and PD-L1, thereby restoring cytotoxic T-cell function and enhancing tumor-specific immunity. Its clinical efficacy has been validated in melanoma, non-small cell lung cancer, and head and neck cancers, establishing it as a key agent in modern immunotherapy. (8-10)

Nevertheless, the therapeutic effectiveness of Keytruda varies significantly depending on several factors, including PD-L1 expression levels in the tumor microenvironment, tumor mutation burden (TMB), and immune cell infiltration. (11) In OSCC, fewer than 20% of patients demonstrate a meaningful response to Keytruda treatment, and those with advanced disease often show poor outcomes with conventional chemotherapy treatment. (12) These limitations have prompted growing interest in combination therapies to enhance the efficacy of immune checkpoint blockade. One emerging strategy involves the use of non-thermal plasma (NTP) therapy.

NTP exerts selective cytotoxicity in cancer cells through the generation of high levels of reactive oxygen species (ROS), while sparing healthy tissues from significant damage. (13) Its anticancer mechanisms include the induction of increased oxidative stress, DNA damage, and apoptosis promotion. (14, 15) Moreover, recent studies suggest that NTP can trigger immunogenic cell death (ICD), thereby enhancing antitumor immune responses and offering potential synergy with immunotherapeutic agents. (16)

In the present study, the authors aimed to evaluate the antitumor efficacy of combining Keytruda with NTP in an OSCC xenograft mouse model established using FADU cells. Tumors were induced by xenografting the cancer cells into immunodeficient mice, followed by treatment with either Keytruda alone or in combination with NTP. The study assessed tumor growth suppression and compared the effects of combination therapy to those of Keytruda monotherapy, while also investigating the underlying mechanisms of action. These findings may provide preclinical evidence supporting the use of Keytruda in combination with NTP as a novel therapeutic strategy for OSCC and contribute foundational data for future clinical applications.

Ⅱ. MATERIALS and METHODS

1. Chemicals

The Keytruda used in this experiment was purchased from Merck (Merck Millipore, Billerica, MA, USA) and dissolved in dimethyl sulfoxide (DMSO) to make a 20 mg/ml stock solution for further use.

2. Non-thermal Atmospheric Pressure Plasma Supplier

The non-thermal plasma (NTP) machine utilized in this study, developed by Feagle Company (Yangsan-si, Kyeongsangnam-do, Korea), consists of one dielectric and two electrodes: a stainless-steel inner electrode surrounded by an alumina cylinder (10mm in dia.), and an outer electrode covered in copper tape. Argon gas flows at 2 standard L/min between the internal electrode and alumina cylinder, generating plasma between electrodes. The plasma temperature was maintained below 35°C for 10 minutes. Although ultraviolet emissions were undetected, the generated plasma primarily contained OH radicals, as previously detailed in other research(16). Voltage was continuously monitored to maintain consistent plasma conditions and prevent unintended cell death.

3. Animal Care

This study was approved by the Institutional Animal Care and Use Committee of Pusan National University (Approval number: PNU-2021-2967). Forty 5-week-old nude mice (BALB/c strain) weighing an average of 19 ± 4 g were purchased from Samtaco BIO KOREA (Osan, Korea). Animals were housed in an SPF animal facility at Pusan National University Yangsan Campus at 23 ± 2℃, 55 ± 5% humidity, with a 12-hour light/dark cycle, and provided free access to food and water. Special care was taken to minimize stress to the animals.

4. Xenograft mouse model

A xenograft nude mouse tumor model using an FADU cell line was employed to assess antitumor effects. Animals were anesthetized using isoflurane inhalation (4–5% induction, 1–2% maintenance) delivered via an isofluranesoaked cotton placed in a cylinder. A mixture of 5 × 10⁶ FADU cells and Matrigel (1:1 ratio, total volume of 100 μl) was subcutaneously injected into the dorsal region of each mouse using a 29G insulin syringe, with each mouse receiving a single injection to form one tumor. Tumor size was measured twice weekly using Vernier calipers, and experiments began when tumors reached 5 mm in diameter. Body weights were regularly monitored.

5. Experimental Design (Grouping by Tumor Induction and Treatment)

Tumor-bearing mice were divided into one control group and three experimental groups. Animal body weights were recorded twice weekly throughout the study.

The control group received phosphate-buffered saline (PBS) injections after tumor formation. Experimental Group 1 received intraperitoneal injections of Keytruda (0.2 mg/dose in RPMI-1640), administered three times at 3-day intervals after tumor formation. Experimental Group 2 was treated with a combination of Keytruda (intraperitoneal) and NTP (applied three times weekly), with plasma exposure lasting 5 minutes per session. Experimental Group 3 received a combination of Keytruda (intratumoral) and NTP (also applied three times weekly), with plasma exposure lasting 5 minutes per session (Figure 1).

Tumor dimensions (length [longest] and width [shortest]) were measured using Vernier calipers twice weekly, and tumor volumes were calculated with the following formula: Tumor volume = 1/2 × length (longest) × width (shortest)² The entire experiment was repeated twice, and the data were statistically analyzed.

6. Animal Euthanasia, Tumor Collection, and Analysis

Fourteen days after treatment initiation, animals were euthanized, grouped by treatment, photographed, and tumors along with vital organs were harvested and weighed. Tumor tissues were embedded in paraffin blocks for histological analysis. Tissue sections were stained with Hematoxylin & Eosin (H&E), TUNEL staining for apoptosis detection, and immunohistochemical staining for caspase 3 and Ki-67 as markers of apoptosis and proliferation, respectively.

7. TUNEL Assay

Apoptotic cells were identified in formalin-fixed, paraffin- embedded tumor sections using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay. Tissue sections (4 μm thickness) were processed according to the manufacturer's instructions, with 3,3'-Diaminobenzidine (DAB) used as the chromogen and hematoxylin as a counterstain. Cells with brown-stained nuclei were classified as TUNEL-positive, whereas those with only blue nuclei were negative.

8. Immunohistochemistry for Caspase 3 and Ki-67

Paraffin-embedded tumor tissue sections (4 μm thickness) were mounted on slides, dried, and deparaffinized three times in xylene, then rehydrated through a series of ethanol washes (100%, 90%, and 70%). Antigen retrieval was performed using a citric acid-based buffer solution (pH 9.0) for 20 minutes. Endogenous peroxidase activity was blocked for 10 minutes. Sections were incubated overnight at 4℃ with primary antibodies against caspase 3 and Ki-67 (Abcam, UK; dilution 1:100). Slides were subsequently incubated with REAL EnVision-HRP horseradish peroxidase (Dako, Carpinteria, CA) for 10 minutes at room temperature, visualized with DAB, counterstained with Mayer’s hematoxylin, and mounted.

9. Quantification of In Situ Cell Death (TUNEL Assay and Immunohistochemistry)

The number of positively stained nuclei was counted in 10 randomly selected, non-overlapping fields at 400× magnification, and the percentage of positive cells was recorded. Staining positivity was graded on a scale from 0 to 4: 0 = no staining; 1 = 1%–25%; 2 = 26%–50%; 3 = 51%– 75%; 4 = 76%–100%. Statistical analysis was conducted using SPSS version 27 (IBM SPSS, Chicago, IL, USA). Differences between the control and experimental groups were analyzed using two-tailed Student’s t-tests, with p-values <0.05 considered statistically significant. Results were graphically presented using GraphPad Prism (version 9.5; GraphPad Software, San Diego, CA, USA).

Ⅲ. RESULTS

1. The Changes of Body Weight Following Keytruda and Plasma Treatment

Body weights were measured on experimental days 1, 8, 12, 15, and 19 following administration of Keytruda, plasma, or their combination. On day 8, all groups exhibited a slight increase in body weight, followed by a gradual decrease as treatment progressed. No statistically significant differences in body weights were observed among the control group, Keytruda-treated group, Keytruda (intraperitoneal) + NTP-treated group, and the group treated with a combination of Keytruda (intratumoral) +NTP (Figure 2). Although no significant difference was found between the Keytruda-alone group and the combined treatment group, the latter showed a comparatively smaller reduction in body weight.

2. Tumor formation and histological evaluation

The first palpable tumor appeared on day 5 following the injection of FADU cells, with xenografted tumors forming in all experimental animals throughout the study. Tumors identified by visual or palpation examination presented either pedunculated or sessile growth patterns with surfaces varying from smooth to irregular. Tumor sizes at the end of the experiment ranged from 1.2 to 2.5 cm in diameter.

Histological examination revealed tumor masses comprising neoplastic cells arranged in solid sheets interspersed with vascular proliferation. Marked cellular pleomorphism and increased mitotic activity were noted. Neither keratin pearl formation nor significant invasive characteristics were observed (Figure 3).

3. Tumor growth inhibition by Keytruda and combined Keytruda+NTP treatment

This experiment aimed to assess whether the Keytruda monotherapy and combined administration of Keytruda and NTP could effectively inhibit tumor growth at lower dosages. Tumor weights were measured on days 8, 12, 15, and 19 as follows:

The control group showed the fastest tumor growth. Slight tumor growth inhibition was observed in groups treated with Keytruda alone or combined Keytruda and NTP.

The Keytruda only-treated tumor weights on day 19 were significantly lower compared to the control group, which exhibited the most pronounced tumor-suppressive effect (Figure 4).

4. Apoptosis analysis via caspase-3 immunoexpression in xenograft tumor tissue

Immunohistochemical analysis assessed cleaved caspase- 3 expression, an apoptosis marker, across treatment groups. Although no significant differences were observed in the Keytruda monotherapy group compared to control, significantly elevated cleaved caspase-3 expression was found in groups receiving combined intraperitoneal or intratumoral Keytruda and NTP treatment. Tumor tissues from these groups displayed extensive activated caspase-3 staining compared to controls (Figure 5). These results indicate the combined treatment increased apoptosis, suggesting partial apoptosis induction contributing to tumor regression in xenograft model.

5. Apoptosis evaluation using TUNEL assay in xenograft tumor tissue

The TUNEL assay identified apoptotic cells in tumor tissues from the control and Keytruda-treated group, and intraperitoneal or intratumoral Keytruda and NTP treated groups, indicated by condensed, dark brown chromatin staining. Although a rise in apoptotic cells was observed in the Keytruda (intratumoral) + NTP combination group, this increase was not statistically significant compared to other groups (Figure 6).

6. Ki-67 expression in xenograft tumor tissue

Ki-67 immunohistochemistry evaluated cellular proliferation across treatment groups. While no significant differences were observed in the Keytruda only and Keytruda (intraperitoneal) + NTP groups, the group treated with combined Keytruda (intratumoral) + NTP demonstrated significantly decreased Ki-67-positive cells. The tumors from this group had markedly fewer Ki-67-positive nuclei compared to control tumors (Figure 7), indicating the treatment effectively inhibited cellular proliferation in vivo.

Ⅳ. DISCUSSION

This study evaluated the combined therapeutic effects of Keytruda and NTP treatment on body weight, tumor growth, apoptosis, and cell proliferation in a xenograft mouse model using FADU cells. Body weights of experimental annimals remained stable across all groups, with the combination treatment group exhibiting the least weight loss, suggesting that the regimen was well tolerated. Macroscopic and histological examinations revealed solid tumor formation in all groups.

Regarding the inhibition of tumor growth, both the Keytruda-only treatment group and the combination (Keytruda+NTP) treatment group exhibited reduced tumor weights compared to control group. Notably, the greatest tumor inhibition was observed in the group receiving intratumoral Keytruda in conjunction with NTP, indicating a synergistic therapeutic effect. Immunohistochemical analysis for cleaved caspase-3 revealed significant increase in apoptosis in the combination treatment group, suggesting enhanced activation of apoptotic pathways. Consistently, Ki-67 staining revealed a marked reduction in cancer cell proliferation in the same group.

Keytruda, a PD-1 immune checkpoint inhibitor, enhances T-cell-mediated tumor destruction by blocking the PD-1 and PD-L1 interaction. While it has demonstrated efficacy in various type of cancers, its theraeutic impact in OSCC remains limited, with response rates below 20%, particularly in advanced, treatment-resistant OSCC cases.(12) This study therefore investigated whether the combination of NTP could improve the efficacy of Keytruda in OSCC.

Recently, NTP is an emerging, selective anti-cancer modality that induces apoptosis and stimulates immune responses specifically in cancer cells. It generates reactive oxygen species (ROS) preferentially in cancerous cells compared to healthy cells, triggering oxidative stress that leads to cell death, proliferation arrest, and apoptosis, further amplifying anti-tumor immunity.(17) Oxidative stress exceeding critical thresholds disrupts cellular functions, halts proliferation, and promotes apoptosis. Indeed, the production of ROS increases with plasma irradiation time, and the anti- tumor effects of plasma are reportedly offset when antioxidants are added to the culture system to eliminate oxidative stress.(18) In addition, Zucker et al. reported that when keratinocytes and malignant melanoma cells were cultured and exposed to NTP, the level of cell death in melanoma cells significantly increased compared to keratinocytes, and the mechanism of this selective cell death was identified as apoptosis.(19) Furthermore, in a study using a xenograft model with U87 cells (a high-grade glioma cell line) in experimental animals, NTP was shown to significantly reduce the size of the xenografted tumors, thereby contributing to prolonged survival of the animals.(20) Based on these findings, it is expected that combined treatment with Pembrolizumab and NTP in xenograft tumors of experimental animals may provide superior therapeutic effects in OSCC.

Moreover, previous studies suggested that the combination of NTP exposure with PD-L1-related regimen upregulated PD-L1 expression in breast cancer cells, thereby enhancing the effectiveness of PD-L1 inhibitors.(21) Consequently, the availability of the PD-L1 inhibitor increased, and when NTP and the PD-L1 inhibitor were co-administered, the cell survival rate significantly decreased. This suggests the potential of NTP as an adjunctive treatment for immunotherapy in triple-negative breast cancer (21). A similar results were reported in OSCC cell lines, where PD-L1 antibody-conjugated gold nanoparticles combined with NTP induced significantly higher cancer cell death compared to monotherapy.(17) Consistent with these findings, our results demonstrated superior tumor suppression with the combined Keytruda-NTP treatment, significantly surpassing the efficacy of either treatment alone. The observed synergistic effect is likely attributable to enhanced ROS production and cell death induced by NTP, augmenting Keytruda's immune-mediated therapeutic effects. The results of this study are consistent with previous reports on the pro-apoptotic effects of NTP and provide preliminary experimental evidence suggesting that the combination of the these treatment modalities may be more effective for the treatment of OSCC.

Increased cleaved caspase-3 expression coupled with reduced Ki-67 levels in the Keytruda-NTP combination group confirmed effective suppression of cancer cell proliferation and promotion of apoptosis. It is highly likely that NTP complemented the limited apoptosis activation of pembrolizumab monotherapy, thereby enhancing the activation of apoptotic signaling pathways more effectively. NTP treatment likely compensates for Keytruda's limited apoptotic induction, amplifying apoptosis. Although TUNEL assays indicated a trend toward increased apoptosis, the lack of statistical significance suggests potential sensitivity limitations. Future studies employing more sensitive and specific methods are warranted for definitive confirmation. Nonetheless, cleaved caspase-3 results strongly support effective apoptosis induction by the Keytruda-NTP combined treatment.

This study provides preliminary experimental evidence supporting the combined use of Keytruda and NTP for treating OSCC, showing enhanced tumor suppression via increased apoptosis and reduced proliferation. The combined approach may beneficially alter the tumor microenvironment, facilitating stronger anti-tumor immune responses.

Limitations of this study include a small sample size, two experimental replications, relatively short experiment duration, and absence of detailed molecular analyses. As this study utilizes an animal model, additional experiments are essential for thorough characterization of tumor microenvironment changes prior to clinical application. Future investigations should elucidate the precise underlying mechanisms of this combination therapy to optimize its clinical applicability.

In conclusion, the combination of Keytruda and NTP treatment demonstrates superior therapeutic potential for OSCC compared to monotherapy with either Keytruda or NTP alone, highlighting its promise as an innovative clinical treatment strategy.