Research ArticleExperimental Studies

An In Vivo Study of Local Administration of Low-dose Anti-PD-1 Antibody Using an Oral Cancer Cell Line

KANAME SAKUMA, TOMOYUKI KII, HARUKA TAKAHASHI, SUSUMU SUZUKI, KAZUHIRO YOSHIKAWA, TETSUYA OGAWA and AKIRA TANAKA
Anticancer Research September 2022, 42 (9) 4293-4303; DOI: https://doi.org/10.21873/anticanres.15929

Abstract

Background/Aim: The immunotherapy approach using anti-programmed cell death 1 (PD-1) antibody has been demonstrated in oral cancer treatment. However, serious immune-related adverse events (irAE) have been reported. If local administration of small doses of anti-PD-1 antibody via intraarterial chemoradiotherapy could have the same antitumor effect of systemic administration, this can reduce irAE and medical expenses. In this study, we investigate the antitumor effects of local and systemic administration of a small amount of anti-PD-1 antibody, the overall survival (OS), and the immune environment around cancer. Materials and Methods: A mouse buccal mucosal oral squamous cell carcinoma cell line (Sq-1979) was used, and anti-mouse PD-1 was used as the drug. The cell line was transplanted to mouse, and the drug was locally (30 mg/body) and systemically (300 mg/body) administered in a dose. The tumor shrinkage rate and antitumor effect were examined 21 and 29 days after the start of administration. The OS was also compared in each of the groups. Furthermore, the subcutaneous growth of the tumors was inhibited, and the expressions of PD-L1, CD8T cells, perforin, and granzyme B were examined. Results: We found that the local low-dose and systemic groups had the same antitumor effect, OS also showed a significant prolongation. In addition, indicated that the expression of granzyme B was higher in the local low-dose group. Conclusion: Local low-dose administration of anti-PD-1 antibody showed the same antitumor effect and OS as systemic normal-dose administration. Therefore, local low-dose administration in oral cancer can be useful.

Key Words:

Surgical therapy, radiotherapy and pharmacotherapy are the three pillars of the treatment strategy for head and neck cancer, which can also be combined in multimodal treatments in advanced cancer treatment (1, 2). In the past years, retrograde superselective intraarterial chemoradiotherapy has been employed to avoid surgery and preserve functions in the treatment of advanced head and neck cancers. In particular, it has proven to be highly effective as small volumes of concentrated anticancer agents can be directly administered to tumor-feeding arteries via the superficial temporal artery (STA) (1-3). However, osteoradionecrosis and severe mucositis often occur because of the combination of radiotherapy and chemotherapy (4, 5). In 2017 in Japan, nivolumab, an immune checkpoint inhibitor (CPI), was approved for recurrent/metastatic head and neck cancer treatment, thus expanding the range of treatment options, and became the fourth pillar (5, 6).

CPIs exert anticancer effects mainly via the activation of T cells, which recognize tumor cells. However, immune-related adverse events (irAEs) have been identified as major issues (7). irAEs, which are similar to autoimmune diseases, reflect excessive immune reactions to normal cells as a result of immune activation by CPIs. Severe irAEs include adrenal insufficiency, fulminant type 1 diabetes mellitus, myasthenia gravis and immune-related myocarditis. In some cases, irAEs have occurred up to 2 years following CPI administration (7). Here, we conducted a basic study to determine whether local administration of low-dose CPI can reduce adverse drug reactions while inducing strong immune responses to maximize therapeutic efficacy. Tumor-bearing mice were prepared by transplanting cells from a mouse oral squamous carcinoma cell line to the back after the cells were confirmed to express programmed cell death-ligand 1 (PD-L1). The mice then received either a low-dose local administration or a normal-dose systemic administration of an anti-programmed cell death 1 (anti-PD-1) antibody. In these two groups of mice, we evaluated the antitumor efficacy, OS rate, body-weight changes, and immune microenvironment in resected tumors via immunohistology and real-time PCR.

Materials and Methods

Cell culture and mouse handling. The C3H murine buccal mucosal oral squamous cell line (Sq-1979) was purchased from the RIKEN BioResource Research Center (Ibaraki, Japan). The cell line was subcultured in Dulbecco’s Modified Eagle’s Medium (Nihon Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal bovine serum (Life Technologies, Van Allen Way, CA, USA), 0.1% MEM non-essential amino acid solution (Life Technologies), 1% penicillin–streptomycin (Life Technologies), and 0.1% fungizone (Life Technologies) using plastic petri dish (35 mm in the major axis length) and placed in an incubator at 37°C, 95% humidity, and 5% CO2. The anticancer agent used was InVivoPlus anti-mouse PD-1 (CD279) (Bio X Cell, Tokyo). Five-week-old female C3H/HeN (same strain as the cell line) mice (CLEA Japan, Tokyo, Japan) were reared under the following conditions: room temperature of 20°C-26°C, humidity of 40%-60%, HEPA air filter, ventilation frequency of 10-15 times/h, wind velocity of 15-20 cm/s, atmospheric pressure of 5-8 mmH2O positive pressure compared with the outside pressure, illumination intensity of 200 lux/FL + 800 mm and light–dark cycle of 12 h.

Cell line characterization. The PD-L1 expression in a mouse oral cancer cell line (Sq-1979) was confirmed via flow cytometry. Sq-1979 cells (1×106) were stained with phycoerythrin (PE) conjugated rat anti-mouse PD-L1 monoclonal antibody (clone; MIH5, BD Pharmingen, San Jose, CA, USA) or PE conjugated rat isotype IgG2a (clone; R35-95, BD Pharmingen) for 20 min at 4oC. The cells were then washed once with wash buffer (PBS containing 0.2% human albumin and 2-mmol/L EDTA) and then analyzed on a BD Fortessa (BD Biosciences, San Jose, CA, USA) with the aid of the FlowJo software (Tree Star, Inc., Ashland, OR, USA).

Comparison of the antitumor efficacy between local low-dose administration and systemic normal-dose administration of an anti-PD-1 antibody. Because anti-PD-1 antibodies are cross-reactive with mouse PD-1, the experiments were conducted in a syngeneic murine tumor model, in which mice with normal immunity were used as hosts. Sq-1979 cells (1×106) diluted in 0.5-mL Hank’s solution were subcutaneously transplanted using a 23G Terumo Syringe® into the backs of 5-week-old female C3H/HeN mice, as previously described (8). When the mean tumor weight (TW) (1/2 × major axis × minor axis2) reached approximately 100-150 mm3, each group was divided into six mice. Based on a report by Ajiona et al. (9) and Cmax in humans receiving nivolumab, the following anti-PD-1 antibody dosing regimens were used: 100 μg/dose was administered intraperitoneally on days 1, 5, and 8 in the intraperitoneal systemic normal-dose administration group, and 10 μg/dose was directly administered topically to the tumor in the back using a 23G Terumo Syringe® on days 1, 5, and 8 in the local low-dose administration group (Figure 1). The groups were as follows: the intraperitoneal systemic normal-dose administration group (total of 300 μg/ml) and the local low-dose administration group (total of 30 μg/ml), which represent the treatment group (Treat: T), and the local administration group with PBS, which is the control group (control: C). The tumor volume (TV) was measured using a caliper every 3 days. The antitumor efficacy of the anticancer agent was evaluated according to the method described by Geran et al. (10). The relative TW of the C group and each T group was measured on day 21, and the T/C value was calculated as an index of the antitumor efficacy. T/C values of ≤50% indicated that the treatment was effective and were used to compare the antitumor efficacy between the local low-dose and intraperitoneal systemic normal-dose administration groups. The mice were sacrificed on day 29 with an intraperitoneal injection of somnopentyl (200 mg/kg), and the growth rates of transplanted tumors were compared before the tumors were resected. The body-weight changes over time were also measured to check for adverse drug reactions in the local low-dose administration and intraperitoneal systemic administration groups. The animal experiments in this study were conducted with approval from the animal experiment ethics board of the Nippon Dental University School of Life Dentistry at Niigata (approval no. 207).

Figure 1.
Figure 1.

The oral cancer cell line transplanted in mice. We divided the experiments into local low-dose administration, intra-abdominal systemic administration and control groups. Experiments were performed on the excised tumor tissue.

Comparison of OS between local low-dose administration and systemic normal-dose administration of an anti-PD-1 antibody. Sq-1979 cells (1×106) diluted in 0.5-mL Hank’s solution were subcutaneously transplanted using a 23G Terumo Syringe® into the backs of 5-week-old female C3H/HeNJcl mice in a similar manner to the comparison of the antitumor efficacy. When the mean TW (1/2 × major axis × minor axis2) reached approximately 100-150 mm3, the mice were divided into groups (three/group). The groups were as follows: the intraperitoneal systemic normal-dose administration group (total of 300 μg/ml), local low-dose administration group (total of 30 μg/ml), and local administration control group (PBS). The anti-PD-1 antibody was administered as follows: intraperitoneally at 100 μg/dose on days 1, 5, and 8 in the intraperitoneal systemic normal-dose administration group and directly topically at 10 μg/dose to the tumor in the back using a 23G Terumo Syringe® on days 1, 5, and 8 in the local low-dose administration group. The endpoints were as follows: TW ≥10% of body weight, TV >2,000 mm3, tumor diameter ≥20 mm, tumor ulceration/necrosis, impaired walking, and impaired drinking/eating. The mice were sacrificed when any of the endpoints were met.

Histopathological examination. The resected tumors in each group were fixed in 10% formalin-neutral buffer solution (Wako Pure Chemical Industries, Osaka, Japan) at 4°C for 7 days. Following a stepwise dehydration in ethanol, the tissue was soaked in xylene and embedded in paraffin. The water in the tissue was replaced with ethanol by gradually transferring from the low concentration ethanol tank to the 100% ethanol tank. It was gradually replaced with 70, 95, and 100% ethanol, and soaked in each solution for 2 to 3 min. After dehydration, xylene was replaced and soaked 3 times for 10 min each to replace the ethanol in the tissue with xylene and embedded in paraffin. For hematoxylin and eosin staining, 5-μm sections were then prepared.

Immunohistochemical staining. Samples from each group were immunohistochemically stained for PD-L1 (NBP-1-76769, NOVUS), CD8a (361 003, Synaptic Systems), granzyme B (14-8822-80, Thermo Fisher Scientific), and perforin (ab16074, Abcam) expression. The stained samples were observed under an optical microscope (BZ-9000; KEYENCE Co., LTD., Osaka, Japan).

Real-time PCR of perforin and granzyme B gene expression. Resected tumor samples (local low-dose administration, systemic normal-dose and control) were homogenized using BioMasher® III. ISOGEN II (Nippon Gene, Tokyo) was used for total RNA extraction according to the manufacturer’s protocol. The high-capacity cDNA reverse transcription kit (Life Technologies) was used to synthesize cDNA from 1 μg of the total extracted RNA. After a Real-Time cycle (25°C for 10 min, 37°C for 60 min, 85°C for 5 min and 4°C indefinitely), the Platinum PCR SuperMix (Life Technologies) and perforin and granzyme B primers were used for 35 cycles of PCR amplification (denaturation at 94°C for 30 s; annealing at 55°C for 30 s and extension at 72°C for 60 s) (Table I). As a standard, glyceraldehyde-3phosphate dehydrogenase (GAPDH) was PCR-amplified under the same conditions as mentioned above. The amplified PCR products were separated via electrophoresis on 2% agarose gels (Nippon Gene, Tokyo, Japan) and visualized with ethidium bromide; the gels were documented with a UV gel imager.

View this table:
Table I.

Primer sequence and PCR conditions.

Quantitative comparison of perforin and granzyme B expression levels via real-time PCR. Similarly to the RT-PCR method, ISOGEN II (Nippon Gene) was used for total RNA extraction from resected tumor samples according to the manufacturer’s protocol. The perforin and granzyme B expression levels were determined via real-time PCR using PrimeScript RT™ reagent kit with gDNA Eraser (Perfect Real-Time) according to the manufacturer’s protocol. The real-time PCR results were normalized based on the GAPDH expression levels and are presented as fold-change values in comparison with the control group results. Each tissue was measured six times.

Statistical analysis. The RT-PCR results were normalized based on GAPDH expression, and the percentage of change compared with the results of the control group was determined. Each OSCC cell line was measured six times. We used ΔΔCT for analysis and one-way analysis of variance (ANOVA) to compare Perforin and granzyme B levels. The statistical analysis software used was Bell Curve (Social Survey Research Information Co., Ltd., Tokyo, Japan). The antitumor effect was determined by the Mann-Whitney U-test (Excel bell curve). OS was determined by the log-rank test that was employed for the statistical analysis of Kaplan–Meier survival curves. Differences with p<0.05 were considered statistically significant. The asterisks indicate confidence intervals (*p<0.05, **p<0.01, ***p<0.001). The Bell Curve for Excel was used for statistical analysis.

Results

Comparison of antitumor efficacy, tumor diameter and body weight after administration of local low-dose and intraperitoneal normal-dose of anti-PD-1 antibody. Flow cytometry revealed that PD-L1 was expressed in Sq-1979 cells (Figure 2).

Figure 2.
Figure 2.

Flow cytometry analysis. From the flow cytometry results, it can be seen that PD-L1 is highly expressed in the Sq-1979 cell line before application of the drug.

The T/C% of the intraperitoneal normal-dose group was 44.1% compared with the control group, indicating the antitumor efficacy of the systemically administered anti-PD-1 antibody (Figure 3A). On the other hand, the T/C% of the local low-dose group was 38.3% compared with the control group, indicating a slightly higher antitumor efficacy of the locally administered anti-PD-1 antibody than the systemically administered agent (Figure 3B). The anti-PD-1 antibody exhibited antitumor effectiveness in the local low-dose and intraperitoneal normal-dose groups in comparison with the control group, with significant differences (p<0.05) (Figure 4A). However, no significant differences in the tumor diameter were observed between the two groups (Figure 4A). During the measurement, body-weight changes of ≥20% were not observed in any of the groups (Figure 4B). The OS in the local low-dose and intraperitoneal normal-dose administration groups were significantly longer than in the control group (p<0.001) (Figure 5A). However, the difference in the OS duration between the two groups was not significant (Figure 5B).

Figure 3.
Figure 3.

Antitumor effects of local low-dose vs. intraperitoneal normal-dose anti-PD-1 antibody administration (T/C%). (A) The T/C% of the intraperitoneal normal-dose group is 44.1%, indicating an antitumor effect. (B) The T/C% of the local low-dose group is 38.3%, indicating an antitumor effect.

Figure 4.
Figure 4.

Changes in tumor volume and body weight. The antitumor effect significantly differed between the local low-dose administration group vs. the intraperitoneal normal-dose administration group and the control group (*p<0.05). No significant differences in the tumor diameter were observed between the local low-dose administration and intraperitoneal normal-dose administration groups (A). No clear changes in body weight were observed throughout the measurement period in any of the groups (B).

Figure 5.
Figure 5.

Comparison of overall survival between local low-dose and intraperitoneal systemic administration. The OS durations in the local low-dose and intraperitoneal normal-dose administration groups were significantly longer than in the control group (***p<0.001) (A). However, the difference in the OS duration between the local low-dose and intraperitoneal normal-dose groups was not significant (B).

Histopathological and immunohistochemical findings. All of the samples from the local low-dose, intraperitoneal normal-dose, and control groups contained a high number of tumor-infiltrating lymphocytes (Figure 6). A <10% PD-L1 expression was observed in tumor samples from all groups, with no clear differences among the groups (Figure 7A). Infiltrating CD8+ T cells were found within the tumor and stroma. The local low-dose and intraperitoneal normal-dose groups had higher infiltrating CD8+ T cell counts than the control group. The local low-dose group had a comparable infiltrating CD8+ T cell count with the intraperitoneal normal-dose group (Figure 7B). The perforin expression levels in the tumor tissue were low, with no clear differences among the local low-dose, intraperitoneal normal-dose, and control groups (Figure 7C). The granzyme B expression levels in the tumor and stroma were higher in the local low-dose and intraperitoneal normal-dose groups than in the control group. The expression level in the local low-dose group was comparable to that in the intraperitoneal normal-dose group (Figure 7D).

Figure 6.
Figure 6.

Hematoxylin- and eosin-staining in excised tumor tissue. A large number of tumor-infiltrating lymphocytes were observed in the local low-dose (30 μg/body), intraperitoneal (300 μg/body), and control groups. Scale bars represent 100 μm.

Figure 7.
Figure 7.

Immunochemical staining in excised tumor tissue. <10% PD-L1 expression in tumors was observed in all groups, with no differences among the local low-dose, intraperitoneal, and control groups (A). Infiltrating CD8+ T cells were found in the tumor and stroma, and the local low-dose and intraperitoneal groups had more infiltrating CD8+ T cells than the control group (B). Perforin was expressed at very low levels in tumor tissues, with no differences among the local low-dose, intraperitoneal, and control groups (C). The granzyme B expression levels in the tumor and stroma were higher in the local low-dose and intraperitoneal administration groups than in the control group (D). Local low dose: 30 μg/body. Intraperitoneal administration: 300 μg/body. Scale bars represent 100 μm.

Perforin and granzyme B gene expression analysis via real-time PCR. Granzyme B and perforin were expressed in all three groups (Figure 8A). The perforin expression levels were lower in the local low-dose and intraperitoneal normal-dose administration groups than in the control group (p<0.05), whereas the granzyme B expression levels were significantly higher in the local low-dose and intraperitoneal normal-dose administration groups than in the control group (p<0.01). The granzyme B expression level was higher in the local low-dose group than in the intraperitoneal normal-dose group (Figure 8B).

Figure 8.
Figure 8.

RT-PCR results (A) and real-time PCR results (B) in excised tumor tissue. Granzyme B and perforin are expressed in the control, PD-1 local low-dose, and PD-1 intraperitoneal normal-dose group. a: GAPDH, b: Perforin, c: Granzyme B. A slight decrease in perforin was observed in the local low-dose group and the intraperitoneal normal-dose group compared with the control group. A high expression of granzyme B was observed with a clear significant difference. In addition, local low-dose administration had higher expression than systemic administration. Significant differences at *p<0.05 and **p<0.01, (n=6).

Discussion

The anti-PD-1 antibody, nivolumab, has been approved as a therapeutic agent for recurrent/metastatic head and neck cancer (6). PD-1 is a receptor expressed on the surface of T cells, and its ligand, PD-L1, is expressed on the surface of cancer cells (11). Normally, cytotoxic T lymphocytes (CTLs), which are activated T cells, invade tumors, and eliminate cancer cells (11). However, when PD-L1 expressed on cancer cells interacts with PD-1 expressed on CTLs, the cytotoxic activity of CTLs is reduced, and they are unable to eliminate cancer cells. Among them, anti-PD-1 antibodies target PD-1. However, the response rates to anti-PD-1 antibodies are approximately 30%, and anti-PD-1 antibodies are not always effective in all patients (11). Moreover, because anti-PD-1 antibodies are systemically administered intravenously, they are often associated with severe irAEs (12). In this study, we tested a local low dose of anti-PD-1 antibody treatment in mice bearing oral squamous cell carcinomas to determine how effective direct local administration is compared with systemic administration and demonstrated that local low dose of anti-PD-1 antibody can lead to a reduction of irAE and medical expenses.

Several reports have described local treatment strategies with CPIs to induce antitumor effects against pancreatic adenocarcinoma and bladder cancer mainly using cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) antibodies (13-16). Furthermore, an anti-PD-1 antibody-based preparation with a high affinity for extracellular matrix has been studied in mouse models with malignant melanoma and breast cancer (17). In addition, Kumagai et al. compared systemic administration and intraperitoneal topical administration of anti-PD-1 antibody for peritoneal metastasis from gastric cancer, and found antitumor effect, however there was no significant difference in antitumor effect between the two groups (18).

There have been no reports of local low-dose CPI administration in oral cancer, and no previous studies have compared survival between mice receiving systemic administration and local low-dose administration. In the present study, both the local low-dose and intraperitoneal systemic normal-dose anti-PD-1 antibody regimens were used and compared in mice bearing oral squamous cell carcinomas. The results indicated antitumor efficacy against the oral cancer cell line (T/C%=38.3% and 44.1%, respectively), and PD-1 inhibition could also be evaluated in the system, suggesting that the tumor model used here is susceptible to PD-1 inhibition. In the comparison of the antitumor efficacy based on the TV, a significant antitumor effect was observed in the local low-dose group compared with the control group (p<0.05), and the observed antitumor effect in the local low-dose group was comparable to that in the intraperitoneal normal-dose systemic administration group. Furthermore, the OS duration was extended equally in mice receiving local low-dose treatment and mice receiving intraperitoneal systemic administration, despite the fact that the dose in the former was 1/10 that of the latter, with significant differences in OS in the control group (p<0.001). This result indicates that 1/10 of a dose of anti-PD-1 antibody administered locally is as effective as intraperitoneal systemic administration. The properties of anti-PD-1 antibodies may be a major factor underlying this finding. Kurino et al. evaluated the antitumor effects of an anti-PD-1 antibody and an anti-PD-L1 antibody in mice bearing colon cancer MC38 cells or mouse breast cancer MM48 cells as tumor grafts. The result indicated that the anti-PD-1 antibody was effective, whereas the anti-PD-L1 antibody was not, suggesting that the observed effectiveness of an anti-PD-1 antibody does not predict the effectiveness of all other CPIs (19). Moreover, anti-PD-1 and anti-PD-L1 antibodies also differ in terms of dosage and drug metabolism. While anti-PD-1 antibodies remain in the circulating blood ~20 times longer than the PD-L1 antibody, anti-PD-L1 antibodies rapidly migrate to various organs, such as the liver and spleen, and are rapidly eliminated from the blood (19). Furthermore, anti-PD-1 antibody migration to tumors was found to be approximately 10 times that of anti-PD-L1 antibody migration (19). Hooren et al. demonstrated that in mice with bladder tumors, the systemic antibody level after a local injection of a CPI was at least 1/10 that after the systemic treatment (16). This result indicates the efficacy of using intratumor injection instead of systemic administration to improve the benefit-to-risk ratio through lower circulating antibody levels without adversely interfering with the antitumor response. Moreover, it demonstrates that the local low-dose administration is a potential way to increase tumor and para-tumor tissue migration amounts while decreasing systemic and peripheral blood levels of an anti-PD-1 antibody, which, when systemically administered, remains for a long time in the blood posing a higher risk of affecting various organs.

In other studies, histological examinations of head and neck cancer samples after treatment with an anti-PD-1 antibody have suggested differences in the efficacy between PD-L1-positive (≥1%) and PD-L1-negative (<1%) cases. However, anti-PD-1 antibody treatment was also successful in PD-L1-negative cases, albeit with a lower response rate than in PD-L1-positive cases, and long-term follow-up data have demonstrated that nivolumab efficacy can be expected in PD-L1-negative cases (6, 20).

The results in the present study were obtained using a cell line that expresses PD-L1 at a high level. However, a sufficient effect can be expected as anti-PD-1 antibodies are likely to exert an antitumor effect even in PD-L1-negative cases of head and neck cancer. Immunohistology examinations of resected tumor tissue samples revealed that infiltrating CD8-positive T cell counts were higher in the local low-dose and intraperitoneal systemic administration groups than in the control group. Real-time PCR results also indicated that granzyme B expression levels were significantly higher in the local low-dose and intraperitoneal normal-dose groups than in the control group as well as in the local low-dose group than in the systemic administration group. Granzyme B is highly expressed in activated CTLs and released by cytotoxic T cells, and recent studies have demonstrated that the granzyme B pathway plays a major role in cytotoxic T cells killing tumor cells in humans (21, 22). The granzyme B expression/non-expression status is effective in predicting the therapeutic effectiveness of CPIs in non-small cell lung cancer (22), although there are no reports on therapeutic outcome prediction in oral cancer. A recent study suggested that granzyme B changes could be used as a biomarker to predict the effectiveness of CPI monotherapy (22). In this study, the granzyme B expression in tumor tissue was significantly elevated after local low-dose administration, which is consistent with the antitumor effect. This result indicates that a low-dose anti-PD-1 antibody locally administered permeates the tumor, attracting CTLs and promoting granzyme-mediated apoptosis.

Overall, the findings of this study demonstrate that local administration of anti-PD-1 antibodies is useful for the treatment of oral cancer. However, the administration method becomes an issue when this strategy is used in humans. Superselective intraarterial administration has been reported to be extremely useful in the treatment of oral cancer as it allows for the direct and selective local administration of a high-concentration drug to tumor-feeding arteries via a catheter inserted retrogradely from the STA (1, 2, 22). Daily preoperative chemotherapy with intraarterial cisplatin (CDDP) administration combined with radiotherapy was highly effective for fighting advanced oral cancer, including cervical lymph node metastasis (23). However, because radiotherapy is employed in combination with chemotherapy, osteoradionecrosis is a major posttreatment complication occurring in 4.2%-17.9% of treated patients, including those whose tumors were completely cured (4, 24). The superselective local low-dose CPI administration studied here demonstrates an antitumor effect against oral cancer without radiotherapy, thus representing a promising way to avoid osteoradionecrosis.

The number of studies conducted to reduce adverse reactions to CPIs is far less than the number of studies conducted to increase the anticancer efficacy of CPIs.

The significance of this study is the demonstration of reduced toxicity and increased anticancer efficacy of a low-dose CPI directly administered to tumor-feeding arteries. However, this strategy is restricted to cancer in regions where local arterial administration is possible. Therefore, this administration method may be indicated for a limited number of diseases. In-depth studies of drug distribution and degradation in humans also have limitations. Organ distribution and susceptibility to degradation in tumor tissues affect drug efficacy and are important predictors of drug efficacy. In the future, we intend to accumulate knowledge from studies in model animals, such as tumor-bearing mouse models, with a focus on differences from humans, thus contributing to the establishment of better cancer treatments.

Acknowledgements

This work was supported by JSPS KAKENHI Grant Number JP19K19214.

Footnotes

  • Authors’ Contributions

    HT, SS and KT made substantial contributions to the conception and design of the study as well as in the acquisition, analysis, and interpretation of the data. YK, OT, and AT were involved in the drafting and critical revision of the manuscript for important intellectual content.

  • Conflicts of Interest

    No competing financial interests exist. The Authors declare that there are no conflicts of interest related to the publication of this study.

  • Received June 27, 2022.
  • Revision received July 20, 2022.
  • Accepted July 21, 2022.

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An In Vivo Study of Local Administration of Low-dose Anti-PD-1 Antibody Using an Oral Cancer Cell Line
KANAME SAKUMA, TOMOYUKI KII, HARUKA TAKAHASHI, SUSUMU SUZUKI, KAZUHIRO YOSHIKAWA, TETSUYA OGAWA, AKIRA TANAKA
Anticancer Research Sep 2022, 42 (9) 4293-4303; DOI: 10.21873/anticanres.15929
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