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Most patients with head and neck cancers struggle with their treatment, particularly those with recurrent cancer. However, there is no consensus on effective treatments for recurrent head and neck cancer. Recurrent cases are often challenging to treat because performing both reirradiation and surgical intervention can occasionally be difficult.This report describes the effective cisplatin intra-arterial chemotherapy with oral S-1 for a recurrent case of maxillary gingival cancer (rT4bN1M0, rStage ⅣB). The patient who had undergone chemoradiotherapy 13 years ago and achieved complete response (CR) was referred to us due to recurrence. His recurrent lesions were located on the left maxillary bone, and a metastatic cervical lymph node was detected. We approached the feeder of the locoregional recurrence site using Seldinger's technique and repeated the cisplatin intra-arterial chemotherapy 5 times. As a result, we achieved a complete response, including the regional metastatic lymph node, without radiation or surgery. Notably, although we infused the anticancer drug into the feeder of the locoregional metastatic area, we noticed its effect on the metastatic cervical lymph node. Initially, neck dissection following intra-arterial chemotherapy had been planned; however, owing to the high effectiveness of the treatment, the subsequent surgery was deemed unnecessary. No evidence of recurrence has been observed during the 2.5-year follow-up period. In the future, intra-arterial chemotherapy can be an alternative option for patients with recurrent head and neck cancers, and our result seems to be enough to indicate that possibility.

Background Chondrosarcoma of the maxilla is extremely rare, requiring complete surgical resection with clear margins. Various surgical approaches have been used for wide surgical resection of chondrosarcomas in the head and neck region. 3D printed bone models are useful surgical and educational tools.Case A 69-year-old man presented with a grade II chondrosarcoma of the maxilla. This tumor was resected by otolaryngologists and dental surgeons using an anterior segmental maxillary osteotomy and open rhinoplasty approach with the assistance of a 3D-printed bone model.Conclusion Chondrosarcomas can be differentiated from chondromas by their higher mean apparent diffusion coefficient (ADC) on diffusion-weighted magnetic resonance imaging. The anterior segmental maxillary osteotomy and rhinoplasty approach, with ideal osteotomy and a good visual field, resulted in wide surgical safety margins, improving patient prognosis.

Abstract The tumor microenvironment plays a pivotal role in cancer development. We recently reported that in oral squamous cell carcinoma (OSCC), adipocyte enhancer-binding protein 1 (AEBP1) is abundantly expressed in cancer-associated fibroblasts (CAFs), leading to CAF activation and inhibition of CD8 + T cell infiltration. In the present study, we investigated whether AEBP1 contributes to the destruction and atrophy of muscle tissues in OSCC. By analyzing human skeletal muscle myoblasts (HSMMs), we found that AEBP1 is downregulated during muscle cell differentiation. Transcriptome analysis revealed that AEBP1 knockdown significantly upregulates myogenesis-related genes in HSMMs, and qRT-PCR and western blot analyses confirmed the induction of muscle-related genes, including MYOG, in HSMMs after AEBP1 knockdown. Conversely, ectopic expression of AEBP1 strongly suppressed myogenesis-related genes in HSMMs. Notably, indirect co-culture of HSMMs with OSCC cells led to AEBP1 upregulation and robust suppression of muscle-related genes in HSMMs. Treatment with TGF-β1 also upregulated AEBP1 and suppressed expression of muscle-related genes in HSMMs. Our findings suggest that AEBP1 is a negative regulator of skeletal muscle cell differentiation and that OSCC cells inhibit muscle cell differentiation, at least in part, by inducing AEBP1.

Abstract Oral lichen planus is a chronic inflammatory condition that adversely affects the oral mucosa; however, its etiology remains elusive. Consequently, therapeutic interventions for oral lichen planus are limited to symptomatic management. This study provides evidence of the accumulation of senescent mesenchymal cells, CD8 + T cells, and natural killer cells in patients with oral lichen planus. We profiled the patients’ tissues using the National Center for Biotechnology Information Gene Expression Omnibus database and found that senescence-related genes were upregulated in these tissues by gene set enrichment analysis. Immunohistochemical analysis showed increased senescent mesenchymal cells in the subepithelial layer of patients with oral lichen planus. Single-cell RNA-seq data retrieved from the Gene Expression Omnibus database of patients with oral lichen planus revealed that mesenchymal cells were marked by the upregulation of senescence-related genes. Cell-cell communication analysis using CellChat showed that senescent mesenchymal cells significantly influenced CD8 + T cells and natural killer cells via CXCL12-CXCR4 signaling, which is known to activate and recruit CD8 + T cells and NK cells. Finally, in vitro assays demonstrated that the secretion of senescence-associated factors from mesenchymal cells stimulated the activation of T cells and natural killer cells and promoted epithelial cell senescence and cytotoxicity. These findings suggest that the accumulation of mesenchymal cells with senescence-associated secretory phenotype may be a key driver of oral lichen planus pathogenesis.

Cell culture methods are indispensable strategies for studies in biological sciences and for drug discovery and testing. Most cell cultures have been developed using two-dimensional (2D) culture methods, but three-dimensional (3D) culture techniques enable the establishment of in vitro models that replicate various pathogenic conditions and they provide valuable insights into the pathophysiology of various diseases as well as more precise results in tests for drug efficacy. However, one difficulty in the use of 3D cultures is selection of the appropriate 3D cell culture technique for the study purpose among the various techniques ranging from the simplest single cell type-derived spheroid culture to the more sophisticated organoid cultures. In the simplest single cell type-derived spheroid cultures, there are also various scaffold-assisted methods such as hydrogel-assisted cultures, biofilm-assisted cultures, particle-assisted cultures, and magnet particle-assisted cultures, as well as non-assisted methods, such as static suspension cultures, floating cultures, and hanging drop cultures. Since each method can be differently influenced by various factors such as gravity force, buoyant force, centrifugal force, and magnetic force, in addition to non-physiological scaffolds, each method has its own advantages and disadvantages, and the methods have different suitable applications. We have been focusing on the use of a hanging drop culture method for modeling various non-cancerous and cancerous diseases because this technique is affected only by gravity force and buoyant force and is thus the simplest method among the various single cell type-derived spheroid culture methods. We have found that the biological natures of spheroids generated even by the simplest method of hanging drop cultures are completely different from those of 2D cultured cells. In this review, we focus on the biological aspects of single cell type-derived spheroid culture and its applications in in vitro models for various diseases.

Abstract Background Aberrant Notch signaling pathway has been related with the tumorigenesis in head and neck region, involving oral cavity. Here, we report the correlation between mutations in the Notch signaling pathway and CD8+ T‐cell infiltration via PD‐L1, which lead to enhanced antitumor immunity and may target for immune‐checkpoint inhibitors (ICIs) therapy. Methods This retrospective study analyzed the results of immunohistochemical staining for PD‐L1 and CD8+ T‐cell infiltration in 10 patients and whole‐exome sequencing (WES) was conducted on five of these patients to identify frequently mutated genes. Results Four of 10 patients were positive for PD‐L1 and CD8+ T. By analyzing WES in three of these four patients, we notably identified the mutations of NOTCH1, FBXW7, and noncoding RNA intronic mutation in NOTCH2NLR in two of these three patients. This study may enable better selection of ICI therapy with CD8+ T‐cell infiltration via PD‐L1 expression for oral squamous cell carcinoma patients with mutations in Notch signaling pathway.

Abstract Background The CXCL12/CXCR4 axis plays a pivotal role in the progression of various malignancies, including oral squamous cell carcinoma (OSCC). In this study, we aimed to clarify the biological and clinical significance of CXCL12 in the tumor microenvironment of OSCCs. Methods Publicly available single‐cell RNA‐sequencing (RNA‐seq) datasets were used to analyze CXCL12 expression in head and neck squamous cell carcinomas (HNSCC). Immunohistochemical analysis of CXCL12, α‐smooth muscle antigen (α‐SMA), fibroblast activation protein (FAP) and CD8 was performed in a series of 47 surgically resected primary tongue OSCCs. Human skeletal muscle cells were co‐cultured with or without OSCC cells, after which CXCL12 expression was analyzed using quantitative reverse‐transcription PCR. Results Analysis of the RNA‐seq data suggested CXCL12 is abundantly expressed in stromal cells within HNSCC tissue. Immunohistochemical analysis showed that in grade 1 primary OSCCs, CXCL12 is expressed in both tumor cells and muscle cells. By contrast, grade 3 tumors were characterized by disruption of muscle structure and reduced CXCL12 expression. Quantitative analysis of CXCL12‐positive areas within tumors revealed that reduced CXCL12 expression correlated with poorer overall survival. Levels of CXCL12 expression tended to inversely correlate α‐SMA expression and positively correlate with infiltration by CD8+ lymphocytes, though these relations did not reach statistical significance. CXCL12 was significantly upregulated in muscle cells co‐cultured with OSCC cells. Conclusion Our results suggest that tongue OSCC cells activate CXCL12 expression in muscle cells, which may contribute to tumor progression. However, CXCL12 is reduced in advanced OSCCs due to muscle tissue destruction.

Abstract Immune checkpoint inhibitor‐based cancer immunotherapy has provided an additional therapeutic option for oral squamous cell carcinoma (OSCC) with recurrence or distant metastases. However, further improvement of OSCC treatment is required to develop the optimal combination or order for chemoradiotherapy and immunotherapy. Along with the accumulation of clinical knowledge and evidence, it is also essential to clarify the biological impact of chemo‐radiotherapeutic agents on the cancer immune microenvironment. In this study, we investigated the effects of cisplatin (CDDP), a key therapeutic agent for OSCC, on programmed death‐ligand 1 (PD‐L1) expression in OSCC lines. Although CDDP treatment increased the surface levels of PD‐L1 on OSCC cell lines, the gene and total protein expression levels of PD‐L1 were not altered. We also demonstrated that the phosphorylation of heat shock factor 1 and heat shock protein 90 was involved in this process. In addition, CDDP‐induced PD‐L1 attenuated the target‐specific cytotoxic T lymphocyte reaction to OSCC. These results provide an immunobiological basis for the response of OSCC to CDDP and will contribute to our biological understanding of the action of novel combination therapy including immunotherapy together with platinum‐based chemotherapy for OSCC.

Key Clinical Message We report a case of diverticulum of the buccal mucosa. A 56‐year‐old man had a small pouch‐shaped lesion behind the parotid papilla that caused pain and food impaction. After resection, the lesion was histopathologically diagnosed as diverticulum without buccal muscle tear. There has been no recurrence during 1 year postoperatively.

The current study’s objective was to elucidate some currently unknown biological indicators to evaluate the biological nature of cancer-associated fibroblasts (CAFs). For this purpose, four different CAFs, CAFS1, CAFS2, SCC17F and MO-1000, were established using surgical specimens from oral squamous cell carcinomas (OSCC) with different clinical malignant stages (CAFS1 and CAFS2, T2N0M0, stage II; SCC17F and MO-1000, T4aN2bM0, stage IVA). Fibroblasts unrelated to cancer (non-CAFs) were also prepared and used as controls. Initially, confirmation that these four fibroblasts were indeed CAFs was obtained by their mRNA expression using positive and negative markers for the CAF or fibroblasts. To elucidate possible unknown biological indicators, these fibroblasts were subjected to a cellular metabolic analysis by a Seahorse bioanalyzer, in conjugation with 3D spheroid cultures of the cells and co-cultures with a pancreas ductal carcinoma cell line, MIA PaCa-2. The mitochondrial and glycolytic functions of human orbital fibroblasts (HOF) were nearly identical to those of Graves’-disease-related HOF (GOF). In contrast, the characteristics of the metabolic functions of these four CAFs were different from those of human conjunctival fibroblasts (HconF), a representative non-CAF. It is particularly noteworthy that CAFS1 and CAFS2 showed markedly reduced ratios for the rate of oxygen consumption to the extracellular acidification rate, suggesting that glycolysis was enhanced compared to mitochondrial respiration. Similarly, the physical aspects, their appearance and stiffness, of their 3D spheroids and fibroblasts that were induced effects based on the cellular metabolic functions of MIA PaCa-2 were also different between CAFs and non-CAFs, and their levels for CAFS1 or SCC17F were similar to those for CAFS2 or MO-1000 cells, respectively. The findings reported herein indicate that cellular metabolic functions and the physical characteristics of these types of 3D spheroids may be valuable and useful indicators for estimating potential biological diversity among various CAFs.

Abstract Long noncoding RNAs (lncRNAs) are deeply involved in cancer development. We previously reported that DLEU1 (deleted in lymphocytic leukemia 1) is one of the lncRNAs overexpressed in oral squamous cell carcinoma (OSCC) cells, where it exhibits oncogenic activity. In the present study, we further clarified the molecular function of DLEU1 in the pathogenesis of OSCC. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis revealed that DLEU1 knockdown induced significant changes in the levels of histone H3 lysine 4 trimethylation (H3K4me3) and H3K27 acetylation (H3K27ac) in OSCC cells. Notably, DLEU1 knockdown suppressed levels of H3K4me3/ H3K27ac and expression of a number of interferon-stimulated genes (ISGs), including IFIT1, IFI6 and OAS1, while ectopic DLEU1 expression activated these genes. Western blot analysis and reporter assays suggested that DLEU1 upregulates ISGs through activation of JAK-STAT signaling in OSCC cells. Moreover, IFITM1, one of the ISGs induced by DLUE1, was frequently overexpressed in primary OSCC tumors, and its knockdown inhibited OSCC cell proliferation, migration and invasion. These findings suggest that DLEU1 exerts its oncogenic effects, at least in part, through activation of a series ISGs in OSCC cells.

Mixed reality refers to all technologies that combine real and virtual spaces to create a new space in which real and virtual objects interact in real time. Recently, the development of technology for superimposing three-dimensional computer graphics on real space has been dramatic. This technology has also been applied to oral surgery. This report describes five diverse cases of oral surgery that were treated using the latest mixed reality device (Microsoft®HoloLens2). Case 1 underwent temporomandibular joint mobilization for ankylosis. Case 2 underwent alveolar bone grafting for cheilognathopalatoschisis. Case 3 underwent genioplasty for jaw deformity. Case 4 underwent resection of a mandibular calcifying epithelial odontogenic tumor. Case 5 underwent open reduction and internal fixation for a mandibular fracture. In each case, three-dimensional images were created from the digital imaging and communications in medicine data of computed tomography images using the three-dimensional Slicer software, the images were edited using the Blender software, an application for HoloLens2 created using the Unity software, and HoloLens2 was worn by the surgical assistant to display the three-dimensional image in the surgical field through manual operations. The visibility of the three-dimensional images was good, and preoperative image information could be observed in real time.

Introduction In early-stage oral tongue squamous cell carcinoma (OTSCC), elective neck dissection (END) is recommended when occult lymph node metastasis is suspected; however, there is no unanimous consensus on the risks and benefits of END in such cases. The management of clinically node-negative (cN0) OTSCC remains controversial. This study, therefore, aimed to evaluate the efficacy of END and its impact on the quality of life (QoL) of patients with cN0 OTSCC.Methods and analysis This is a prospective, multicentre, nonrandomised observational study. The choice of whether to perform END at the same time as resection of the primary tumour is based on institutional policy and patient preference. The primary endpoint of this study is 3-year overall survival. The secondary endpoints are 3-year disease-specific survival, 3-year relapse-free survival and the impact on patient QoL. Propensity score-matching analysis will be performed to reduce selection bias.Ethics and dissemination This study was approved by the Clinical Research Review Board of the Nagasaki University. The protocol of this study was registered at the University Hospital Medical Information Network Clinical Trials Registry. The datasets generated during the current study will be available from the corresponding author on reasonable request. The results will be disseminated internationally, through scientific and professional conferences and in peer-reviewed medical journals.Trial registration number UMIN000027875.

Steroid has recently been reported as a treatment for new coronavirus disease (COVID-19). The incidence of oropharyngeal candidiasis due to the inhaled steroid ciclesonide is lower than that due to other inhaled steroids. We report the first case of oral candidiasis with COVID-19 pneumonia using ciclesonide. A 75-year-old man was hospitalized for COVID-19 pneumonia. After admission, an oral combination of lopinavir/ritonavir was administered, and ciclesonide was inhaled for 7 days. On the 14th day of hospitalization, white plaque was found in his oral mucosa. Candida albicans was identified by oral bacterial tests, and amphotericin B was initiated. On the 35th hospital day, negative result for C. albicans was confirmed. Intraoral monitoring and intervention by dental care workers are considered important for the prevention of infectious complications induced by corticosteroids.

Abstract Background Cases of diverticula of the buccal mucosa are extremely rare. Literature searches of databases such as PubMed/MEDLINE for this condition have revealed only 10 case reports. In this case report, we describe our experience in the management of this rare condition and review the previous 10 previously reported cases. Case presentation A 66-year-old man presented with a pouch containing inspissated food debris located posterior to the papilla of the parotid duct in his left buccal mucosa. The diagnosis of a diverticulum arising from the buccal mucosa was confirmed based on clinical and radiographic findings. Gross examination of the locally resected tissue specimen revealed a pouch measuring 14 mm in diameter and 8 mm in depth, that was whitish in color and had an elastic, soft, and smooth surface. Microscopic examination revealed a cyst-like lesion lined by stratified squamous epithelium and granulation tissue, with a chronic inflammatory infiltration in the peripheral stromal tissue of the epithelial layer. After surgical excision of the lesion, there was no recurrence during the follow-up period of 5 years and 10 months. Conclusions We have presented a rare case of a diverticulum of the buccal mucosa. This is the first report of a case confirmed not only by the clinicopathological findings, but also by computed tomography and magnetic resonance imaging findings. From the magnetic resonance imaging and intraoperative findings, we inferred that the diverticulum was caused by an idiopathic developmental anomaly due to a partial defect of the buccinator muscle.

BACKGROUND:Tumor-infiltrating lymphocytes include tumor-reactive lymphocytes and regulatory T-cells. However, the prognostic value of tumor-infiltrating lymphocytes in oral squamous cell carcinoma (OSCC) remains unclear. METHODS:We used immunohistochemistry to evaluate the presence of tumor-infiltrating FoxP3⁺ T-cells and CTLA-4⁺ cells in four distinct histological compartments (tumor parenchyma and stroma at the tumor center, and parenchyma and stroma at the invasive front) and assessed the association between the prevalence of these cells and the histopathological status of 137 patients with OSCC. RESULTS:Five-year overall survival, disease-specific survival, and recurrence-free survival were favorable in patients with high numbers of FoxP3⁺ T-cells in the parenchyma of the invasive front. Recurrence-free survival and metastasis-free survival were decreased in patients with high numbers of CTLA-4⁺ cells in the parenchyma of the invasive front. CONCLUSIONS:The presence of FoxP3⁺ T-cells in the parenchyma of the invasive front may be a useful prognostic factor. Our results indicate that FoxP3⁺ T-cells may exert site-specific anti-tumor effects but may not play an immunosuppressive role in OSCC. In addition, our results suggest that CTLA-4+ cells suppress the function of FoxP3+ T-cells and promote anti-tumor immunity in OSCC.

Pathologically proven regional lymph node metastasis affects the prognosis in early stage oral cancer. Therefore we investigated invasive tumor patterns predicting nodal involvement and survival in patients with clinically node-negative T1 and T2 oral squamous cell carcinoma (cT1,2N0M0 OSCC). Ninety-one cases of cT1,2N0M0 OSCC treated with transoral resection of the primary tumor were assessed based on 3 types of invasive tumor patterns on histopathologic and pancytokeratin-stained immunohistological sections: the mode of invasion, worst pattern of invasion (WPOI), and tumor budding. The correlations among invasive tumor patterns, regional metastasis, and disease-free survival were analyzed. Of the 91 cases, 22 (24%) had pathologically proven regional metastasis. The mode of invasion (p