Tumor microenvironment dynamics in oral cancer: unveiling the role of inflammatory cytokines in a syngeneic mouse model.

The process of cervical lymph node metastasis is dependent on the phenotype of the tumor cells and their interaction with the host microenvironment and immune system; conventional research methods that focus exclusively on tumor cells are limited in their ability to elucidate the metastatic mechanism. In cancer tissues, a specialized environment called the tumor microenvironment (TME) is established around tumor cells, and inflammation in the TME has been reported to be closely associated with the development and progression of many types of cancer and with the response to anticancer therapy. In this study, to elucidate the mechanism of metastasis establishment, including the TME, in the cervical lymph node metastasis of oral cancer, we established a mouse-derived oral squamous cell carcinoma cervical lymph node highly metastatic cell line and generated a syngeneic orthotopic transplantation mouse model. In the established highly metastatic cells, epithelial-mesenchymal transition (EMT) induction was enhanced compared to that in parental cells. In the syngeneic mouse model, lymph node metastasis was observed more frequently in tumors of highly metastatic cells than in parental cells, and Cyclooxygenase-2 (COX-2) expression and lymphatic vessels in primary tumor tissues were increased, suggesting that this model is highly useful. Moreover, in the established highly metastatic cells, EMT induction was enhanced compared to that in the parent cell line, and CCL5 and IL-6 secreted during inflammation further enhanced EMT induction in cancer cells. This suggests the possibility of a synergistic effect between EMT induction and inflammation. This model, which allows for the use of two types of cells with different metastatic and tumor growth potentials, is very useful for oral cancer research involving the interaction between cancer cells and the TME in tumor tissues and for further searching for new therapeutic agents.

CDDP-induced desmoplasia-like changes in oral cancer tissues are related to SASP-related factors induced by the senescence of cancer cells.

The tumor microenvironment (TME) concept has been proposed and is currently being actively studied. The development of extracellular matrix (ECM) in the TME is known as desmoplasia and is observed in many solid tumors. It has also been strongly associated with poor prognosis and resistance to drug therapy. Recently, cellular senescence has gained attention as an effect of drug therapy on cancer cells. Cellular senescence is a phenomenon wherein proliferating cells become resistant to growth-promoting stimuli, secrete the SASP (senescence-associated phenotypic) factors, and stably arrest the cell cycle. These proteins are rich in pro-inflammatory factors, such as interleukin (IL)-6, IL-8, C-X-C motif chemokine ligand 1, C-C motif chemokine ligand (CCL)2, CCL5, and matrix metalloproteinase 3. This study aimed to investigate the desmoplasia-like changes in the TME before and after cancer drug therapy in oral squamous cell carcinomas, evaluate the effect of anticancer drugs on the TME, and the potential involvement of cancer cell senescence. Using a syngeneic oral cancer transplant mouse model, we confirmed that cis-diamminedichloroplatinum (II) (CDDP) administration caused desmoplasia-like changes in cancer tissues. Furthermore, CDDP treatment-induced senescence in tumor-bearing mouse tumor tissues and cultured cancer cells. These results suggest CDDP administration-induced desmoplasia-like structural changes in the TME are related to cellular senescence. Our findings suggest that the administration of anticancer drugs alters the TME of oral cancer cells. Additionally, oral cancer cells undergo senescence, which may influence the TME through the production of SASP factors.

Risk factors for impacted lower third molar root perforation through the undercut-shaped lingual plate: preoperative insights from panoramic radiography.

This study aimed to identify risk factors associated with perforation of the undercut (U)-shaped lingual plate (LP) by the lower third molar (LM3) root using panoramic radiography (PAN). We retrospectively examined 468 impacted LM3s from 468 individuals, categorizing LM3-LP associations and LP morphology in the coronal section of cone-beam computed tomography as perforation or nonperforation and U-type or non-U-type, respectively. The outcome was the combination of perforation and U-type, and study variables included patient demographics (age and sex) and PAN-associated features (Winter's classification, Pell-Gregory classification, and two major Rood signs). Multivariate logistic regression methods were used for analysis. Perforated and U-type LPs were observed in 205 (43.8%) and 212 (45.3%) cases, respectively. The double-positive outcome was observed in 126 LM3s (26.9%). In the multivariate model, age ≥ 26 years [odds ratio (OR), 2.66; p = 0.002], men (OR, 2.01; p = 0.002), mesioangular (OR, 2.74; p = 0.038) and horizontal impaction (OR, 3.05; p = 0.019), and root darkening (OR, 1.73; p = 0.039) were independently associated with the risk. Class III impaction (OR, 0.35; p = 0.021) and interruption of the white line (OR, 0.55; p = 0.017) were negatively correlated with the risk. In conclusion, this study highlights the importance of identifying the higher probability of U-type LP perforation by the LM3 root in men aged over their midtwenties with Class I/II impaction and mesioangularly or horizontally impacted LM3s, along with root darkening and no interruption of the white line on PAN.

Deep learning model for the automated evaluation of contact between the lower third molar and inferior alveolar nerve on panoramic radiography.

Background/purpose: In lower third molar (LM3) surgery, panoramic radiography (PAN) is important for the initial assessment of the anatomical association between LM3 and the inferior alveolar nerve (IAN). This study aimed to develop a deep learning model for the automated evaluation of the LM3-IAN association on PAN. Further, its performance was compared with that of oral surgeons using original and external datasets. Materials and methods: In total, 579 panoramic images of LM3 from 384 patients in the original dataset were utilized. The images were divided into 483 images for the training dataset and 96 for the testing dataset at a ratio of 83:17. The external dataset comprising 58 images from an independent institution was used for testing only. The LM3-IAN associations on PAN were categorized into direct or indirect contact based on cone-beam computed tomography (CBCT). The You Only Look Once (YOLO) version 3 algorithm, a fast object detection system, was applied. To increase the amount of training data for deep learning, PAN images were augmented using the rotation and flip techniques. Results: The final YOLO model had high accuracy (0.894 in the original dataset and 0.927 in the external dataset), recall (0.925, 0.919), precision (0.891, 0.971), and f1-score (0.908, 0.944). Meanwhile, oral surgeons had lower accuracy (0.628, 0.615), recall (0.821, 0.497), precision (0.607, 0.876), and f1-score (0.698, 0.634). Conclusion: The YOLO-driven deep learning model can help oral surgeons in the decision-making process of applying additional CBCT to confirm the LM3-IAN association based on PAN images.

External validation of cone-beam computed tomography- and panoramic radiography-featured prediction models for inferior alveolar nerve injury after lower third molar removal: proposal of a risk calculator.

We previously developed basic and extended models to predict inferior alveolar nerve injuries (IANI) after lower third molar (LM3) removal based on cone-beam computed tomography (CBCT) images. Although these models comprised predictors, including increased age and inferior alveolar canal-related CBCT factors, external validations were lacking. Therefore, this study externally validated these models and compared them with other related models based on their performance. Original and newly validated samples included patients who underwent LM3 removal following CBCT. Subsequently, 39 and 25 patients with IANI, then 457 and 295 randomly selected patients without IANI were chosen of the observed 1573 and 1052 patients, respectively. CBCT- and panoramic radiograph (PAN)-featured models were validated. Then, models' discrimination and calibration abilities were assessed using C-statistics and calibration plots, respectively. Brier scores were also quantified, after which logistic recalibration was achieved to optimize calibration, and a risk calculator was developed. During the external validation, the extended model exhibited the best C-statistic (0.822) and Brier score (0.064), whereas two CBCT- and two PAN-featured models showed lower performances with C-statistics (0.764, 0.706, 0.584, and 0.627) and Brier scores (0.069, 0.074, 0.075, and 0.072). Besides, all models showed a tendency to overpredict its high-risk range. However, recalibration of the extended model resulted in excellent calibration performance. CBCT-featured models, especially the extended model, conclusively showed a superior predictive performance to PAN models. Therefore, the risk calculator on the extended CBCT model is proposed to be a clinical decision-aid tool that preoperatively predicts IANI risk.

GATA6 regulates expression of annexin A10 (ANXA10) associated with epithelial-mesenchymal transition of oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) can disturb oral function and quality of life and is associated with poor survival, likely due to the development of cervical lymph node metastases. Epithelial-mesenchymal transition (EMT) is a process in which cells acquire molecular alterations that facilitate cell motility and invasion, and has been associated with tumor metastasis. EMT changes also play important roles in the induction of lymph node metastasis in OSCC. GATA6 is known as the earliest marker of the primitive endoderm lineages. GATA6 inhibits de-differentiation and EMT in human pancreatic ductal adenocarcinoma cells and promotes EMT. However, in OSCC, the expression and function of GATA6 in EMT and lymph node metastasis remains unclear. Therefore, this study aimed to clarify the targets of GATA6 in OSCC cells and whether the change in GATA6 expression affects EMT in OSCC cells, as well as the association between GATA6 and lymph node metastasis. The results showed that GATA6 knockdown OSCC cells promoted EMT and increased lymph node metastasis compared with control cells, whereas the overexpression of GATA6 inhibited the induction of EMT and reduced lymph node metastasis. In addition, annexin A10 (ANXA10) which is the largest type of Ca2+-regulated phospholipid-binding protein in eukaryotic cells was detected as a target gene for GATA6 and ANXA10 suppressed Vimentin expression in EMT in OSCC. Therefore, the GATA6/ANXA10 cascade may be a potential therapeutic approach for the treatment of lymph node metastases in OSCC patients.

Predictive scoring model for inferior alveolar nerve injury after lower third molar removal based on features of cone-beam computed tomography image.

INTRODUCTION: This study aimed to construct a predictive scoring system for inferior alveolar nerve injury (IANI) following lower third molar (LM3) surgery based on cone-beam computed tomography (CBCT) images. MATERIAL AND METHODS: Of the 1573 patients who underwent LM3 removal following the CBCT, 39 with IANI and 457 randomly selected patients without IANI were enrolled. We collected information regarding the demographic characteristics of the patients, surgical situations, and inferior alveolar canal (IAC)-related CBCT factors. The association with IANI-risk was evaluated with a backward stepwise logistic regression model as per the Akaike information criterion. Scoring models' abilities of discrimination (area under the curve) and calibration (Hosmer-Lemeshow test and calibration plots) were assessed, followed by evaluation of the clinical usefulness using decision curve analysis. RESULTS: As per the multivariate analysis, the coronal positioned IAC on the enlarged root (odds ratio [OR], 3.78; P = 0.001), the length of perforated IAC (>3.4 mm) (OR, 3.05; P = 0.012), lingual/inter-radicular position of the IAC (OR, 3.96; P = 0.001), multiple roots closed to the perforated IAC (OR, 2.78; P = 0.025), and age >30 y (OR, 2.31; P = 0.076) were identified in the extended scoring model ranging from 0 to 12. This model was compared with our previously constructed baseline model that involved the latter three variables mentioned above, resulting in superior performance than that of the baseline model. CONCLUSION: The extended model would be a useful tool for reliable determination of the preoperative probability of IANI.

Anesthetic Management of a Juvenile Hyaline Fibromatosis Patient With Trismus and Cervical Movement Limitation.

Juvenile hyaline fibromatosis (JHF) is a rare autosomal recessive disease characterized by the presence of tissue nodules, joint contractures, and gingival hyperplasia. With a 1-year-9-month-old female patient scheduled for a gingivectomy and excision of a lower lip mass under general anesthesia, it was anticipated that airway management would be difficult because of trismus and limited cervical movement. Intubation with video-laryngoscopic assistance could not be achieved because gingival hyperplasia and trismus prevented blade insertion and manipulation. Therefore, 2 endotracheal tubes were used: 1 used as a nasopharyngeal airway for assisted ventilation, and 1 used for intubation along with a flexible fiberoptic scope. This case demonstrated a useful method for managing ventilation and intubation in patients with JHF, particularly when the use of oral airway devices is difficult.

Longitudinal study of risk for facial nerve injury in mandibular condyle fracture surgery: marginal mandibular branch-traversing classification of percutaneous approaches.

OBJECTIVE: This study aimed to longitudinally assess the risk of facial nerve injury (FNI) in the surgical repair of mandibular condylar neck and subcondylar fractures (CN/SCFs) and to explore its predictors. MATERIALS AND METHODS: In a retrospective cohort study, the outcome was defined as FNI at 1 week and 1, 3, and 6 months postoperatively. Potential predictors included age, sex, etiology, fracture site and pattern (dislocation/non-dislocation), concomitant facial fractures, interval to surgery, surgeons' experience, plate types, and the marginal mandibular branch-traversing approach (deep/superficial group). We employed generalized estimating equations (GEEs) for repeated measurements throughout the 6-month follow-up period. RESULTS: Among 102 patients with 114 fractures, 27 patients (26.5%) developed FNI within 1 week. Prolonged FNI (≥ 1 month) occurred in 19 (19.2%) of 99 patients. Multivariate GEE analyses revealed that deep surgical approaches (i.e., traditional submandibular and retroparotid approaches; odds ratio [OR], 18.90; p = 0.011), fractures with dislocation (OR, 3.60; p = 0.025), and female gender (OR, 2.71; p = 0.040) were independently associated with the overall FNI risk. Additionally, the deep approaches (OR, 15.91; p = 0.014) and female gender (OR, 3.41; p = 0.035) were correlated with a prolonged FNI risk. Sensitivity analyses for the outcomes identified the same predictors. CONCLUSION: The predictors longitudinally associated with FNI in CN/SCF surgeries included a deep MMB-traversing approach, dislocated fracture, and female gender. CLINICAL RELEVANCE: The superficial surgical approaches (i.e., transparotid, transmasseteric anteroparotid, and high perimandibular approaches) should be adopted for CN/SCF treatment to minimize postoperative morbidity, especially for female patients with dislocated condyles.

Risk stratification against inferior alveolar nerve injury after lower third molar extraction by scoring on cone-beam computed tomography image.

The study aimed to stratify the risk of inferior alveolar nerve injury (IANI) after lower third molar (LM3) surgery with a scoring system using identified predictive factors based on cone-beam computed tomography (CBCT) images. In a case-control study, the primary outcome was IANI occurrence. The control group included randomly selected patients without IANI. Predictor variables included patient demographics, surgical situations, Pell-Gregory classification, and inferior alveolar canal (IAC)-associated factors on CBCT. Study variables were analyzed using logistic regression models. Risk stratification was assessed by a scoring system that was constructed using independent predictors. The 858 patients who underwent LM3 surgery (1177 teeth) after CBCT scan were divided into case (25 patients, 2.9%, 27 teeth) and control (235 patients, 300 teeth) groups. In the multivariate model, lingual/inter-radicular position of IAC [odds ratio (OR) 7.21; P < 0.001; assigned score, 2], multiple roots closed to the IAC with cortical perforation (OR 3.72; P = 0.015; 1), and age > 30 years (OR 4.99; P = 0.008; 2) were associated with an increased IANI-risk. The IANI-risk scoring system could be stratified into low- and high-risk groups at a cutoff score of 3 (sensitivity, 68.0%; specificity, 90.6%; positive predictive value, 17.8%; positive likelihood ratio, 7.23). In conclusion, the high-risk group of IANI after LM3 surgery corresponded to individuals with multiple factors: lingual/inter-radicular IAC position to LM3, multiple roots with perforated IAC, and increased age (> 30 years). Raising awareness of the higher probability for IANI is needed for patients with multiple aforementioned factors.

[Clinicopathological Evaluation of Preoperative Chemotherapy Containing S-1 as a Treatment for Patients with Oral Squamous Cell Carcinoma during Waiting Time for Surgery].

BACKGROUND: In our department, patients with oral squamous cell carcinoma(OSCC)received preoperative chemotherapy containing S-1 to prevent the growth and dissemination of tumors during the waiting time before definitive surgery. We retrospectively evaluated the usefulness of this treatment. PATIENTS AND METHODS: One hundred and five patients comprising stages T1(26), T2(64), T3(7), and T4(8 cases)were enrolled in this study from July 2001 to June 2013. In principle, patients were administered S-1(80mg/m / 2/day, days 1-14)and followed by a drug holiday(days 15-21), continuing until 1 week before surgery. RESULTS: The median administration period was 14 days(256 days). Ninety-eight patients underwent definitive surgery, but 7 patients who revealed clinical CR underwent only biopsy and showed histological CR. The histological responses of all patients were CR(24), PR(22), and NC(59), and the response rate was 43.8%. Almost all adverse effects were Grade 1 or 2, except 1 case of neutropenia(Grade 3)and 1 case of urticaria(Grade 3). The 5-year overall survival rates were 86.7% in all cases, 95.3% in CR/PR cases, and 79.7% in NC cases. CONCLUSION: Preoperative S-1 administration during the waiting time was a safe and very effective method and was considered beneficial for patients with OSCC.

Lipoma Rarely Involving Multiple Suprahyoid Fascial Spaces: A Large Lesion Removed With Bimanual Transcervical Finger Dissection Using a "Push-Pull Down" Maneuver.

Lipoma involving multiple fascial spaces is extremely rare and poses a challenge to surgeons using less invasive procedures. Although blunt dissection using a finger is often used in excisional surgeries as a supplementary maneuver, few cases have been described using the bimanual technique for the removal of these extensive lesions.Herein, the authors present a large lipoma of the oral floor extending to the unilateral submandibular and parapharyngeal spaces, which was excised only via a submandibular approach using the "push-pull down" maneuver, blunt finger dissection of the tumor with counter-pushing on the overlying mucosa to the submandibular direction facilitated tumor separation. The tumor was consequently pulled down and removed without an additional intraoral incision.Based on accurate preoperative examinations, this maneuver, provides a less invasive surgery for well-encapsulated benign tumors involving multiple fascial spaces.

Gingival Neurofibroma With Teardrop-Shaped Defects of the Interdental Alveolar Bone: An Unusual Oral Manifestation of Neurofibromatosis Type 1.

Gingival enlargement, although frequently encountered in clinical settings, is rarely associated with systemic diseases or syndromes. Among the diverse pathological conditions of neurofibromatosis type 1 (NF-1), minor manifestations in the orofacial region are occasionally overlooked. Herein, the authors present an unusual case of gingival neurofibroma in a patient with NF-1 associated with characteristic osseous defects in the alveolus in the long-term course of 17 years from the first examination.A 5-year-old boy with NF-1 was referred for the evaluation of gingival enlargement in the posterior left maxilla. An incisal biopsy led to the diagnosis of neurofibroma. At 22 years of age, the patient was referred again with a complaint of bleeding and pain in the same region refractory to periodontal therapy. The gingiva and tuberosity were swollen, and the second molar was affected by the tumor. Radiography revealed a low level of the interdental septum beneath the tumor with a relatively intact periodontal cortical bone, exhibiting a teardrop-shaped bone defect. The lesion was completely resected with the periosteum.Gingival neurofibroma in NF-1 may be associated with osseous and dental abnormalities and can be mistaken for periodontitis. Raising awareness of this clinical entity can lead to proper management of the esthetic and functional problems in the oral and maxillofacial region.

Autophagy as a Survival Mechanism for Squamous Cell Carcinoma Cells in Endonuclease G-Mediated Apoptosis.

Safingol, L- threo-dihydrosphingosine, induces cell death in human oral squamous cell carcinoma (SCC) cells through an endonuclease G (endoG) -mediated pathway. We herein determined whether safingol induced apoptosis and autophagy in oral SCC cells. Safingol induced apoptotic cell death in oral SCC cells in a dose-dependent manner. In safingol-treated cells, microtubule-associated protein 1 light chain 3 (LC3)-I was changed to LC3-II and the cytoplasmic expression of LC3, amount of acidic vesicular organelles (AVOs) stained by acridine orange and autophagic vacuoles were increased, indicating the occurrence of autophagy. An inhibitor of autophagy, 3-methyladenine (3-MA), enhanced the suppressive effects of safingol on cell viability, and this was accompanied by an increase in the number of apoptotic cells and extent of nuclear fragmentation. The nuclear translocation of endoG was minimal at a low concentration of safingol, but markedly increased when combined with 3-MA. The suppressive effects of safingol and 3-MA on cell viability were reduced in endoG siRNA- transfected cells. The scavenging of reactive oxygen species (ROS) prevented cell death induced by the combinational treatment, whereas a pretreatment with a pan-caspase inhibitor z-VAD-fmk did not. These results indicated that safingol induced apoptosis and autophagy in SCC cells and that the suppression of autophagy by 3-MA enhanced apoptosis. Autophagy supports cell survival, but not cell death in the SCC cell system in which apoptosis occurs in an endoG-mediated manner.

Entry of Oncolytic Herpes Simplex Virus into Human Squamous Cell Carcinoma Cells by Ultrasound.

Low-intensity ultrasound is a useful method to introduce materials into cells due to the transient formation of micropores, called sonoporations, on the cell membrane. Whether oncolytic herpes simplex virus type 1 (HSV-1) can be introduced into oral squamous cell carcinoma (SCC) cells through membrane pores remains undetermined. Human SCC cell line SAS and oncolytic HSV-1 RH2, which was deficient in the 134.5 gene and fusogenic, were used. Cells were exposed to ultrasound in the presence or absence of microbubbles. The increase of virus entry was estimated by plaque numbers. Viral infection was hardly established without the adsorption step, but plaque number was increased by the exposure of HSV-1-inoculated cells to ultrasound. Plaque number was also increased even if SAS cells were exposed to ultrasound and inoculated with RH2 without the adsorption step. This effect was abolished when the interval from ultrasound exposure to virus inoculation was prolonged. Scanning electron microscopy revealed depressed spots on the cell surface after exposure to ultrasound. These results suggest that oncolytic HSV-1 RH2 can be introduced into SAS cells through ultrasound-mediated pores of the cell membrane that are resealed after an interval.