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BackgroundPerineural invasion (PNI) has been shown to be a key pathological feature of several types of cancer, including oral squamous epithelial carcinoma (OSCC). However, the overall clinical and genomic landscape of PNI+ OSCC are still unclear, and the molecular mechanism of PNI remains to be further investigated.Methods279 OSCC samples were extracted from the TCGA database and grouped according to PNI. The clinicopathological information, prognostic and survival analyses were performed. The Cibersort algorithm and ESTIMATE algorithm was used to estimate the impacts on proportion of immune cells, immune score and stromal score by PNI. Immunotherapy prediction analysis was also performed. 167 differentially expressed genes were screened for functional enrichment analysis. Actin α1 (ACTA1) protein, which was significantly upregulated in the PNI+ group, was selected for validation in our OSCC patient’s cohort (n = 70). We next analyzed the ratio and absolute number of key immunocytes in peripheral blood of OSCC patients according to Actin α1 expression by flow cytometry.ResultsPNI was more likely to occur in patients with advanced tumors and worse prognosis. Immunomodulation analyses showed that T cells follicular helper and cells were significantly lower, but M2 macrophages and total stromal score was significantly higher in PNI+ OSCC. Immunotherapy prediction analyses showed that PNI+ OSCC may be more sensitive to CTLA4 inhibitor treatment. 167 differentially expressed genes were identified and enriched in muscle structure and cell movement-related pathway. Among them, Actin α1 (ACTA1) was significantly upregulated in PNI+ advanced OSCC with worse clinical outcome whose had relatively low ratio of CD3+CD8+ circulating cytotoxic T cells.ConclusionPNI+ OSCC patients with upregulated of Actin α1 could benefit from cytotoxic T cell-mediated immunotherapy.

Background With the increasing application of guided endodontics to treat complex root canal treatment, the entire process of root canal treatment has become more precise, reducing damage to tooth structure and improving success rates. However, due to the limitations of the operating space, the use of guided endodontic templates in posterior root canal treatment is less common. This study aims to compare the accuracy and reliability of selective laser melting (SLM) and traditional stereolithography etching (SLA) guided endodontic templates for posterior root canals, providing better treatment strategies for posterior root canal treatment. Methods The teeth were randomly assigned to either SLM or SLA group. Preoperative cone-beam computed tomography (CBCT) and a three-dimensional (3D) scanner were used to establish the 3D root canal system and the accurate occlusal models of the teeth. The virtual access to the canal access was designed using Mimics 19.0 and 3-Matic 11.0. The endodontic access was performed based on either SLM or SLA templates. The accuracy of endodontic preparation was measured in three-dimensions by calculating deviations from planned accesses. The template height and tooth substance loss rates in each group were measured. Results SLM-guided templates have a low average deviation at the entry point and apical portion of the bur of total posterior teeth (including premolars and molars) and individual molars (P < 0.05). Moreover, there was a significant difference in angular deviations and height of template in total posterior teeth and individual molars (P < 0.05). The mean substance loss rate of the SLA group was slightly greater than that of the SLM group, but the difference was not statistically (P > 0.05). Conclusions SLM-guided endodontics provides a more predictable and precise location of root canal orifice for the treatment of posterior teeth.

Abstract An active host adaptive response is characterized by the existence of programmed cell death protein 1 (PD‐1)+/IFN‐γ+ cytotoxic T cells and IFN‐γ‐induced PD‐L1+ tumor cells (TCs), which predicts high response rate to anti‐PD‐1/L1 therapy. Recently, CD161 and its ligand LLT1 (CLEC2D) have been identified as an emerging checkpoint for immunotherapy. Clarifying its heterogeneous clinical expression pattern and its immune landscape is a prerequisite for maximizing the response rate of CD161 blockade therapy in a specific population of oral squamous cell carcinoma (OSCC) patients. Here, we investigated the expression pattern of CD161/LLT1 and its association with major immunocytes (T cells, B cells, NK cells, and macrophages) by multiplex immunofluorescence, immunohistochemistry, and flow cytometry in 109 OSCC tissues and 102 peripheral blood samples. TCs showed higher LLT1 levels than tumor infiltrating lymphocytes (TILs), whereas CD161 was highly expressed in CD8+ T cells at the tumor front, which was decreased in paracancerous tissue. High expression of TC‐derived LLT1 (LLT1TC) conferred poor clinical outcomes, whereas higher CD161+ and LLT1+ TILs were associated with better prognosis. Meanwhile, patients with high LLT1TC showed a decreased ratio of CD8+/Foxp3+ T cells in situ, but CD161+ TILs correlated with more peripheral CD3+ T cells. Interestingly, treatment of OSCC patients with nivolumab (anti‐PD‐1) could restore tumoral CD161/LLT1 signal. Furthermore, an OSCC subgroup characterized by high LLT1+ TCs and low CD161+CD8+ T cells showed fewer peripheral T cells and a higher risk of lymph node metastasis, leading to a shorter 5‐year survival time (29%). More LLT1TC at the invasive front was another risk characteristic of exhausted T cells. In conclusion, in view of this heterogeneity, the LLT1/CD161 distribution pattern should be determined before CD161‐based immunotherapy.

Abstract Aims Different regions of oral squamous cell carcinoma (OSCC) have particular histopathological characteristics, and the individual histological characteristics of the tumors are poorly understood. Therefore, calculating the proportion of tumor cells in different regions that allow assessment of the prognostic outcomes for OSCC patients would be of great clinical significance. Methods and Results We established an open‐source software‐based analytic pipeline that defines the inner tumor and invasive tumor front (ITF) in pancytokeratin‐stained whole slide images (WSIs) and quantifies the tumor‐stroma ratio (TSR) within the two regions. We applied this method to 114 patients with OSCC and predicted patient prognosis by the TSR. The proportion of tumor area in the inner tumor was generally higher than that in the ITF (p

Abstract Background Tumor metastasis is responsible for the high mortality rate of patients with oral squamous cell carcinoma (OSCC). Although many hypotheses have been proposed to elucidate the mechanism of tumor metastasis, the origin of the metastatic tumor cells remains unclear. In this study, we explored the role of cell fusion in the formation of OSCC metastatic tumor cells. Methods Murine OSCC tumor cells and macrophages were fused in vitro, and the cell proliferation, migration, and phagocytosis abilities of hybrid cells and parental cells were compared. Subsequently, we compared the transcriptome differences between hybrid and parental cells. Results Murine OSCC tumor cells and macrophages were successfully fused in vitro. The cytological and molecular experimental results revealed that OSCC tumor cells obtained a migration‐related phenotype after fusion with macrophages, and the migration ability of hybrid cells was related to the activation of the “chemokine signal pathway”. Conclusion After fusion with macrophages, the chemokine signaling pathway in OSCC tumor cells was activated, leading to metastasis.

Head and neck cancer ranks as the sixth most prevalent malignancy, constituting 5 % of all cancer cases. Its inconspicuous onset often leads to advanced stage diagnoses, prompting the need for early detection to enhance patient prognosis. Currently, research into early diagnostic markers relies predominantly on genomics, proteomics, transcriptomics, and other methods, which, unfortunately, necessitate tumor tissue homogenization, resulting in the loss of temporal and spatial information. Emerging as a recent addition to the omics toolkit, spatial metabolomics stands out. This method conducts in situ mass spectrometry analyses on fresh tissue specimens while effectively preserving their spatiotemporal information. The utilization of spatial metabolomics in life science research offers distinct advantages. This article comprehensively reviews the progress of spatial metabolomics in head and neck cancer research, encompassing insights into cancer cell metabolic reprogramming. Various mass spectrometry imaging techniques, such as secondary ion mass spectrometry, stroma-assisted laser desorption/ionization, and desorption electrospray ionization, enable in situ metabolite analysis for head and neck cancer. Finally, significant emphasis is placed on the application of presently available techniques for early diagnosis, margin assessment, and prognosis of head and neck cancer.

IntroductionCurrently, hyperuricemia has shown a surprisingly rising trend, which attracts widespread attention due to potentially major health risks. Considering the inevitable side effects of long-term medicine, probiotics are emerging as potential therapeutics due to their ability to improve uric acid metabolism and superior safety.MethodsIn our study, two strains of probiotics, Lactobacillus gasseri LG08 (LG08) and Leuconostoc mesenteroides LM58 (LM58) isolated from kimchi were evaluated for the prebiotic properties in vitro and uric-lowering effects in vivo. Here, hyperuricemia animal model and 16S rRNA gene amplicons analysis were further studied to investigate whether these probiotics exert different effects in prevention and treatment.ResultsIn vivo indicators and intestinal flora immunity revealed that both LG08 and LM58 significantly prevent the development and progression of hyperuricemia, repair the antioxidant system and maintain intestinal flora balance in healthy rats, especially LM58. After hyperuricemia was formed, although the effect of LG08 and LM58 could decrease the level of uric acid, the effect to reverse and repair antioxidant levels in the body was limited.DiscussionIn our study, these findings have important implications for hyperuricemia prevention and therapy, and provided more mechanistic insights into the effect of probiotics in hyperuricemia.

Worst pattern of invasion (WPOI) is a parameter used to quantify tumor invasiveness of oral squamous cell carcinoma (OSCC). Here the authors show that a fibroblast subset characterized by the expression of the oxytocin receptor is enriched in highly invasive WPOI 4-5 OSCC tumors and can be targeted to reduce the desmoplastic stroma and tumor metastasis.

BackgroundWith the frequent use of Bisphosphonates (BPs), the morbidity of BP-related osteonecrosis of the jaw (BRONJ) is also increasing. However, the prevention and treatment of BRONJ is faced with enormous challenges. This study aimed to illuminate the influence of BP administration in the rat mandible and explore the feasibility of discriminating BRONJ lesion bone with Raman spectroscopy.Materials and methodsFirst, we explored the time- and mode-dependent effects of BP administration on the rat mandible with Raman spectroscopy. Second, the BRONJ rat model was constructed, and the lesion and healthy bone components were analyzed using Raman spectroscopy.ResultsWhen only BPs were administered, no rats showed BRONJ symptoms, and no difference could be found in the Raman spectra. However, when combined with local surgery, six (6/8) rats showed BRONJ symptoms. The Raman spectra also showed a significant difference between the lesion and healthy bone.ConclusionIn the progression of BRONJ, BPs and local stimulation play an essential role. Both BPs administration and local stimulation need to be controlled to prevent BRONJ. Moreover, BRONJ lesion bone in rats could be discriminated with Raman spectroscopy. This novel method would become a complement in the treatment of BRONJ in the future.

Recurrent aphthous ulcer (RAU), one of the most common diseases in humans, has an unknown etiology and is difficult to treat. Thalidomide is an important immunomodulatory and antitumor drug and its effects on the gut microbiota still remain unclear. We conducted a metagenomic sequencing study of fecal samples from a cohort of individuals with RAU, performed biochemical assays of cytokines, immunoglobulins and antimicrobial peptides in serum and saliva, and investigated the regulation effects of thalidomide administration and withdrawal. Meanwhile we constructed the corresponding prediction models. Our metagenome-wide association results indicated that gut dysbacteriosis, microbial dysfunction and immune imbalance occurred in RAU patients. Thalidomide regulated gut dysbacteriosis in a species-specific manner and had different sustainable effects on various probiotics and pathogens. A previously unknown association between gut microbiota alterations and RAU was found, and the specific roles of thalidomide in modulating the gut microbiota and immunity were determined, suggesting that RAU may be affected by targeting gut dysbacteriosis and modifying immune imbalance. In-depth insights into sophisticated networks consisting of the gut microbiota and host cells may lead to the development of emerging treatments, including prebiotics, probiotics, synbiotics, and postbiotics.

Abstract The purpose of this study was to design silver nanoparticles (AgNPs) poloxamer thermoreversible gel (AgNPs-PL) and investigate whether this gel could provide sustained antibacterial activity against Enterococcus faecalis (E. faecalis) in the root canal. The gels fabricated were characterized in terms of gelatin temperature, particle size, in-vitro Ag+ release, and elemental content. Cytotoxicity of AgNPs-PL on primary human periodontal ligament fibroblasts (HPDLFs) was examined by CCK-8 assay. Characterization of AgNPs-PL gel revealed that it contained particles existing as large clumps/fused aggregates of different shapes, with a mean diameter of 21.624 ± 14.689 nm, exhibited sustained release of Ag+ for 9 days, and non-toxic to HPDLFs at a low dose (4–32 μg/mL) through 24, 48, and 72 h exposures. The antibacterial effect of 16 and 32 μg/mL concentrations of AgNPs-PL was compared with blank poloxamer gel (PL) and calcium hydroxide (CH) using three methods: (I) agar counting plate, (II) scanning electron microscope (SEM) observations, and (III) confocal laser scanning microscope (CLSM) analysis. AgNPs-PL at the two doses above was more effective than PL and CH in removing E. faecalis biofilm at 1, 3, 9 days. Thus, AgNPs-PL exhibits strong activity against E. faecalis and is easy to produce, with a continuous release profile of Ag+. AgNPs-PL gel may be a candidate for a new root canal disinfection.

Abstract The survival rate of oral squamous cell carcinoma (OSCC) can be greatly improved if intervention could be initiated as early as possible. This poses a technical demand for developing a sensitive screening and specific in vitro diagnosis method for OSCC. Herewith, a large cohort consisting of 241 healthy contrast (HC) and 578 OSCC patients were recruited for conducting the rapid metabolic profiling of trace volume serum using conductive polymer spray ionization mass spectrometry (CPSI‐MS). Statistical analysis picked out 65 metabolite ions as potential characteristic markers for differentiating OSCC from HC. With the aid of a supporting vector machine (SVM), OSCC can be distinguished from HC with an accuracy of 98.0% by cross‐validation in the discovery cohort and 89.6% accuracy in the validation cohort. Furthermore, orthogonal partial least square‐discriminant analysis (OPLS‐DA) also initially showed the potential for OSCC staging, especially between T1/T2 and T3/T4 stages with an accuracy of 90.1%. CPSI‐MS combined with SVM or OPLS‐DA can not only quickly distinguish OSCC from HC but also predict the OSCC progression from T1/2 to T3/4 stages in a few minutes, making it a promising tool for both screening and diagnosing high‐risk population. Key points Sixty‐five characteristic metabolite ions significantly changed in OSCC serum metabolic profile compared to that in the HC group. CPSI‐MS combined with SVM achieved 89.6% accuracy on the validation cohort for OSCC prediction. CPSI‐MS/OPLS‐DA can distinguish T1/T2 from T3/T4 stages with an accuracy of 90.1% by cross‐validation.

BackgroundPeriodontitis is considered a risk factor for atherosclerosis, but the mechanism is not clear. It was reported that oral administration of Porphyromonas gingivalis altered the gut microbiota in mice. Gut dysbiosis and the intestinal metabolite trimethylamine N-oxide (TMAO) were verified to be associated with atherosclerosis. Therefore, the possible TMAO-related mechanism between periodontitis and atherosclerosis needs to be explored.MethodsExperimental periodontitis was established by oral administration of P. gingivalis for 2 months in ApoE−/− mice. Mouse hemi-mandibles were scanned using Micro-CT. Quantification of TMAO was performed using liquid chromatography–tandem mass spectrometry. Mouse feces were collected and the bacterial DNA was extracted, then the gut microbiota was analyzed using 16S rRNA genes. Atherosclerotic lesion areas were quantified. Livers, small intestines, and large intestines were analyzed for gene expression.ResultsAggravated atherosclerosis plaques were found in experimental periodontitis mice. Plasma TMAO, a pathogenic factor of atherosclerosis, was initially found to be increased in periodontitis mice. Changes in the composition and abundance of the intestinal microflora of periodontitis mice were found. Flavin monooxygenase 3 (FMO3), the catalyzing enzyme of TMAO in the liver, was significantly increased, accompanied by an increase of IL-6 in liver, the abnormal intestinal integrity and enhanced plasma LPS. The IL-6 and LPS were verified to be able to increase FMO3 in HepG2 cells.ConclusionOur research discovered that experimental periodontitis in ApoE−/− mice induced gut dysbiosis and an increase in TMAO. These results suggest a possible mechanism by which periodontitis may accelerate atherosclerosis by influencing the intestinal microbes and the metabolism, which were triggered by inflammation of the liver and intestine.

As a key histopathological characteristic of tumor invasion, perineural invasion (PNI) assists tumor dissemination, whereas the current definition of PNI by dichotomy is not accurate and the prognostic value of PNI has not reached consensus. To define PNI status in each patient when mixed types of PNI occurred simultaneously, we here further subclassified the traditional PNI in 183 patients with oral squamous cell carcinoma (OSCC). The spatial localization of nerves in OSCC microenvironment was thoroughly evaluated and successfully concluded into four types of PNI: 0, tumor cells away from nerves; 1, tumor cells encircling nerves less than 33%; 2, tumor cells encircling nerves at least 33%; and 3, tumor cells infiltrating into nerve sheathes. Sequentially, patients were stratified by single and mixed types of PNI. Traditionally, types 0 and 1 were defined as PNI−, while types 2 and 3 were PNI+, which predicted shorter survival time. When multiple types of PNI existed within one tumor, patients with higher score of PNI types tended to have a relatively worse prognosis. Therefore, to define the status of PNI more precisely, the new variable worst pattern of PNI (WPNI) was proposed, which was taken as the highest score of PNI types present in each patient no matter how focal. Results showed that patients with WPNI 1 had longest survival time, and WPNI 2 correlated with better overall survival (p = 0.02), local-regional recurrence-free survival (p = 0.03), and distant metastasis-free survival (p = 0.046) than WPNI 3. Multivariate Cox analysis confirmed that only WPNI 3 could independently predict patients’ prognosis, which could be explained by a more damaged immune response in WPNI 3 patients with less CD3+CD8+ T cells and CD19+ B cells. Conclusively, WPNI by trichotomy provide more meticulous and precise pathological information for tumor-nerve interactions in OSCC patients.

Cigarette smoking could have certain effects on gut microbiota. Some pioneering studies have investigated effects of active smoking on the microbiome in local segments of the digestive tract, while active smoking-induced microbiome alterations in the whole digestive tract have not been fully investigated. Here, we developed a rat model of active smoking and characterized the effects of active smoking on the microbiota within multiple regions along the digestive tract. Blood glucose and some metabolic factors levels, the microbial diversity and composition, relative abundances of taxa, bacterial network correlations and predictive functional profiles were compared between the control group and active smoking group. We found that active smoking induced hyperglycemia and significant reductions in serum insulin and leptin levels. Active smoking induced region-specific shifts in microbiota structure, composition, network correlation and metabolism function along the digestive tract. Our results demonstrated that active smoking resulted in a reduced abundance of some potentially beneficial genera (i.e. Clostridium, Turicibacter) and increased abundance of potentially harmful genera (i.e. Desulfovibrio, Bilophila). Functional prediction suggested that amino acid, lipid, propanoate metabolism function could be impaired and antioxidant activity may be triggered. Active smoking may be an overlooked risk to health through its potential effects on the digestive tract microbiota, which is involved in the cause and severity of an array of chronic diseases.

Background: Nicotinamide N-methyltransferase (NNMT) is a metabolic enzyme that catalyzes the methylation of nicotinamide (NAM) to generate 1-methyl nicotinamide (MNAM). Although previous studies have shown that NNMT is frequently dysregulated to promote the onset and progression of many malignancies, its expression profile, prognostic value and function in oral squamous cell carcinoma (OSCC) are still unknown. Methods: We used untargeted metabolomics based on mass spectrometry to analyze potential metabolite differences between tumors and matched adjacent normal tissues in 40 OSCC patients. Immunohistochemistry (IHC) was used to analyze the NNMT expression profile in OSCC, and the diagnostic and prognostic values of NNMT were evaluated. Next, qPCR and Western blot were used to compare the expression of NNMT in five OSCC cell lines. Stable transfected cell lines were constructed, and functional experiments were carried out to elucidate the effects of NNMT on the proliferation and migration of OSCC cells. Finally, gene set enrichment analysis (GSEA) was performed using The Cancer Genome Atlas (TCGA) data to investigate the potential functional mechanisms of NNMT in OSCC. Results: We found that the nicotinamide metabolic pathway was abnormally activated in OSCC tumor tissues compared with normal tissues. NNMT was expressed ubiquitously in tumor cells (TCs) and fibroblast-like cells (FLCs) but was absent in tumor-infiltrating lymphocytes (TILs). OSCC patients with highly expressed NNMT in TCs had higher risk of lymph node metastasis and showed a worse pattern of invasion (POI). Moreover, patients with highly expressed NNMT were also susceptible to postoperative recurrence. Highly expressed NNMT can independently predict shorter disease-free survival and recurrence-free survival. Functionally, we demonstrated that the ectopic expression of NNMT promoted OSCC tumor cell proliferation and migration in vitro. Conversely, silencing exerted significantly opposite effects in vitro. In addition, GSEA showed that highly expressed NNMT was mainly enriched in the epithelial–mesenchymal transformation (EMT) pathway, which displayed a significant positive correlation with the six classic EMT markers. Conclusions: Our study uncovered that NNMT may be a critical regulator of EMT in OSCC and may serve as a prognostic biomarker for OSCC patients. These findings might provide novel insights for future research in NNMT-targeted OSCC metastasis and recurrence therapy.

Background: Although there is consensus that the optimal safe margin is ≥ 5mm, obtaining clear margins (≥5 mm) intraoperatively seems to be the major challenge. We applied a molecular diagnostic method at the lipidomic level to determine the safe surgical resection margin of OSCC by desorption electrospray ionisation mass spectrometry imaging (DESI-MSI). Methods: By overlaying mass spectrometry images with hematoxylin-eosin staining (H&E) from 18 recruited OSCC participants, the mass spectra of all pixels across the diagnosed tumour and continuous mucosal margin regions were extracted to serve as the training and validation datasets. A Lasso regression model was used to evaluate the test performance. Findings: By leave-one-out validation, the Lasso model achieved 88.6% accuracy in distinguishing between tumour and normal regions. To determine the safe surgical resection distance and margin status of OSCC, a set of 14 lipid ions that gradually decreased from tumour to normal tissue was assigned higher weight coefficients in the Lasso model. The safe surgical resection distance of OSCC was measured using the developed 14 lipid ion molecular diagnostic model for clinical reference. The overall accuracy of predicting tumours, positive margins, and negative margins was 92.6%. Interpretation: The spatial segmentation results based on our diagnostic model not only clearly delineated the tumour and normal tissue, but also distinguished the different status of surgical margins. Meanwhile, the safe surgical resection margin of OSCC on frozen sections can also be accurately measured using the developed diagnostic model. Funding: This study was supported by Nanjing Municipal Key Medical Laboratory Constructional Project Funding (since 2016) and the Centre of Nanjing Clinical Medicine Tumour (since 2014).

BackgroundReelin, an extracellular glycoprotein, is expressed on neuronal cells and participates in neuronal migration during brain development. Recently, Reelin also has a vital role in carcinogenesis. However, its role in oral squamous cell carcinoma (OSCC) remains to be explored. The purpose of this study was to explore the roles of Reelin in OSCC.MethodsThe expression of Reelin in cancer-associated fibroblasts (ReelinCAF) and tumor cells (ReelinTC) was analyzed by the Gene Expression Omnibus (GEO) database. Immunohistochemistry (IHC) was used to detect the spatial pattern of Reelin in 75 OSCCs. The diagnostic and prognostic values of Reelin were evaluated and also verified by The Cancer Genome Atlas (TCGA) database. Primary CAFs from 13 OSCC patients were isolated to confirm Reelin expression. Thirty-nine OSCC peripheral blood samples were used to analyze the change of immunocytes based on Reelin levels by flow cytometry. The relationship between Reelin and tumor immune microenvironment in head and neck squamous cell carcinoma (HNSCC) tissues was determined by TISIDB and the Tumor Immune Estimation Resource (TIMER) database.ResultsIn breast cancer, pancreatic cancer and rectal cancer, Reelin in CAFs was significantly upregulated compared with Reelin in TCs. The IHC results in OSCC also showed that Reelin levels were higher in CAFs. Upregulated ReelinTC was related to a decreased pN stage and distant metastasis. Strikingly, patients with enhanced ReelinCAF had a high risk of lymph node metastasis, poor worst pattern of invasion (WPOI), and distant metastasis, but showed comparable Ki-67 level in all OSCC patients, resulting in shorter overall survival (OS) and disease-specific survival (DSS). Unexpectedly, Reelin in tumor-infiltrating lymphocytes (ReelinTIL) was correlated with postoperative relapse. Patients with high ReelinTIL, but not ReelinTC and ReelinCAF, had poor cytotoxicity of CD8+ T cells and higher ratio of CD4/CD8 in peripheral blood. However, Reelin was positively associated with tissue-resident B cells and NK cells in the tumor microenvironment.ConclusionReelin has a versatile function in distinct cell types during the development of OSCC via governing tumor cell and stroma microenvironment.

BackgroundCD38 belongs to the ribosyl cyclase family and is expressed on various hematological cells and involved in immunosuppression and tumor promotion. Although targeting CD38 antibodies has been approved for treatment of multiple myeloma, the function of CD38 in solid tumor, oral squamous cell carcinoma (OSCC) etc., has not been investigated.MethodsThis retrospective study included 92 OSCC samples and analyzed the spatial distribution of CD38 by immunohistochemistry (IHC). The values of diagnosis and prognosis of CD38 were evaluated. Additionally, 53 OSCC preoperative peripheral blood samples were used to be analyzed by flow cytometry. Tumor Immune Estimation Resource (TIMER) and cBioPortal databases were used to study CD38 level in various tumors and its correlation with tumor immune microenvironment in head and neck squamous cell carcinoma (HNSCC).ResultsCD38 ubiquitously presented in tumor cells (TCs), fibroblast-like cells (FLCs), and tumor-infiltrating lymphocytes (TILs). Patients with highly expressed CD38 in TCs (CD38TCs) had higher TNM stage and risk of lymph node metastasis. Upregulation of CD38 in FLCs (CD38FLCs) was significantly associated with poor WPOI. Escalated CD38 in TILs (CD38TILs) led to higher Ki-67 level of tumor cells. Moreover, patients with enhanced CD38TCs were susceptible to postoperative metastasis occurrence, and those with highly expressed CD38TILs independently predicted shorter overall and disease-free survival. Strikingly, patients with highly expressed CD38TILs, but not CD38TCs and CD38FLCs, had significantly lower CD3+CD4+ T cells and higher ratio of CD3−CD16+CD56+NK cells. The imbalance of immune system is attributed to dysregulated immune checkpoint molecules (VISTA, PD-1, LAG-3, CTLA-4, TIGIT, GITR) as well as particular immune cell subsets, which were positively correlated with CD38 expression in HNSCC.ConclusionCD38 is a poor prognostic biomarker for OSCC patients and plays a vital role in governing immune microenvironment and circulating lymphocyte homeostasis. Co-expression between CD38 and immune checkpoint molecules provides new insight into immune checkpoint therapy.

Abstract Background Local resident normal fibroblasts (NFs) are the major source of cancer-associated fibroblasts (CAFs), which are distinguishable from NFs by their tumor-supportive properties. However, the mechanism and the effects underlying the transition of NFs to CAFs in oral squamous cell carcinoma (OSCC) remain unclear. Methods Five pairs of matching primary NFs and CAFs derived from OSCC patients were sent for RNA sequencing. Epiregulin (EREG) expression was analyzed by IHC in fibroblasts from OSCC patients. The role of EREG in the NF-CAF transition and the consequential effects on OSCC progression were examined by upregulation/downregulation of EREG in NFs/CAFs both in vitro and in vivo. Results Here, we identified epiregulin (EREG) as the most remarkably upregulated gene in CAFs. High EREG expression in CAFs correlated with higher T stage, deeper invasion and inferior worst pattern of invasion (WPOI) in OSCC patients and predicted shorter overall survival. Overexpression of EREG in NFs activated the CAF phenotype. Mechanistically, the JAK2/STAT3 pathway was enhanced by EREG in parallel with increased IL-6 expression, which could be inhibited by the JAK2 inhibitor AG490. Recombinant IL-6 upregulated the JAK2/STAT3/EREG pathway in a feedback loop. Moreover, EREG-induced CAF activation promoted the epithelial-mesenchymal transition (EMT) necessary for migration and invasion, which was dependent on JAK2/STAT3 signaling and IL-6. In vivo, EREG expression in stroma fibroblasts promoted tumor growth with high stromal α-SMA, phospho-JAK2/STAT3, and IL-6 expression and upregulated EMT in HSC3 cells. Conclusions EREG is essential for the NF-CAF transformation needed to induce EMT of tumor cells in a JAK2-STAT3- and IL-6-dependent manner in OSCC.

Dysregulated amino acids metabolism reciprocally interplays with evolutionary phenotypic characteristics of cancer cells to enhance metastasis. The high metastasis potential of oral squamous cell carcinoma (OSCC) can manifest with perineural invasion (PNI). We here aimed to determine the role of amino acids metabolism in OSCCs with different PNI statuses. Targeted metabolomics was used to quantify 48 amino acids in 20 fresh OSCC samples and 25 amino acids were successfully detected, within which 9 were significantly up-regulated in PNI positive (PNI+) samples. As its highest area under the curve value (0.9063), l-asparagine was selected as the biomarker to distinguish PNI+ from PNI negative (PNI−). Then, the key enzyme of l-asparagine, asparagine synthetase (ASNS), was investigated using immunohistochemistry with 86 OSCC patients. The results showed that ASNS mainly expressed in tumor epitheliums and positively correlated with lymph node metastasis and PNI. Moreover, subgroup survival analysis revealed that ASNS expression combined with PNI status significantly improved their prognostic value, which was confirmed by the TCGA OSCC cohort (n = 279). To validate whether ASNS promotes PNI, we determined ASNS expression levels in five OSCC cell lines and one normal oral keratinocyte, and HSC3 showed the lowest ASNS level but CAL33 had the highest. Therefore, HSC3 and CAL33 (or PBS as control) were selected and injected separately into sciatic nerves to construct the in vivo PNI mouse models. Although both models eventually developed the hind-limb paralysis, nerve dysfunction in the CAL33 model progressed significantly earlier than HSC3 (Day 9 vs. Day 24). Besides, CAL33 migrated significantly farther than HSC3 in the nerve microenvironment (P = 0.0003), indicating high ASNS expression is indispensable for OSCC progression, especially PNI formation, through l-asparagine metabolism alteration. This study provides novel insights into how amino acids metabolism disorders alter tumor neurotropism which helps cancer metastasis.

The 5-year survival rate for oral squamous cell carcinoma (OSCC), one of the most common head and neck cancers, has not improved in the last 20 years. Poor prognosis of OSCC is the result of failure in early and precise diagnosis. Metabolic reprogramming, including the alteration of the uptake and utilisation of glucose, amino acids and lipids, is an important feature of OSCC and can be used to identify its biomarkers for early and precise diagnosis. In this review, we summarise how recent findings of rewired metabolic networks in OSCC have facilitated early and precise diagnosis of OSCC.

Abstract FADD, a classical apoptotic signaling adaptor, was recently reported to have non‐apoptotic functions. Here, we report the discovery that FADD regulates lipid metabolism. PPAR‐α is a dietary lipid sensor, whose activation results in hypolipidemic effects. We show that FADD interacts with RIP140, which is a corepressor for PPAR‐α, and FADD phosphorylation‐mimic mutation (FADD‐D) or FADD deficiency abolishes RIP140‐mediated transcriptional repression, leading to the activation of PPAR‐α. FADD‐D‐mutant mice exhibit significantly decreased adipose tissue mass and triglyceride accumulation. Also, they exhibit increased energy expenditure with enhanced fatty acid oxidation in adipocytes due to the activation of PPAR‐α. Similar metabolic phenotypes, such as reduced fat formation, insulin resistance, and resistance to HFD‐induced obesity, are shown in adipose‐specific FADD knockout mice. Additionally, FADD‐D mutation can reverse the severe genetic obesity phenotype of ob/ob mice, with elevated fatty acid oxidation and oxygen consumption in adipose tissue, improved insulin resistance, and decreased triglyceride storage. We conclude that FADD is a master regulator of glucose and fat metabolism with potential applications for treatment of insulin resistance and obesity.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase critically involved in cancer metastasis. We found an elevation of FAK expression in highly metastatic melanoma B16F10 cells compared with its less metastatic partner B16F1 cells. Down-regulation of the FAK expression by either small interfering RNA or dominant negative FAK (FAK Related Non-Kinase, FRNK) inhibited the B16F10 cell migration in vitro and invasiveness in vivo. The mechanism by which FAK activity is up-regulated in highly metastatic cells remains unclear. In this study, we reported for the first time that one of the Est family proteins, PEA3, is able to transactivate FAK expression through binding to the promoter region of FAK. We identified a PEA3-binding site between nucleotides -170 and +43 in the FAK promoter that was critical for the responsiveness to PEA3. A stronger affinity of PEA3 to this region contributed to the elevation of FAK expression in B16F10 cells. Both in vitro and in vivo knockdown of PEA3 gene successfully mimicked the cell migration and invasiveness as that induced by FAK down-regulation. The activation of the well-known upstream of PEA3, such as epidermal growth factor, JNK, and ERK can also induce FAK expression. Furthermore, in the metastatic human clinic tumor specimens from the patients with human primary oral squamous cell carcinoma, we observed a strong positive correlation among PEA3, FAK, and carcinoma metastasis. Taking together, we hypothesized that PEA3 might play an essential role in the activation of the FAK gene during tumor metastasis.

Here we report two unrelated Chinese families with congenital missing teeth inherited in an X-linked manner. We mapped the affected locus to chromosome Xp11-Xq21 in one family. In the defined region, both families were found to have novel missense mutations in the ectodysplasin-A (EDA) gene. The mutation of c.947A>G caused the D316G substitution of the EDA protein. The mutation of c.1013C>T found in the other family resulted in the Thr to Met mutation at position 338 of EDA. The EDA gene has been reported responsible for X-linked hypohidrotic ectodermal dysplasia (XLHED) in humans characterized by impaired development of hair, eccrine sweat glands, and teeth. In contrast, all the affected individuals in the two families that we studied here had normal hair and skin. Structural analysis suggests that these two novel mutants may account for the milder phenotype by affecting the stability of EDA trimers. Our results indicate that these novel missense mutations in EDA are associated with the isolated tooth agenesis and provide preliminary explanation for the abnormal clinical phenotype at a molecular structural level.

Background: Ki67 has been a key role for the treatment options and prognosis evaluation in some kinds of tumors; however, the spatial expression of Ki67 in oral squamous cell carcinoma (OSCC) has not been fully-evaluated. Therefore, in the present study, we aimed to elucidate the prognosis value of Ki67 spatial expression including in different cell types and at different compartments of tumor in OSCC patients. Methods: Immunohistochemical expression of Ki67 in tumor cells (TCs) and fibroblast like cells (FLCs) at center of tumor (CT) and invasive front (IF) was evaluated in 109 OSCC patients. Then correlations of Ki67 expressions with clinicopathological parameters were analyzed by Chi-square test, and survival curves were evaluated by Kaplan-Meier methods. Furthermore, univariate and multivariate analysis were performed to assess the diagnostic values of Ki67 expression by the Cox regression model. Results: Ki67 expression in TCs was much higher than in FLCs both at CT and IF compartments, but Ki67 expression in TCs was simultaneously higher at CT than that at IF (P=0.0004), which was converse to Ki67 expression in FLCs (P<0.0001). Additionally, high Ki67 expression in FLCs at IF was significantly associated with poor tumor differentiation (P=0.003), worse depth of invasion (DOI, P=0.027) and worst pattern of invasion (WPOI, P=0.041), but Ki67 expression in TCs had no correlation with clinical parameters no matter at CT or IF. Moreover, patients with higher Ki67 expression in TCs at CT had significantly increased risk for OS (overall survival; HR:1.935, 95% CI: 1.181-4.823, P=0.0395) and DFS (disease-free survival; HR: 2.974, 95% CI:1.189-5.023, P=0.046). On contrary, higher Ki67 expression in FLCs at IF was correlated with better OS (HR: 0.15, 95% CI: 0.018-0.846, P=0.0396) and DFS (HR: 0.15, 95% CI: 0.018-0.947, P=0.0445). Whereas, Ki67 expression both at TCs in IF and at FLCs in CT had no significant prognostic value for OS and DFS. Furthermore, Cox multivariate analysis revealed that Ki67 expression in FLCs at IF could not be an independent prognostic factor for OSCC patients. Conclusion: These results show that higher Ki67 expression in FLCs at IF indicated better clinical outcomes for OSCC patients. [ABSTRACT FROM AUTHOR]

Our study proved that intracellular NOD2 in DCs could be activated by MDP, which could promote maturation and the ability to prime Th0 cells to Th2 cells of DCs exposed to P. gingivalis-LPS. Background: To study the roles of muramyl dipeptide (MDP) in dendritic cells (DCs) exposed to Porphyromonas gingivalis lipopolysaccharide (P. gingivalis-LPS) on maturation and antigen-presenting functions of DCs. To explore the possible mechanism of DCs in periodontitis. Methods: Flow cytometry was used to detect CD11c, MHC-II, CD80, CD86, and CD40 expression, and ELISA was used to detect IL-12, IFN-γ, IL-10, and IL-13 secreted by DCs. RT-PCR analysis was used to detect NOD2, TLR2, and TLR4 mRNA expression in DCs. CCK8 was used to assay CD4+T cell proliferation. Results: MDP had a weak ability to stimulate DC maturation but MDP could promote DC maturation stimulated by P. gingivalis-LPS. MDP could facilitate TLR2 mRNA expression in DCs exposed to P. gingivalis-LPS. The ability of MDP to promote DCs secreting cytokines was far below P. gingivalis-LPS but MDP could promote the functions of Th2 cell-promoting DCs induced by P. gingivalis-LPS. MDP could promote CD4+T cell proliferation primed by DCs exposed to P. gingivalis-LPS and elevate the ability of DCs exposed to P. gingivalis-LPS to prime Th0 cells to Th2 cells. Conclusions: Intracellular NOD2 in DCs could be activated by MDP, which could promote maturation and the ability to prime Th0 cells to Th2 cells of DCs exposed to P. gingivalis-LPS. [ABSTRACT FROM AUTHOR]

Stromal carcinoma-related fibroblasts (CAFs) are the main type of non-immune cells in the tumor microenvironment (TME). CAFs interact with cancer cells to promote tumor proliferation. Long non-coding RNAs (lncRNAs) are known to regulate cell growth, apoptosis and metastasis of cancer cells, but their role in stromal cells is unclear. Using RNA sequencing, we identified a stromal lncRNA signature during the transformation of CAFs from normal fibroblasts (NFs) in oral squamous cell carcinoma (OSCC). We uncovered an uncharacterized lncRNA, FLJ22447, which was remarkably up-regulated in CAFs, referred to LncRNA-CAF (Lnc-CAF) hereafter. Interleukin-33 (IL-33) was mainly located in the stroma and positively co-expressed with Lnc-CAF to elevate the expression of CAF markers (a-SMA, vimentin and N-cadherin) in fibroblasts. In a co-culture system, IL-33 knockdown impaired Lnc-CAF-mediated stromal fibroblast activation, leading to decreased proliferation of tumor cells. Mechanistically, Lnc-CAF up-regulated IL-33 levels and prevented p62-dependent autophagy-lysosome degradation of IL-33, which was independent of LncRNA-protein scaffold effects. Treatment with the autophagy inducer, rapamycin, impaired the proliferative effect of Lnc-CAF/IL-33 by promoting IL-33 degradation. In turn, tumor cells further increased Lnc-CAF levels in stromal fibroblasts via exosomal Lnc-CAF. In patients with OSCC, high Lnc-CAF/IL-33 expression correlated with high TNM stage (n = 140). Moreover, high Lnc-CAF expression predicted poor prognosis. In vivo, Lnc-CAF knockdown restricted tumor growth and was associated with decreased Ki-67 expression and a-SMA+ CAF in the stroma. In conclusion, we identified a stromal lncRNA signature, which reprograms NFs to CAFs via Lnc-CAF/IL-33 and promotes OSCC development. [ABSTRACT FROM AUTHOR]

Objectives: Margin status and invasion pattern are prognostic factors for oral tongue squamous cell carcinoma (OTSCC). Current methods to identify these factors are limited to 2D observation; it is necessary to explore 3D reconstruction with whole-mount sample to improve the accuracy of analysis. This study aimed to study the tissue preparation, section generation, and 3D reconstruction with whole-mount OTSCC specimen.Study Design: Two OTSCC samples were retrieved from Nanjing Stomatological Hospital, Medical School of Nanjing University. One sample was sliced into 3 equal-sized pieces and subjected to different processing schedules to determine the best method. The second sample was processed accordingly. Serial whole-mount sections of the second sample were generated, stained with HE/anticytokine antibody in intersection manner, and scanned into digital images. Digital images were aligned and reconstructed into 3D images with Hetero Genius Medical Image Manager 3D Pathology Add-On [HGMIM3D].Results: Successful serial whole-mount sections of comparable quality to traditional sections were generated. Three-dimensional images with serial whole-mount sections were successfully generated.Conclusions: Whole-mount histopathological 3D reconstruction of OTSCC was successfully generated, providing a solid foundation for comprehensive margin and invasion analysis. Although future study and improvement were needed, whole-mount histopathological 3D reconstruction proved to be a promising method in OTSCC study. [ABSTRACT FROM AUTHOR]

Objectives: To delineate clinical and pathological features and determine the prognostic factors of primary intraosseous squamous cell carcinoma (PIOSCC). Materials and methods: Patients diagnosed with PIOSCC, attending the department of oral and maxillofacial surgery, Nanjing stomatological hospital between 2005 and 2015, were identified and retrospectively reviewed for clinical and pathological characteristics. Therapeutic modalities were measured and related follow-up data recorded, in order to determine prognostic factors of PIOSSC. Results: A total of 77 patients with PIOSCC were included in the study. Mean age at diagnosis was 58.8 years, (range, 37−81 years). Of the 77 patients, there were 58 men and 19 women. The most common location of disease was the mandible (71.42%), particularly the posterior mandible. The common presenting symptoms included jaw swelling (79.2%) and ulceration (42.65%). The estimated 2-year and 5-year overall survival were 68.9% and 38.8%, respectively. Univariate analysis identified the following as negative prognostic factors: histological grade, N classification, nodal status and treatment modalities. However, multivariate analysis determined positive nodal status, high histological grade and advanced N classification as the independent significant prognostic factors. Conclusion: Our results demonstrate several clinical and pathological features of PIOSCC and identify important prognostic factors associated with overall survival in PIOSCC. These prognostic factors include nodal status, histological grade, N classification, and treatment modalities, all of which are important for patient counseling and may be useful for the development of new treatment approaches. [ABSTRACT FROM AUTHOR]

Gelsolin (GSN) is one of the most abundant actin-binding proteins, and is involved in several pathological processes, including Alzheimer's disease, cardiac injury and cancer. The aim of the present study was to assess the effect of GSN on the growth and motility of oral squamous cell carcinoma Tca8113 cells. The overexpression vector pcDNA3.1-GSN was transfected into Tca8113 cells and the stable GSN overexpression cell line was identified based on G418 antibiotic selection. The effect of GSN overexpression on the proliferation, apoptosis, migration and invasion of Tca8113 cells was examined using a cell counting kit-8 assay, flow cytometry and Transwell assays. The results revealed that GSN overexpression significantly promoted the cell proliferation and apoptosis of Tca8113 cells. In addition, Transwell assays demonstrated that the migration and invasion abilities of Tca8113 cells were enhanced by GSN overexpression. Therefore, the upregulation of GSN promotes cell growth and motility, indicating that it may perform a vital function in the progression of human oral cancers. [ABSTRACT FROM AUTHOR]

The successful treatment of cancer with dendritic cell (DC) tumor vaccine is highly dependent on the efficacy of antigen presentation and T cell activation. In the present study, a novel vaccine of DCs fused with autologous tumor cells was introduced, which had a marked ability to suppress head and neck carcinoma. DCs generated from the bone marrow of mice were fused with an autologous tumor cell line using polyethylene glycol (PEG). To observe the fused cells, confocal microscopy and FACS analysis were performed. Subsequently, the activation and proliferation of T cells, as well as animal experiments, were examined. The efficiency of DC/tumor fusion was 18.03% and T cells were well-activated by the hybrids. The volumes of tumors on the tumor-bearing mice were controlled, survival time of tumor-bearing mice was prolonged and the level of IFN-γ in serum was signiicantly increased compared with the control group and lysate-pulsed DC group. The results indicate that the DC/tumor fusion vaccine appears to be more effective than DCs pulsed with tumor lysate for the treatment of head and neck carcinoma, which may be useful in future clinical studies. [ABSTRACT FROM AUTHOR]

The current dental curriculum in China was developed from the system in the Soviet Union in the 1950s. This curriculum is outdated and must be reformed to keep pace with the development of modern dental education. The new dental educational system should be customized to China's needs: care for a large population with poor overall oral health, operating within a government-owned, centralized health care delivery system. Chine e research universities have a mission to produce competent dentists who will also be educators and researchers. To efficiently train academic dentists who can al o meet the clinical need of today's Chinese population, a new dental curriculum wa developed at the Institute and Ho pi tal of Dentistry, anjing University Medical School. This curriculum ha four main features: I) a two-year general higher education plu five-year dental education ("2+5") model; 2) improved integration of didactic and practical learning; 3) improved integration of dental education with research training; and 4) improved overall equencing of the entire curriculum. This article de cribe the details ofthi new dental curriculum [ABSTRACT FROM AUTHOR]

FADD (Fas-associated death domain) has been widely expressed in various tissues and its expression has been recently demonstrated to correlate with tumour progression and prognosis. Currently, measurement of FADD expression mainly depends on Western-blot or immunohistochemical approaches. To develop a conventional sandwich ELISA avenue for the detection of FADD protein to supplement Western blotting or immunohistochemistry, a series of mAbs (monoclonal antibodies) specific for FADD protein, designated 3A3, 3F9, 3G4, 4B9, 4G1, 7A8, 7B8 and 7F4, were produced by fusing mouse s/p20 myeloma cells with the spleen cells of a mouse immunized with the Escherichia coli-expressed recombinant His6-FADD protein. On the basis of the characterization of these mAbs, purified 3F9 was selected as the capture antibody and the biotin-conjugated 3A3 was selected as the detection antibody in sandwich ELISA. The limit of detection for the ELISA was 0.3 ng of purified His6–FADD (FADD tagged with hexahistidine), and it could detect both recombinant and native human FADD protein. Furthermore, the positive reaction of the ELISA could be blocked by rabbit anti-FADD sera. All of these results indicated that the ELISA developed in the present paper could be a promising tool for detection of FADD protein. [ABSTRACT FROM AUTHOR]