Integrative analysis reveals associations between oral microbiota dysbiosis and host genetic and epigenetic aberrations in oral cavity squamous cell carcinoma.

Dysbiosis of the human oral microbiota has been reported to be associated with oral cavity squamous cell carcinoma (OSCC) while the host-microbiota interactions with respect to the potential impact of pathogenic bacteria on host genomic and epigenomic abnormalities remain poorly studied. In this study, the mucosal bacterial community, host genome-wide transcriptome and DNA CpG methylation were simultaneously profiled in tumors and their adjacent normal tissues of OSCC patients. Significant enrichment in the relative abundance of seven bacteria species (Fusobacterium nucleatum, Treponema medium, Peptostreptococcus stomatis, Gemella morbillorum, Catonella morbi, Peptoanaerobacter yurli and Peptococcus simiae) were observed in OSCC tumor microenvironment. These tumor-enriched bacteria formed 254 positive correlations with 206 up-regulated host genes, mainly involving signaling pathways related to cell adhesion, migration and proliferation. Integrative analysis of bacteria-transcriptome and bacteria-methylation correlations identified at least 20 dysregulated host genes with inverted CpG methylation in their promoter regions associated with enrichment of bacterial pathogens, implying a potential of pathogenic bacteria to regulate gene expression, in part, through epigenetic alterations. An in vitro model further confirmed that Fusobacterium nucleatum might contribute to cellular invasion via crosstalk with E-cadherin/ß-catenin signaling, TNFα/NF-κB pathway and extracellular matrix remodeling by up-regulating SNAI2 gene, a key transcription factor of epithelial-mesenchymal transition (EMT). Our work using multi-omics approaches explored complex host-microbiota interactions and provided important insights into genetic and functional basis in OSCC tumorigenesis, which may serve as a precursor for hypothesis-driven study to better understand the causational relationship of pathogenic bacteria in this deadly cancer.

Nutritional Status and Quality of Life in Patients with Oral Squamous Cell Carcinoma Before and After Surgical Oncological Treatment: A Single-Center Retrospective Study.

BACKGROUND Oral squamous cell carcinoma (OSCC) is the most common of head and neck malignancies in well-developed countries. In most cases, patients with OSCC experience a degree of nutritional status disturbances and decreased quality of life (QoL). This study aimed to compare nutritional status and QoL in 51 patients before and after surgery for OSCC. MATERIAL AND METHODS Fifty-one eligible patients with OSCC were followed during a 3-year period (2019-2022). For all patients, we determined body mass index (BMI), serum albumin (ALB), prognostic nutritional index, and nutritional risk index before and after treatment. Also, all patients completed a standardized QoL questionnaire before and after treatment. The obtained data were compared between the groups by using the t test and multivariate Cox regression. RESULTS The values of BMI and NRI were statistically significantly different between the preoperative and postoperative measurements (24.1 kg/m² vs 21.1 kg/m², and 103 vs 100.1, respectively, P=0.001), while values of ALB and prognostic nutritional index did not differ significantly (41.35 g/L vs 39.1 g/L, and 48.5 vs 46.2, respectively). Dysphagia (P=0.03) and chewing problems (P=0.04) were found to be the 2 most important factors decreasing the QoL of patients. CONCLUSIONS Based on our results, BMI and NRI were the most sensitive parameters of nutritional status. Dysphagia and chewing problems were the 2 most important factors affecting the QoL in patients with OSCC.

Expression of Toll-like receptors in oral squamous cell carcinoma.

Almost 380,000 new cases of oral cancer were reported worldwide in 2020. Oral squamous cell carcinoma (OSCC) accounts for 90% of all types of oral cancers. Emerging studies have shown association of Toll-like receptors (TLRs) in carcinogenesis. The present study aimed to investigate the expression levels and tissue localization of TRL1 to TRL10 and NF-κB between OSCC and healthy oral mucosa, as well as effect of Candida colonization in TRL expression in OSCC. Full thickness biopsies and microbial samples from 30 newly diagnosed primary OSCC patients and 26 health controls were collected. The expression of TLR1 to TLR10 and NF-κB was analyzed by immunohistochemistry. Microbial samples were collected from oral mucosa to detect Candida. OSCC epithelium showed lower staining intensity of TRL1, TRL2 TRL5, and TRL8 as compared to healthy controls. Similarly, staining intensity of TRL3, TRL4, TRL7, and TRL8 were significantly decreased in basement membrane (BM) zone. Likewise, OSCC endothelium showed lower staining intensity of TLR4, TLR7 and TLR8. Expression of NF-κB was significantly stronger in normal healthy tissue compared to OSCC sample. Positive correlation was found between the expression of NF-κB, TRL9 and TRL10 in basal layer of the infiltrative zone OSCC samples (P = 0.04 and P = 0.002, respectively). Significant increase in TRL4 was seen in BM zone of sample colonized with Candida (P = 0.01). According to the limited number of samples, our data indicates downregulation of TLRs and NF-κB in OSCC, and upregulation of TLR4 expression with presence of Candida.

Maackia amurensis seed lectin (MASL) and soluble human podoplanin (shPDPN) sequence analysis and effects on human oral squamous cell carcinoma (OSCC) cell migration and viability.

Maackia amurensis lectins serve as research and botanical agents that bind to sialic residues on proteins. For example, M. amurensis seed lectin (MASL) targets the sialic acid modified podoplanin (PDPN) receptor to suppress arthritic chondrocyte inflammation, and inhibit tumor cell growth and motility. However, M. amurensis lectin nomenclature and composition are not clearly defined. Here, we sought to definitively characterize MASL and its effects on tumor cell behavior. We utilized SDS-PAGE and LC-MS/MS to find that M. amurensis lectins can be divided into two groups. MASL is a member of one group which is composed of subunits that form dimers, evidently mediated by a cysteine residue in the carboxy region of the protein. In contrast to MASL, members of the other group do not dimerize under nonreducing conditions. These data also indicate that MASL is composed of 4 isoforms with an identical amino acid sequence, but unique glycosylation sites. We also produced a novel recombinant soluble human PDPN receptor (shPDPN) with 17 threonine residues glycosylated with sialic acid moieties with potential to act as a ligand trap that inhibits OSCC cell growth and motility. In addition, we report here that MASL targets PDPN with very strong binding kinetics in the nanomolar range. Moreover, we confirm that MASL can inhibit the growth and motility of human oral squamous cell carcinoma (OSCC) cells that express the PDPN receptor. Taken together, these data characterize M. amurensis lectins into two major groups based on their intrinsic properties, clarify the composition of MASL and its subunit isoform sequence and glycosylation sites, define sialic acid modifications on the PDPN receptor and its ability to act as a ligand trap, quantitate MASL binding to PDPN with KD in the nanomolar range, and verify the ability of MASL to serve as a potential anticancer agent.

[Proposal for the delineation of postoperative primary clinical target volumes in maxillary sinus and nasal cavity cancers]. / Proposition de délinéation des volumes cibles anatomocliniques postopératoires de la tumeur primitive des cancers du sinus maxillaire et des cavités nasales.

In this article, we propose a consensus delineation of postoperative clinical target volumes for the primary tumour in maxillary sinus and nasal cavity cancers. These guidelines are developed based on radioanatomy and the natural history of those cancers. They require the fusion of the planning CT with preoperative imaging for accurate positioning of the initial GTV and the combined use of the geometric and anatomical concepts for the delineation of clinical target volume for the primary tumour. This article does not discuss the indications of external radiotherapy (nor concurrent systemic treatment) but focuses on target volumes when there is an indication for radiotherapy.

The role of CXCL2-mediated crosstalk between tumor cells and macrophages in Fusobacterium nucleatum-promoted oral squamous cell carcinoma progression.

Dysbiosis of the oral microbiota is related to chronic inflammation and carcinogenesis. Fusobacterium nucleatum (Fn), a significant component of the oral microbiota, can perturb the immune system and form an inflammatory microenvironment for promoting the occurrence and progression of oral squamous cell carcinoma (OSCC). However, the underlying mechanisms remain elusive. Here, we investigated the impacts of Fn on OSCC cells and the crosstalk between OSCC cells and macrophages. 16 s rDNA sequencing and fluorescence in situ hybridization verified that Fn was notably enriched in clinical OSCC tissues compared to paracancerous tissues. The conditioned medium co-culture model validated that Fn and macrophages exhibited tumor-promoting properties by facilitating OSCC cell proliferation, migration, and invasion. Besides, Fn and OSCC cells can recruit macrophages and facilitate their M2 polarization. This crosstalk between OSCC cells and macrophages was further enhanced by Fn, thereby amplifying this positive feedback loop between them. The production of CXCL2 in response to Fn stimulation was a significant mediator. Suppression of CXCL2 in OSCC cells weakened Fn's promoting effects on OSCC cell proliferation, migration, macrophage recruitment, and M2 polarization. Conversely, knocking down CXCL2 in macrophages reversed the Fn-induced feedback effect of macrophages on the highly invasive phenotype of OSCC cells. Mechanistically, Fn activated the NF-κB pathway in both OSCC cells and macrophages, leading to the upregulation of CXCL2 expression. In addition, the SCC7 subcutaneous tumor-bearing model in C3H mice also substantiated Fn's ability to enhance tumor progression by facilitating cell proliferation, activating NF-κB signaling, up-regulating CXCL2 expression, and inducing M2 macrophage infiltration. However, these effects were reversed by the CXCL2-CXCR2 inhibitor SB225002. In summary, this study suggests that Fn contributes to OSCC progression by promoting tumor cell proliferation, macrophage recruitment, and M2 polarization. Simultaneously, the enhanced CXCL2-mediated crosstalk between OSCC cells and macrophages plays a vital role in the pro-cancer effect of Fn.

Improved recurrence rates and progression-free survival in primarily surgically treated oral squamous cell carcinoma - results from a German tertiary medical center.

OBJECTIVES: This study aimed to explore survival and recurrence patterns in patients undergoing primarily surgical treatment for oral squamous cell carcinoma (OSCC) at a high-volume tertiary medical center in Germany. MATERIALS AND METHODS: The study included 421 patients with primary OSCC who underwent radical tumor resection, neck dissection, and reconstruction with a free flap. Prognostic relevance of clinicopathological characteristics was assessed using Cox proportional-hazards models. Kaplan-Meier method estimated local recurrence-free survival, progression-free survival (PFS), and overall survival (OS), while the log-rank test compared survival outcomes between groups. RESULTS: Recurrence manifested in 16.63% of the patients (70 patients), encompassing local recurrence in 54 patients (77.14%) and distant metastasis in 24 patients (34.28%). Neck recurrence occurred in only 1 patient (0.24%) on the contralateral side. The majority of recurrences occurred within the initial twelve months following primary tumor surgery (64.29%). Overall, the 5-year OS stood at 58.29%, while the 5-year PFS reached 72.53%. Patients with early recurrence within ≤ 12 months showed the least favorable prognosis (log-rank, all p < 0.001). CONCLUSIONS: Our findings show a significant decrease in recurrence rates and enhanced PFS at a high-volume tertiary medical center in Germany compared to previous studies. Local recurrence was the primary form observed, with most recurrences happening within the initial twelve months post-surgery. Opting for treatment at a high-volume center and devising therapy plans in interdisciplinary tumor boards may not only enhance OS but also contribute to improved PFS. CLINICAL RELEVANCE: These findings offer valuable insights for physicians regarding the post-treatment care of patients with OSCC. The results underscore the importance of frequent follow-up appointments, particularly during the initial year, and highlight the critical need for vigilance in monitoring for local recurrence.

Survival analysis of Sudanese oral squamous cell carcinoma patients with field of cancerization.

BACKGROUND: The late presentation and diagnosis of OSCC account for the large number of patients with the advanced form of the disease. In Sudan, cases with delayed presentation, particularly those with risk factors such as Toombak dipping and alcohol consumption, frequently present with extensive lesions and a wide area of Field cancerization which characterized by the presence of genetic and epigenetic changes in histologically normal-appearing tissues, and have increased risk for recurrent and second primary tumors. This necessitates more aggressive treatment and is usually associated with poorer outcomes. The present study aims to investigate the survival of oral squamous cell carcinoma patients with a wide field of cancerization. METHODS: This prospective longitudinal study includes ninety-three oral cancer patients with extensive fields of cancerization who underwent surgical treatment at Khartoum Teaching Dental Hospital (KTDH) conducted from 2019 to 2023. These patients were regularly assessed for clinical changes such as recurrence, the development of second primary tumours, and overall survival over a period of one year. RESULTS: Out of the 93 patients, 57 (61.3%) were males, and 36 (38.7%) were females. The majority of the patients (82%) had stage IV tumours, and 62.3% had nodal metastasis. Twenty-eight (30%) patients developed recurrences, and 14 (15%) developed second primary tumours. The overall one-year survival rate was 89%, and all deceased patients passed away within 12 months. The survival rate for patients with different types of recurrences varied, with patients who had regional, local, and locoregional recurrences having survival rates of 87%, 74%, and 72%, respectively. Patients who did not experience a recurrence had a one-year survival rate of 92%. Patients who developed second primary tumours had an 86% survival rate. The survival rates for OSCC patients at stages III, IVa, and IVb were 90%, 90%, and 71%, respectively. CONCLUSION: In this study, 62% of patients had nodal metastasis, 30% developed recurrence, and 15% developed second primary tumours. The overall one-year survival rate was 89%, although the development of recurrences and second primary tumours had a negative impact on the survival rate.

Transcutaneous carbon dioxide suppresses skeletal muscle atrophy in a mouse model of oral squamous cell carcinoma.

Cancer cachexia causes skeletal muscle atrophy, impacting the treatment and prognosis of patients with advanced cancer, but no treatment has yet been established to control cancer cachexia. We demonstrated that transcutaneous application of carbon dioxide (CO2) could improve local blood flow and reduce skeletal muscle atrophy in a fracture model. However, the effects of transcutaneous application of CO2 in cancer-bearing conditions are not yet known. In this study, we calculated fat-free body mass (FFM), defined as the skeletal muscle mass, and evaluated the expression of muscle atrophy markers and uncoupling protein markers as well as the cross-sectional area (CSA) to investigate whether transcutaneous application of CO2 to skeletal muscle could suppress skeletal muscle atrophy in cancer-bearing mice. Human oral squamous cell carcinoma was transplanted subcutaneously into the upper dorsal region of nude mice, and 1 week later, CO2 gas was applied to the legs twice a week for 4 weeks and FFM was calculated by bioimpedance spectroscopy. After the experiment concluded, the quadriceps were extracted, and muscle atrophy markers (muscle atrophy F-box protein (MAFbx), muscle RING-finger protein 1 (MuRF-1)) and uncoupling protein markers (uncoupling protein 2 (UCP2) and uncoupling protein 3 (UCP3)) were evaluated by real-time polymerase chain reaction and immunohistochemical staining, and CSA by hematoxylin and eosin staining. The CO2-treated group exhibited significant mRNA and protein expression inhibition of the four markers. Furthermore, immunohistochemical staining showed decreased MAFbx, MuRF-1, UCP2, and UCP3 in the CO2-treated group. In fact, the CSA in hematoxylin and eosin staining and the FFM revealed significant suppression of skeletal muscle atrophy in the CO2-treated group. We suggest that transcutaneous application of CO2 to skeletal muscle suppresses skeletal muscle atrophy in a mouse model of oral squamous cell carcinoma.

The metabolic reprogramming of γ-aminobutyrate in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common head and neck malignancy. The oncometabolites have been studied in OSCC, but the mechanism of metabolic reprogramming remains unclear. To identify the potential metabolic markers to distinguish malignant oral squamous cell carcinoma (OSCC) tissue from adjacent healthy tissue and study the mechanism of metabolic reprogramming in OSCC. We compared the metabolites between cancerous and paracancerous tissues of OSCC patients by 1HNMR analysis. We established OSCC derived cell lines and analyzed their difference of RNA expression by RNA sequencing. We investigated the metabolism of γ-aminobutyrate in OSCC derived cells by real time PCR and western blotting. Our data revealed that much more γ-aminobutyrate was produced in cancerous tissues of OSCC patients. The investigation based on OSCC derived cells showed that the increase of γ-aminobutyrate was promoted by the synthesis of glutamate beyond the mitochondria. In OSCC cancerous tissue derived cells, the glutamate was catalyzed to glutamine by glutamine synthetase (GLUL), and then the generated glutamine was metabolized to glutamate by glutaminase (GLS). Finally, the glutamate produced by glutamate-glutamine-glutamate cycle was converted to γ-aminobutyrate by glutamate decarboxylase 2 (GAD2). Our study is not only benefit for understanding the pathological mechanisms of OSCC, but also has application prospects for the diagnosis of OSCC.

SLC2A3 promotes tumor progression through lactic acid-promoted TGF-ß signaling pathway in oral squamous cell carcinoma.

BACKGROUNDS: Oral squamous cell carcinoma is a malignant tumor of the head and neck, and its molecular mechanism remains to be explored. METHODS: By analyzing the OSCC data from the TCGA database, we found that SLC2A3 is highly expressed in OSCC patients. The expression level of SLC2A3 was verified by RT-PCR and western blotting in OSCC cell lines. The function of SLC2A3 in OSCC cell lines and Lactic acid in SLC2A3-knockdown OSCC cells were detected by colony formation, CCK8, transwell, and wound healing assays. The effect of SLC2A3 on tumor growth and metastasis was tested in vivo. GSEA and Western blot were used to analyze and validate tumor phenotypes and signaling pathway molecules. RESULTS: We analyzed OSCC datasets in the TCGA database and found that SLC2A3 had abnormally high expression and was associated with poor prognosis. We also found that oral squamous cell carcinoma cells had increased proliferation, migration, invasion, EMT phenotype, and glycolysis due to SLC2A3 overexpression. Conversely, SLC2A3 knockdown had the opposite effect. Our in vivo experiments confirmed that SLC2A3 overexpression promoted tumor growth and metastasis while knockdown inhibited it. We also observed that high SLC2A3 expression led to EMT and the activation of the TGF-ß signaling pathway, while knockdown inhibited it. Interestingly, exogenous lactic acid restored the EMT, proliferation, migration, and invasion abilities of oral cancer cells inhibited by knocking down SLC2A3. CONCLUSIONS: Our study reveals that SLC2A3 expression was up-regulated in OSCC. SLC2A3 activates the TGF-ß signaling pathway through lactic acid generated from glycolysis, thus regulating the biological behavior of OSCC.

Expression of PTGS2 along with genes regulating VEGF signalling pathway and association with high-risk factors in locally advanced oral squamous cell carcinoma.

BACKGROUND: PTGS2 encodes cyclooxygenase-2 (COX-2), which catalyses the committed step in prostaglandin synthesis. Various in vivo and in vitro data suggest that COX-2 mediates the VEGF signalling pathway. In silico analysis performed in TCGA, PanCancer Atlas for head and neck cancers, demonstrated significant expression and co-expression of PTGS2 and genes that regulate VEGF signalling. This study was designed to elucidate the expression pattern of PTGS2 and genes regulating VEGF signalling in patients with locally advanced oral squamous cell carcinoma (OSCC). METHODOLOGY: Tumour and normal tissue samples were collected from patients with locally advanced OSCC. RNA was isolated from tissue samples, followed by cDNA synthesis. The cDNA was used for gene expression analysis (RT-PCR) using target-specific primers. The results obtained were compared with the in silico gene expression of the target genes in the TCGA datasets. Co-expression analysis was performed to establish an association between PTGS2 and VEGF signalling genes. RESULTS: Tumour and normal tissue samples were collected from 24 OSCC patients. Significant upregulation of PTGS2 expression was observed. Furthermore, VEGFA, KDR, CXCR1 and CXCR2 were significantly upregulated in tumour samples compared with paired normal samples, except for VEGFB, whose expression was not statistically significant. A similar expression pattern was observed in silico, except for CXCR2 which was highly expressed in the normal samples. Co-expression analysis showed a significant positive correlation between PTGS2 and VEGF signalling genes, except for VEGFB which showed a negative correlation. CONCLUSION: PTGS2 and VEGF signalling genes are upregulated in OSCC, which has a profound impact on clinical outcomes.

Cholesterol depletion induces mesenchymal properties in oral squamous cell carcinoma cell line.

BACKGROUND: Cholesterol in cell membranes is crucial for cell signaling, adhesion, and migration. Membranes feature cholesterol-rich caveolae with caveolin proteins, playing roles in epithelial-mesenchymal transition and cancer progression. Despite elevated cholesterol levels in tumors, its precise function and the effects of its depletion in oral squamous cell carcinoma remain unclear. The aim of this study was to evaluate the influence of cholesterol depletion in oral squamous cell carcinoma cell line and epithelial-mesenchymal transition process. METHODS: Cholesterol depletion was induced on SCC-9 cells by methyl-ß-cyclodextrin and cell viability, proliferation, apoptosis, and colony formation capacities were evaluated. Gene and protein expressions were evaluated by reverse transcription polymerase chain reaction (RT-qPCR) and Western Blot, respectively, and cell sublocalization was assessed by immunofluorescence. RESULTS: Cholesterol depletion resulted in alteration of oral squamous cell carcinoma cell morphology at different concentrations of methyl-ß-cyclodextrin, as well as decreased cell proliferation and viability rates. Analysis of CAV1 transcript expression revealed increased gene expression in the treated SCC-9 during the 24 h period, at different concentrations of methyl-ß-cyclodextrin: 5 , 7.5, 10, and 15 mM, in relation to parental SCC-9. CAV1 protein expression was increased, with subsequent dose-dependent decrease. A statistically significant difference was observed in samples treated with 5 mM of methyl-ß-cyclodextrin (p = 0.02, Kruskal-Wallis test). The immunofluorescence assay showed lower cytoplasmic and membrane labeling intensity in the treated samples for CAV1. CONCLUSION: These findings indicate the modulation of cholesterol as a possible mechanism underlying the regulation of these molecules and activation of epithelial-mesenchymal transition in oral squamous cell carcinoma.

Chemerin promotes invasion of oral squamous cell carcinoma by stimulating IL-6 and TNF-α production via STAT3 activation.

AIMS: Elevated levels of adipokine chemerin have been identified in oral squamous cell carcinoma (OSCC) and found to be associated with metastasis to the cervical lymph nodes. The underlying mechanism through which chemerin affects OSCC progression is unclear. The aims of this study were firstly to determine chemerin levels and cytokine concentrations in serum from patients with OSCC and in OSCC cell cultures, and secondly to observe chemerin effects on OSCC cell cytokine secretion, migration, and invasion in vitro. METHODS: Serum samples were collected from 20 patients diagnosed with OSCC, including groups with (LN+) and without (LN-) cervical lymph node metastasis. A Luminex liquid suspension assay was used to quantify serum concentrations of 27 types of cytokines. Correlations between chemerin and cytokines (i.e., IL-6, IL-15, GM-CSF, RANTES, TNF-α, and VEGF) were analyzed. ELISAs (enzyme-linked immunosorbent assays) were used to determine concentrations of chemerin and selected cytokines in serum and in supernatants of OSCC cell cultures (SCC9 and SCC25 cell lines). OSCC cells were stimulated with human recombinant chemerin, STAT3 inhibitor, or IL-6 together with TNF-α neutralizing antibodies. Phosphorylated STAT3 protein levels were measured with western blot analysis. OSCC cell migration and invasion were investigated with Transwell assays. RESULTS: Compared to the LN- group, OSCC patients with cervical lymph node metastasis had higher levels of IL-6 (P = 0.006), IL-15 (P = 0.020), GM-CSF (P = 0.036), RANTES (P = 0.032), TNF-α (P = 0.005), VEGF (P = 0.006), and chemerin (P = 0.001). Patients' serum chemerin levels correlated directly with IL-6, GM-CSF, TNF-α, and VEGF levels in OSCC patients. Exogenous recombinant chemerin treatment promoted secretion of IL-6 and TNF-α via activation of STAT3 in OSCC cells. Chemerin induced OSCC-cell migration and invasion, and these effects were reduced by IL-6 and TNF-α neutralizing antibodies. CONCLUSION: Our findings indicate that chemerin may play a role in advancing OSCC progression by increasing production of IL-6 and TNF-α, perhaps via a mechanism involving STAT3 signaling.

Expression of ß1- and ß2-adrenergic receptors in oral squamous cell carcinoma and their association with psychological and clinical factors.

BACKGROUND: Psychological stressors have been related to tumor progression through the activation of beta-adrenergic receptors (ß-AR) in several types of cancer. PURPOSE: This study aimed to investigate the expressions of ß1- and ß2-AR and their association with psychological and clinicopathological variables in patients with oral squamous cell carcinoma. METHODS: Tumor samples from 99 patients diagnosed with OSCC were subjected to immunohistochemical reaction to detect the expression of ß1-AR and ß2-AR. Anxiety and depression symptoms were assessed using the Beck Anxiety Inventory and Beck Depression Inventory (BDI), respectively. The Brunel Mood Scale was used for measuring affective mood states. RESULTS: Univariate analyzes revealed that higher expression of ß1-AR was associated with increased alcohol consumption (p = 0.032), higher education (p = 0.042), worse sleep quality (p = 0.044) and increased levels of pain related to the primary tumor (p < 0.001). Higher expression of ß2-AR was related with regional metastasis (p = 0.014), increased levels of pain related to the primary tumor (p = 0.044), anxiety (p < 0.001) and depressive (p = 0.010) symptoms and higher mood scores of angry (p = 0.010) and fatigue (p = 0.010). Multivariate analysis identified that patients with advanced clinical stage had lower ß1-AR expression (OR=0.145, 95% CI=0.025-0.828, p = 0.003). Higher anxiety symptoms and higher mood fatigue are independent factors for increased ß2-AR expression (OR=4256, 95% CI=1439-12606, p = 0.009; OR=3816, 95% CI=1258-11,573, p = 0.018, respectively). CONCLUSION: This study reveal that anxiety, fatigue symptoms, and clinical staging are associated with tumor expression of beta-adrenergic receptors in patients with oral cancer.

Association of COX-2, TNF-α, TLR4, and IKKα With Survival of Patients With Oral Squamous Cell Carcinomas: A Systematic Review.

BACKGROUND: This review investigated the association of COX-2, TNF-α, TLR4, and IKKα with the survival of patients with oral squamous cell carcinoma (SCC). METHODS: A systematic search was conducted in the databases PUBMED, Web of Science, LILACS, EMBASE, Scopus, and Cochrane Library. The studies should assess the expression of those proteins in the tumor and survival outcomes. RESULTS: Twenty-one articles were included. The meta-analysis results leaned towards an association of COX-2 overexpression with a lower overall survival. The estimated hazard ratio was 1.51 (95% CI 0.97, 2.33), but not statistically significant (p=0.07). A low heterogeneity was observed (I2=0%). Regarding TNF-α, TLR4, and IKKα, statistically significant results for the association with survival were presented, but there was not enough data to a meta-analysis. CONCLUSION: COX-2 overexpression may be associated with a poorer prognosis in oral SCC. The insufficiency of studies about TNF-α, TLR4, and IKKα restrained their validation as predictors of prognosis.

High P16INK4A Expression, Keratinizing Features, and Surgical Margin-Free Status are Associated with Improved Survival in Patients with Oral Squamous Cell Carcinomas.

BACKGROUND: Oral squamous cell carcinoma (OSCC) continues to prevail as a highly prevalent cancer in Southeast Asia and causes a significant health burden. Stratification of patients with high risks of recurrence and mortality is important in the planning of treatment and surveillance. METHODS: Formalin-fixed paraffin-embedded (FFPE) tissues of OSCC were immuno-stained and analyzed for p16 expression. Risk factors and clinical parameters of OSCC patients were collected and compared to identify factors associated with recurrences and overall survival. RESULTS: After a median follow-up of 32 months, OSCC recurrences and mortality were observed in 82% and 78% of patients (N=60), respectively. Larger and more extensive tumors (T3 and T4) were significantly associated with both recurrences and cancer-associated mortality (OR = 3.967, 95% CI = 1.007-15.618 and OR = 5.885, 95% CI = 1.541-22.47, respectively). P16INK4A positive staining was found in 31% of tumors. Patients with p16INK4A positive staining were significantly associated with better recurrence-free and overall survivals (medians of recurrence-free survivals were 31.2 vs 19.0 months, P=0.038 and overall survivals were 39.0 vs 28.8 months,  P=0.048; respectively). Some other clinical characteristics including early stages, non-keratinizing tumors, negative cervical node, and free-surgical margin were significantly associated with better recurrence-free and overall survivals (log-rank tests, P<0.05). CONCLUSION: P16INK4A positive staining, early stages, negative cervical lymph node infiltration, and free-surgical margins are associated with better prognosis in OSCC patients. The study emphasizes the importance of early detection and the potential use of p16INK4A and other clinical variables to stratify OSCC patients with high risks of recurrence and worse overall survivals.

Development and validation of a cadaveric porcine Pseudotumor model for Oral Cancer biopsy and resection training.

OBJECTIVE: The gold standard of oral cancer (OC) treatment is diagnostic confirmation by biopsy followed by surgical treatment. However, studies have shown that dentists have difficulty performing biopsies, dental students lack knowledge about OC, and surgeons do not always maintain a safe margin during tumor resection. To address this, biopsies and resections could be trained under realistic conditions outside the patient. The aim of this study was to develop and to validate a porcine pseudotumor model of the tongue. METHODS: An interdisciplinary team reflecting various specialties involved in the oncological treatment of head and neck oncology developed a porcine pseudotumor model of the tongue in which biopsies and resections can be practiced. The refined model was validated in a final trial of 10 participants who each resected four pseudotumors on a tongue, resulting in a total of 40 resected pseudotumors. The participants (7 residents and 3 specialists) had an experience in OC treatment ranging from 0.5 to 27 years. Resection margins (minimum and maximum) were assessed macroscopically and compared beside self-assessed margins and resection time between residents and specialists. Furthermore, the model was evaluated using Likert-type questions on haptic and radiological fidelity, its usefulness as a training model, as well as its imageability using CT and ultrasound. RESULTS: The model haptically resembles OC (3.0 ± 0.5; 4-point Likert scale), can be visualized with medical imaging and macroscopically evaluated immediately after resection providing feedback. Although, participants (3.2 ± 0.4) tended to agree that they had resected the pseudotumor with an ideal safety margin (10 mm), the mean minimum resection margin was insufficient at 4.2 ± 1.2 mm (mean ± SD), comparable to reported margins in literature. Simultaneously, a maximum resection margin of 18.4 ± 6.1 mm was measured, indicating partial over-resection. Although specialists were faster at resection (p < 0.001), this had no effect on margins (p = 0.114). Overall, the model was well received by the participants, and they could see it being implemented in training (3.7 ± 0.5). CONCLUSION: The model, which is cost-effective, cryopreservable, and provides a risk-free training environment, is ideal for training in OC biopsy and resection and could be incorporated into dental, medical, or oncologic surgery curricula. Future studies should evaluate the long-term training effects using this model and its potential impact on improving patient outcomes.

Analysis of the effect of CCR7 on the microenvironment of mouse oral squamous cell carcinoma by single-cell RNA sequencing technology.

BACKGROUND: Studies have shown that CCR7, an important inflammatory factor, can promote the proliferation and metastasis of oral squamous cell carcinoma (OSCC), but its role in the tumor microenvironment (TME) remains unclear. This paper explores the role of CCR7 in the TME of OSCC. METHODS: In this work, we constructed CCR7 gene knockout mice and OSCC mouse models. Single-cell RNA sequencing (scRNA-seq) and bioinformatics were used to analyze the differences in the OSCC microenvironment between three CCR7 gene knockout mice (KO) and three wild-type mice (WT). Immunohistochemistry, immunofluorescence staining, and flow cytometry were used to analyze the expression of key genes in significantly different cell types between the KO and WT groups. An in vitro experiment was used to verify the effect of CCR7 on M2 macrophage polarization. RESULTS: In the mouse OSCC models, the tumor growth rate in the KO group was significantly lower than that in the WT group. Eight main cell types (including tumor cells, fibroblasts, macrophages, granulocytes, T cells, endothelial cells, monocytes, and B cells) were identified by Seurat analysis. The scRNA-seq results showed that the proportion of tumor cells was lower, but the proportion of inflammatory cells was significantly higher in the KO group than in the WT group. CellPhoneDB analysis results indicated a strong interaction relationship between tumor cells and macrophages, T cells, fibroblasts, and endothelial cells. Functional enrichment results indicated that the expression level of the Dusp1 gene in the KO group was generally higher than that in the WT group in various cell types. Macrophage subclustering results indicated that the proportion of M2 macrophages in the KO group was lower than that in the WT group. In vitro experimental results showed that CCR7 can promote M2 macrophage polarization, thus promoting the proliferation, invasion and migration of OSCC cells. CONCLUSIONS: CCR7 gene knockout can significantly inhibit the growth of mouse oral squamous cell carcinoma by promoting the polarization of M2 macrophages.

Expression of HIF-1α and Nestin in oral squamous cell carcinoma and its association with vasculogenic mimicry.

AIM: To evaluate and correlate the expression of HIF1-α and Nestin in tumor center and periphery of nonmetastatic, and recurrent oral squamous cell carcinoma (OSCC) and its association with vasculogenic mimicry. MATERIALS AND METHODS: About 60 histopathological proven cases of OSCC with proper tumor center and periphery were collected. Among them 25 are nonmetastatic, 25 metastatic, and 10 recurrent cases of OSCC. Immunohistochemical analysis of HIF, Nestin, and CD31/PAS (periodic acid Schiff) was done. RESULTS: Based on the extent of tumor cells stained, staining intensity and index score, expression of both HIF and Nestin was highly significant in periphery of metastatic OSCC with a P value of 0.003* and 0.001*. The total number of vessels expressed in nonmetastatic, metastatic, and recurrent OSCC was not significant but the overall expression of CD31/PAS was significant in the periphery of the tumor with a P value of 0.024*. Correlating the overall expression, HIF showed a positive relation with Nestin and CD31/PAS with a P value of 0.026* and 0.038* in nonmetastatic OSCC using Pearson's correlation coefficient analysis. CONCLUSION: Based on the above results hypoxia plays a vital role in cancer stem cells maintenance with the formation of vessel-like structures by tumor cells at an early stage of cancer development.