ABSTRACT Background The aim of this study is to establish a novel and reproducible orthotopic mouse model, aiming to facilitate research related to laryngeal cancer. Methods Implantation of mouse squamous cell carcinoma cell line, Meer‐LUC, into the larynx of C57BL/6 mice ( n = 16). Tumor growth and survival were observed using magnetic resonance imaging and optical in vivo three‐dimensional imaging. Laryngeal and cervical lymph node specimens were obtained for HE staining and evaluation. Single‐cell sequencing was performed on laryngeal cancer patients, orthotopic mouse models, and subcutaneous tumor models to investigate the immune microenvironment of laryngeal cancer. Results We successfully established 15 cases of in situ laryngeal cancer mouse models. Tumors maintained steady growth at each observation time point, and histopathological evaluation confirmed the successful construction of the model. Single‐cell sequencing results suggest that the orthotopic model better simulates the immune microenvironment alterations of real laryngeal cancer. Conclusions We have developed a novel and reproducible orthotopic mouse model, which more faithfully mimics the immune system response of the human body and better simulates the biological evolution process of tumors. This model provides an essential animal research framework for investigating the molecular mechanisms underlying the occurrence and development of laryngeal cancer.

Cathepsin L (CTSL) is an important oncogene. However, its mechanism of action in laryngeal cancer is still unclear. This study aims to explore the role of CTSL in laryngeal cancer and its clinical significance. Conducting bioinformatics analysis utilising the Cancer Genome Atlas (TCGA) database and the Gene Expression Omnibus (GEO) database. Performing CCK8 analysis, Western blotting, qRT-PCR, wound healing assay and transwell invasion assay. Additionally, conducting immunoprecipitation experiments and immunohistochemical staining to investigate the impact of CTSL on cell proliferation, autophagy and related signalling pathways. We observed a significant upregulation of CTSL in laryngeal cancer tissues, and its elevated levels are indicative of poor prognosis in laryngeal cancer patients. The proliferation of laryngeal cancer cells relies on the expression of CTSL, with overexpression of this gene enhancing the proliferative capacity of these cells. Concurrently, CTSL is closely associated with the autophagic levels in laryngeal cancer cells. During the autophagic process mediated by CTSL, the IL6-JAK-STAT3 signalling pathway is activated, suggesting that CTSL promotes autophagy through the IL6-JAK-STAT3 pathway. Considering the correlation between CTSL and autophagy, we developed and validated a multi-gene signature. The risk score derived from this signature demonstrates significant potential in predicting various aspects. We found that CTSL upregulates autophagy in laryngeal cancer cells by activating the IL6-JAK-STAT3 signalling pathway. By taking into account the autophagy-regulating role of CTSL, the clinical predictive ability of CTSL in HNSC can be enhanced.

Rationale: LAMP2A is a key translocase in chaperone-mediated autophagy (CMA), and the STING/TBK1 axis is crucial in antitumor immunity. This study explored the complex mechanisms by which CMA regulates the STING/TBK1 degradation and whether targeting LAMP2A could enhance the efficacy of PD-1 monoclonal antibodies. Methods: The expression of STING and TBK1 was detected after treatment with various inhibitors of protein degradation pathways. Confocal microscopy was used to detect the localization of STING and TBK1 in lysosomes. R software was used to analyze LAMP2A expression and prognosis. The biological function of LAMP2A was examined by in vitro and in vivo experiments. Results: Through in vitro and in vivo experiments and a review of clinical specimens, we identified STING/TBK1 as a novel substrate of CMA. Downregulation of LAMP2A enhanced IFNβ production and cellular antiviral response by inhibiting CMA-mediated degradation of STING and TBK1. Based on these observations, further in vivo experiments confirmed that the LAMP2A loss combined with PD-1 monoclonal antibodies significantly stimulated the activation of infiltrating CD8+ T cells, thereby inhibiting tumor growth. Also, non-responder head and neck squamous cell carcinoma (HNSCC) patients undergoing neoadjuvant immuno-chemotherapy had higher levels of LAMP2A and lower levels of PD-L1 expression in their tumor tissues. Conclusions: Our study has revealed a prospective combination therapy, in which diclofenac functioning as a LAMP2A inhibitor, enhances the anti-tumor efficacy of PD-1 monoclonal antibody by inhibiting the degradation of STING and TBK1.

Single-molecule Real-time Isoform Sequencing (Iso-seq) of transcriptomes by PacBio can generate very long and accurate reads, thus providing an ideal platform for full-length transcriptome analysis.A number of computational tools have been developed for long-read sequencing data. However, integrated computational frameworks for analyzing Iso-seq data are still lacking. We present a Toolkit for Analyzing full-length GEne Transcripts (TAGET) for Iso-seq. Starting from polished high-quality transcripts (circular consensus sequences or CCSs), TAGET first aligns transcripts to the reference genome by integrating alignment results from long and short reads and further improves splice site predictions using a Convolutional Neural Network (CNN). TAGET then annotates transcripts by comparing with reference isoform databases and classifies transcripts into seven classes. Finally, TAGET estimates gene or isoform expressions and performs differential expression gene (DEG) and differential isoform usage (DIU) analysis. We evaluate the performance of TAGET using a public Iso-seq dataset and newly sequenced Iso-seq datasets from tumor patients. TAGET gives significantly more precise novel splice site prediction and enables more accurate novel isoform and gene fusion discoveries, as validated by experimental validations and comparisons with RNA-seq data. We identify and experimentally validate a differential isoform usage gene ECM1, and further show that its isoform ECM1b may be a tumor-suppressor in laryngocarcinoma. Our results demonstrate that TAGET provides a valuable computational toolkit and can be applied to many full-length transcriptome studies.

Rationale Metastatic head and neck squamous cell carcinoma (mHNSCC) poses a significant threat to patient survival. Previous studies have identified cathepsin L (CTSL) as a key driver of tumourigenesis, metastasis and chemoresistance. However, the regulatory mechanisms underlying CTSL expression remain poorly understood.Methods A specific deubiquitinase responsible for CTSL expression was identified through treatment with broad-spectrum deubiquitinase inhibitors and mass spectrometry analysis. The colocalization of CTSL and USP20 in the cytoplasm was examined using confocal microscopy. The effects of CTSL or USP20 depletion on tumour biological behaviour were evaluated through various in vitro and in vivo assays.Results We identified USP20 as a specific deubiquitinase of CTSL. USP20 mediates the deubiquitination and stabilization of CTSL, thereby promoting epithelial-to-mesenchymal transition and cancer stem cell renewal, ultimately enhancing metastatic potential and chemoresistance. Notably, USP20 competes with STUB1 for CTSL binding, further driving the malignant phenotype of HNSCC. Analysis of clinical samples revealed that both CTSL and USP20 are highly expressed in metastatic HNSCC tissues, with a positive correlation between their expression levels.Conclusions Our study reveals a novel mechanism in which USP20 competitively interacts with STUB1 to stabilize CTSL and promote tumour progression. These findings provide preclinical evidence supporting USP20 as a potential therapeutic target for overcoming metastasis and chemotherapy resistance in HNSCC.Key points USP20 deubiquitinates and stabilizes CTSL. STUB1 promotes CTSL ubiquitination and degradation. USP20 competitively binds to CTSL in competition with STUB1. Targeting USP20 sensitizes cancer cells to cisplatin or paclitaxel.

Head and neck carcinomas are the sixth most common cancers worldwide, with laryngeal squamous cell carcinoma (LSCC) being the second most prevalent subtype. Improving survival outcomes in LSCC patients remains a critical clinical challenge. This retrospective study aimed to develop a nomogram model integrating tumor-infiltrating lymphocytes (TILs) and clinicopathological characteristics to predict the prognosis of LSCC patients. The nomogram model was constructed using Cox and Lasso regression analyses and was subsequently evaluated through receiver operating characteristic (ROC) curves, calibration curves, and decision curve analysis (DCA). Data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were utilized for model validation and to further elucidate the role of TILs and immune responses in LSCC. This study cohort included LSCC patients diagnosed by pathological examination between 2011 and 2014 at Xiangya Hospital and Harbin Medical University Cancer Hospital. A total of 412 patients were assigned to the training cohort and 140 patients to the test cohort for validation. The final nomogram model integrated TNM stage, TILs, PLR, BMI, age, differentiation and NLR. The area under the curve (AUC) was 0.745, indicating strong calibration and clinical utility. Kaplan-Meier survival curves demonstrated significant discrimination. TILs were positively correlated with immune cell abundance and the expression of immune-related genes. In conclusion, the nomogram model based on TILs and clinicopathological features effectively predicts the prognosis of LSCC patients.

Objective:The aim of this study is to develop a multimodal MRI radiomics-based model for predicting long-term overall survival in hypopharyngeal cancer patients undergoing definitive radiotherapy. Methods:We enrolled 207 hypopharyngeal cancer patients who underwent definitive radiotherapy and had 5-year overall survival outcomes from two major cancer centers in China. Pretreatment MRI images and clinical features were collected. Regions of interest (ROIs) for primary tumors and lymph node metastases (LNM) were delineated on T2 and contrast-enhanced T1 (CE-T1) sequences. Principal component analysis (PCA), support vector machine (SVM), and 5-fold cross-validation were used to develop and evaluate the models. Results:Multivariate Cox regression analysis identified age under 50 years, advanced T stage, and N stage as risk factors for overall survival. Predictive models based solely on clinical features (Model A), single radiomics features (Model B), and their combination (Model C) performed poorly, with mean AUC values in the validation set of 0.663, 0.772, and 0.779, respectively. The addition of multimodal LNM and CE-T1 radiomics features significantly improved prediction accuracy (Models D and E), with AUC values of 0.831 and 0.837 in the validation set. Conclusion:We developed a well-discriminating overall survival prediction model based on multimodal MRI radiomics, applicable to patients receiving definitive radiotherapy, which may contribute to personalized treatment strategies.

Anillin (ANLN) is a highly conserved protein involved in cytokinesis and cytoskeletal remodeling. This study investigates the role of ANLN in head and neck squamous cell carcinoma (HNSCC) progression and its impact on the tumor immune microenvironment, with a focus on the combination of ANLN silencing and anti-programmed cell death protein 1 (PD-1) therapy. Through in vitro and in vivo experiments, along with clinical specimen analysis, we discovered that silencing ANLN not only inhibits the malignant progression of HNSCC but also reduces the activation of the extracellular signal-regulated kinase-mitogen-activated protein kinase (ERK-MAPK) signaling pathway and decreases programmed death ligand-1 (PD-L1) expression. Integrating ANLN silencing with anti-PD-1 monoclonal antibody treatment significantly enhances the activation of infiltrating CD8+ T cells, leading to marked tumor growth suppression. Our findings highlight the potential of ANLN as a therapeutic target in HNSCC, providing a foundation for developing innovative and effective combined treatment strategies.

Head and neck cancer is a malignant tumour with a high mortality rate characterized by late diagnosis, high recurrence and metastasis rates, and poor prognosis. Head and neck squamous cell carcinoma (HNSCC) is the most common type of head and neck cancer. Various factors are involved in the occurrence and development of HNSCC, including external inflammatory stimuli and oncogenic viral infections. In recent years, studies on the regulation of cell death have provided new insights into the biology and therapeutic response of HNSCC, such as apoptosis, necroptosis, pyroptosis, autophagy, ferroptosis, and recently the newly discovered cuproptosis. We explored how various cell deaths act as a unique defence mechanism against cancer emergence and how they can be exploited to inhibit tumorigenesis and progression, thus introducing regulatory cell death (RCD) as a novel strategy for tumour therapy. In contrast to accidental cell death, RCD is controlled by specific signal transduction pathways, including TP53 signalling, KRAS signalling, NOTCH signalling, hypoxia signalling, and metabolic reprogramming. In this review, we describe the molecular mechanisms of nonapoptotic RCD and its relationship to HNSCC and discuss the crosstalk between relevant signalling pathways in HNSCC cells. We also highlight novel approaches to tumour elimination through RCD.

It has been recognized that depth of invasion (DOI) is closely associated with patient survival for most types of cancer. The purpose of this study was to determine the DOI optimal cutoff value and its prognostic value in laryngeal squamous carcinoma (LSCC). Most importantly, we evaluated the prognostic performance of five candidate modified T-classification models in patients with LSCC. LSCC patients from Harbin Medical University Cancer Hospital and Chinese Academy of Medical Sciences Cancer Hospital were divided into training group (n = 412) and validation group (n = 147). The primary outcomes were overall survival (OS) and relapse-free survival (RFS), and the effect of DOI on prognosis was analyzed using a multivariable regression model. We identified the optimal model based on its simplicity, goodness of fit and Harrell’s consistency index. Further independent testing was performed on the external validation queue. The nomograms was constructed to predict an individual’s OS rate at one, three, and five years. In multivariate analysis, we found significant associations between DOI and OS (Depth of Medium-risk invasion HR, 2.631; P < .001. Depth of high-risk invasion: HR, 5.287; P < .001) and RFS (Depth of high-risk invasion: HR, 1.937; P = .016). Model 4 outperformed the American Joint Committee on Cancer (AJCC) staging system based on a low Akaike information criterion score, improvement in the concordance index, and Kaplan-Meier curves. Inclusion of DOI in the current AJCC staging system can improve the differentiation of T classification in LSCC patients.

Single-molecule Real-time Isoform Sequencing (Iso-seq) of transcriptomes by PacBio can generate very long and accurate reads, thus providing an ideal platform for full-length transcriptome analysis. We present an integrated computational toolkit named TAGET for Iso-seq full-length transcript data analyses, including transcript alignment, annotation, gene fusion detection, and quantification analyses such as differential expression gene analysis and differential isoform usage analysis. We evaluate the performance of TAGET using a public Iso-seq dataset and newly sequenced Iso-seq datasets from tumor patients. TAGET gives significantly more precise novel splice site prediction and enables more accurate novel isoform and gene fusion discoveries, as validated by experimental validations and comparisons with RNA-seq data. We identify and experimentally validate a differential isoform usage gene ECM1, and further show that its isoform ECM1b may be a tumor-suppressor in laryngocarcinoma. Our results demonstrate that TAGET provides a valuable computational toolkit and can be applied to many full-length transcriptome studies.

Objective Preoperative evaluation of lateral lymph node metastasis (LLNM) in patients with papillary thyroid carcinoma (PTC) has been one of the major clinical challenges. This study aims to develop and validate iodine nutrition-related nomogram models to predict lateral cervical lymph node metastasis in patients with PTC. Methods This is a retrospective study. Urinary iodine concentration (UIC) and serum iodine concentration (SIC) were measured in 187 LLNM patients and 289 non-LLNM (NLLNM) patients. All patients were randomized 3:1 into the training cohort (n = 355) and the validation cohort (n = 121). Using logistic regression analysis, we analyzed the influence of iodine nutrition-related factors and clinicopathological characteristics on LLNM in PTC patients. Lasso regression method was used to screen risk factors and construct a nomogram for predicting LLNM. The receiver operating characteristic curve (ROC curve), calibration curve, and decision curve analysis (DCA) of the nomogram models were carried out for the training and validation cohorts. Results Gender, SIC, smoking history, drinking history, family history of PTC, multifocality, bilateral or unilateral tumors, TSH, Tg, and tumor size were included in the nomogram model predicting LLNM, with an area under the curve (AUC) of .795. The nomogram model showed good calibration and clinical benefit in both the training and validation cohorts. Conclusion The nomogram model based on iodine nutrition and other clinicopathological features is effective for predicting the lateral lymph node metastasis in PTC patients.

Abstract Background Cuproptosis is a novel type of programmed cell death which plays an important role in the development and progression of cancer. However, there is a limited amount of research on cuproptosis-associated long non-coding RNAs (lncRNAs) in head and neck squamous cell carcinomas (HNSCCs). This study aimed to investigate the predictive value of cuproptosis-related lncRNA signature for HNSCC prognosis. Method Transcriptomic and clinical data of HNSCC patients were obtained from the Cancer Genome Atlas (TCGA). We established a cuproptosis-related lncRNA signature and then constructed a hybrid nomogram based on risk scores and clinical factors. We also performed differential expression genes (DEGs) function, immune cells infiltration, immune checkpoint analysis based on cuproptosis-associated lncRNA signature. Results A signature of 27 cuproptosis-related lncRNAs was performed and the prognosis of patients at high risk is worse compared with patients at low risk based on above signature. A nomogram which integrated risk scores and clinical features also showed favorable predictive power. Furthermore, DEGs in high or low risk group were mainly enriched in immune-related pathways. Anti-tumor immune cells and immune checkpoints were mainly enriched in low risk group compared with high risk group. Conclusion Cuproptosis-related lncRNAs could be regarded as independent indicators for HNSCC prognosis which might be effective targets for HNSCC therapy.

医学人才培养是医疗卫生健康事业的根基,优秀的肿瘤外科医生需要扎实的解剖学基础、较强的实践操作能力、灵活的临床思辨能力和良好的沟通协调能力.为满足肿瘤外科新术式、新技术等发展,需要进一步从临床实际出发,围绕解剖学的学习,培养学生的多学科综合临床思维.

头颈部肿瘤是常见的恶性肿瘤之一,由于颈部解剖结构复杂,解剖学对于头颈外科研究生的培养显得尤为重要.传统的解剖学授课方式枯燥无味,无法帮助学生形成立体化的三维解剖结构,也无法清晰地了解颈部重要的器官、血管和神经的毗邻关系,更无法从单纯的断面解剖理解到复杂的立体解剖.通过超声检查结合PBL(problem-based learning,PBL)、CBL(case-based learning,CBL)教学法[1],可以利用超声技术,了解病变与重要的器官、血管和神经的毗邻关系,建立颈部三维立体化的解剖结构概念[2].本研究以我院肿瘤外科专业型硕士研究生为研究对象,探索超声检查结合PBL、CBL教学法在解剖学教学改革中的应用价值及推广价值.

The main role of platelets is to control bleeding and repair vascular damage via thrombosis. They have also been implicated to promote tumor metastasis through platelet-tumor cell interactions. Platelet-tumor cell interactions promote tumor cell survival and dissemination in blood circulation. Tumor cells are known to induce platelet activation and alter platelet RNA profiles. Liquid biopsies based on tumor-educated platelet biomarkers can detect tumors and correlate with prognosis, personalized therapy, treatment monitoring, and recurrence prediction. Platelet-based strategies for cancer prevention and tumor-targeted therapy include developing drugs that target platelet receptors, interfere with the release of platelet particles, inhibit platelet-specific enzymes, and utilize platelet-derived “nano-platelets” as a targeted drug delivery platform for tumor therapy. This review elaborates on platelet-tumor cell interactions and the molecular mechanisms and discusses future research directions for platelet-based liquid biopsy techniques and platelet-targeted anti-tumor strategies.

Objective: Laryngeal squamous cell carcinoma (LSCC) belongs to head and neck squamous cell carcinoma (HNSCC), with dismal prognosis. Here, this study aims to disclose the role of LINC-PINT in cancer development, which may contribute to improving the clinical outcomes of LSCC treatment. Methods: LINC-PINT expression in LSCC tissues and in TU-177 and Hep-2 cells was quantified, and subsequently, the association between LINC-PINT and LSCC malignancies was analyzed. pcDNA3.1-LINC-PINT or pcDNA3.1-EZH2 was introduced into Hep-2 and TU-177 cells. qRT-PCR and Western blot analyses examined the levels of proteins related to the AKT/mTOR pathway and their phosphorylated proteins in Hep-2 and TU-177 cells. The viability as well as migration and invasion abilities of Hep-2 and TU-177 cells were determined. Also, the distribution of LINC-PINT in Hep-2 cells was investigated as well as the interplay between LINC-PINT and EZH2. The downstream genes that might interact with EZH2 were screened. Results: LINC-PINT expression was inhibited in LSCC tissues and in Hep-2 and TU-177 cells, whose downregulation was associated with unsatisfactory prognosis. LINC-PINT overexpression suppressed the proliferative, migratory and invasive capacities of Hep-2 and TU-177 cells. LINC-PINT, mainly expressing in nuclei, could enrich EZH2 to silence ZEB1. In Hep-2 and TU-177 cells, the inhibition of LINC-PINT or overexpression of ZEB1 could enhance cell proliferation, migration and invasion. The phosphorylated levels of proteins related to the AKT/mTOR pathway were declined in cells with LINC-PINT overexpression, and the levels of these phosphorylated proteins were increased in cells with LINC-PINT inhibition. Conclusion: LINC-PINT enriches EZH2 to silence ZEB1 and thus inhibits the proliferative, migratory, and invasive capacities of Hep-2 and TU-177 cells. In addition, LINC-PINT might exert its biological function through the AKT/mTOR pathway.

BACKGROUND:In the past few years, immunotherapy has changed the way we treat solid tumors. People pay more and more attention to the immune microenvironment of laryngeal squamous cell carcinoma (LSCC). In this study, our immunotherapy research took advantage of the clinical database and focused our in-depth analysis on the tumor microenvironment (TME).METHODS:This study evaluated the relationship between the clinical outcome and the local tissue and overall immune status in 412 patients with primary LSCC. We constructed and validated a risk model that could predict prognosis, assess immune status, identify high-risk patients, and develop personalized treatment plans through bioinformatics. In addition, through immunohistochemical analysis, we verified the differential expression of CTSL and KDM5D genes with the largest weight coefficients in the model in LSCC tissues and their influence on the prognosis and tumor-infiltrating lymphocytes (TILs).RESULTS:We found that interstitial tumor-infiltrating lymphocytes, tumor parenchymal-infiltrating lymphocyte volume, tumor infiltrates lymphocytes of frontier invasion, and the platelet-to-lymphocyte ratio (PLR) were independent factors affecting the prognosis of patients with LSCC. A novel risk model can guide clinicians to accurately predict prognosis, identify high-risk patients, and formulate personalized treatment plans. The differential expression of genes such as CTSL and KDM5D has a significant correlation with the TILs of LSCC and the prognosis of patients.CONCLUSION:Local and systemic inflammatory markers in patients with laryngeal squamous cell carcinoma are reliable prognostic factors. The risk model and CTSL, KDM5D gene have important potential research value.

Long noncoding RNAs (lncRNAs) have multiple functions with regard to the cancer immunity response and the tumor microenvironment. The prognosis of head and neck squamous cell carcinoma (HNSCC) is still poor currently, and it may be effective to predict the clinical outcome and immunotherapeutic response of HNSCC by immunogenic analysis. Therefore, by using univariate COX analysis and Lasso Cox regression, we identified a signature consisting of 21 immune-related lncRNA pairs (IRLPs) that predicted clinical outcome and Immunotherapeutic response in HNSCC. Specifically, it was associated with immune cell infiltration (i.e., T cells CD4 memory resting, CD8 T cells, macrophages M0, M2, and NK cells), and more importantly this signature was strongly related with immune checkpoint inhibitors (ICIs) [such as PDCD1 (r = -0.35, P < 0.001), CTLA4 (r = -0.26, P < 0.001), LAG3 (r = -0.22, P < 0.001) and HAVCR2 (r = -0.2, P < 0.001)] and immunotherapy-related biomarkers (MMR and HLA). The present study highlighted the value of the 21 IRLPs signature as a predictor of prognosis and immunotherapeutic response in HNSCC.

Oropharyngeal squamous cell carcinoma (OPSCC) is one of most common cancers that often brings lots of inconvenience to the patient in swallowing and phonation even after the operation. Moreover, OPSCC is typically as nodal metastases and high recurrence rate due to the high-risk human papillomavirus (HPV) infection for 90% of patients. Obviously, completely curing OPSCC requires simultaneous removal of solid tumor and related pathogenic virus, which is very indispensable but never be realized by any kind of clinical therapy up to now. In this work, we selected the ZrC nanoparticles as difunctional photoactive substance for synchronous generation of hyperthermia and reactive oxygen species (ROS) under NIR excitation. The resultant synergistic photothermal and photodynamic treatment outcome contributed to an excellent anti-tumor effect. The phototherapy of this work was found not only to be able to damage cancer cells directly, but also could trigger the host immunity for further tumor removal and desirable HPV inactivation. An immunologic mechanism of this work was reasonable proposed by monitoring level of shock protein (HSP), calreticulin (CRT), T lymphocytes and dendritic cells (DCs) and immune check point of B7H3, B7H4 and PD-L1 post phototherapy. It was found that tumor-associated antigens of CRT (?eat-me? signal), HSPs and cell debris were released as cancer cell damage, and then the adaptive immune system and the congenital immunity were triggered to activate DCs maturity, antigen presentation to T cells, proliferation of CD4+ and CD8+ T cells, recruiting macrophages and NK cells and so forth immune responses. Being the first example of using phototherapy for virus-related cancer study, this work opens the door for photo-immunotherapy.