Head and neck cancers (HNC) predominantly arise in the mucosal linings of the oral cavity, oropharynx, larynx, nasopharynx, and hypopharynx. Ranking as the sixth most common cancer globally, over 90% of these cases are HNSC (Mery et al., 2016). Current statistics project approximately 54,000 new cases and an estimated 11,000 deaths in 2022 (Siegel et al., 2022). Risk factors for HNSC encompass tobacco and alcohol consumption, betel nut chewing, inadequate oral hygiene, and HPV infection (Leemans et al., 2018). As sexual behaviors evolve and HPV transmission becomes more prevalent, the incidence of HPV-related HNSC is steadily increasing (Wang M. B. et al., 2015). The therapeutic responses to chemoradiotherapy differ significantly between HPV-positive and HPV-negative HNSC patients, with HPV-negative typically facing a poorer prognosis (Shui et al., 2018). The mechanisms responsible for these differences are not yet fully understood. The primary treatment modalities for HNSC include surgery, radiotherapy, and chemotherapy, either as standalone treatments or in combination. Despite the availability of diverse therapeutic options, the overall 5-year survival rate for HNSC patients remains below 50%, posing a significant threat to human health (Park et al., 2021). Apart from cetuximab monotherapy, which targets the epidermal growth factor receptor (EGFR), no other novel targeted therapies have been approved for HNSC treatment in decades. Therefore, there is an urgent need to identify superior molecular markers that can facilitate the development of precision medicine and personalized treatment strategies for HNSC. Notably, research into HPV-related HNSC is particularly significant, offering both clinical relevance and substantial social value.

Claudins (CLDNs), derived from the Latin word ‘claudere’ meaning ‘to close’, were identified by Tsukita in 1998 as the major integral membrane proteins that formed tight junction (TJ) strands (Furuse et al., 1998). CLDN proteins are 20–34 kDa in size and the structure of the various claudins is very similar and includes four transmembrane domains (TM1-4), the intracellular N and C termini, and two extracellular loops (ECL1 and ECL2) (Suzuki et al., 2014). The CLDN protein family comprises 27 members that function as skeleton proteins within tight junctions (TJs) (Singh and Dhawan, 2015). CLDNs possess a barrier function that regulates epithelial polarity and vectorial movement of solutes and fluids in the intercellular space (Tsukita et al., 2019). For instance, the deletion of CLDN1 compromises the epidermal barrier, leading to rapid lethality within 1 day due to excessive water loss (Furuse et al., 2002). Loss of CLDN7 resulted in the aberrant regulation of small organic solute paracellular flux across the colon epithelium, leading to colonic inflammation (Tanaka et al., 2015). Additionally, CLDNs have a fence function that controls the lateral diffusion of proteins within the lipid bilayer (Otani and Furuse, 2020). Apart from the obvious role of creating scaffolds, CLDNs are involved in signal transduction within the cell. For example, a positive feedback regulation between CLDN1 and Wnt-signaling, the key regulator of colon carcinogenesis (Dhawan et al., 2005). CLDN6 is found to be upregulated and interacts with ZO-2/YAP1, activating the Hippo signaling pathway in hepatocellular carcinoma (HCC) cells (Kong et al., 2021). Studies have shown that dysregulation of CLDN-mediated barrier function and signaling is a precursor to cancer pathogenesis (Zihni et al., 2016).

CLDN10, a crucial member of this protein family, is widely expressed in various organs and tissues. In 2017, a study confirmed that mutations in the CLDN10 gene could lead to hypohidrosis and electrolyte imbalance (HELIX) (Bongers et al., 2017), which garnered significant attention. Apart from its involvement in kidney function, CLDN10 has been implicated in various tumor developments. For instance, it promotes the malignant phenotype of osteosarcoma cells through JAK1/Stat1 signaling (Zhang et al., 2019). Additionally, it enhances the metastatic ability of melanoma cells via activation of the ERK pathway (Perez et al., 2017). Furthermore, CLDN10 serves as a novel biomarker for predicting prognosis in patients with ovarian cancer (Li Z. et al., 2020) and metastatic high-grade serous carcinoma (Davidson et al., 2023). Overexpression of CLDN10 suppresses growth and metastasis in human clear cell renal cell carcinoma (Yang et al., 2022), whereas its upregulation is associated with overall survival (OS) in hepatocellular carcinoma patients (Huang et al., 2011). Although several studies have examined the interaction, regulatory mechanisms, and localized expression of claudin-10 with other closely associated molecules, the precise role and mechanisms of CLDN10 in various tumors, particularly HNSC, remain to be fully elucidated.

Therefore, the aim of this study is to utilize a range of bioinformatics approaches, in conjunction with in vitro experiments, to conduct a preliminary investigation into the expression of CLDN10 in HNSC and to explore its potential mechanisms.

HNSC is characterized by a complex anatomical structure and diverse tumor types that often have an insidious onset. The malignancy of HNSC is such that up to 30% of patients experience cancer relapse and treatment failure (Canning et al., 2019). While tumor immunotherapy has recently shown promising results, it is not universally effective due to individual differences and tumor heterogeneity (Ferris et al., 2018). Therefore, it is crucial to analyze prognostic factors and elucidate the molecular mechanisms of HNSC. Concurrently, there is a critical need to search for molecular markers that can facilitate precision medicine approaches, specifically tailored for personalized treatment strategies for HNSC.

A large body of evidence highlights that CLDNs play a role in nearly all aspects of tumor biology and all steps of tumor development, including inflammation, growth, survival, proliferation, epithelial-mesenchymal transition (EMT), metastasis, therapy resistance and cancer stem cell (CSC) renewal (Gowrikumar et al., 2019). For example, CLDN5 can be used to distinguish mesothelioma from angiosarcoma (Nakashima et al., 2020). The expression of CLDN6 is upregulated in hepatocellular carcinoma and can be used as a therapeutic target for HCC lineage plasticity (Kong et al., 2021; Lu et al., 2021). In gastric cancer, high CLDN7 expression correlates with shorter OS, while high CLDN18 expression correlates with longer OS (Yang et al., 2018). Moreover, numerous studies have demonstrated aberrant expression patterns of CLDNs in malignant tumors originating from various epithelial tissues,and they can serve as prognostic markers in lung cancer (Wang Q. et al., 2015), prostate cancer (Kind et al., 2020), cervical cancer (Akimoto et al., 2018), ovarian cancer (Martin de la Fuente et al., 2018), breast cancer (Jaaskelainen et al., 2018), endometrial cancer (Micke et al., 2014), and Gastric Cancer (Hashimoto and Oshima, 2022). These results suggest that claudins can be used as a biomarker to determine diagnosis, prognosis, and treatment. In conclusion, various members of the CLDN family, including CLDN10, have been documented to play a pivotal role in the genesis and progression of multiple malignancies. Currently, targeted therapies against CLDN18.2 have seen successful commercialization, offering hope to patients with advanced gastric cancer. Additionally, chimeric antigen receptor T (CAR-T) cell therapy directed at CLDN6 in solid tumors has achieved new milestones. As a significant member of the CLDN family, we speculate whether CLDN10 can be the next new therapeutic target for malignant tumors. Few relevant studies exist on the role of CLDN10 in malignant tumors, and research on CLDN10 in HNSC remains lacking; therefore, we conducted this study.

The immunohistochemical staining results showed that compared with normal head and neck tissues, the level of CLDN10 in HNSC tissues was significantly decreased, and the expression level was even lower in the HPV-negative subgroup. Survival analysis indicated that low expression of CLDN10 was significantly associated with shortened OS in HNSC patients, and was an independent prognostic factor in the multivariate analysis. Moreover, compared with HPV-positive patients, HPV-negative patients had a worse prognosis. Studies report that HPV-positive patients with tongue squamous cell carcinoma exhibit increased sensitivity to radiotherapy and chemotherapy, which leads to better prognosis (Gillison et al., 2008). Similarly, patients with HPV-associated oropharyngeal squamous cell carcinoma generally have a favorable prognosis (Massano et al., 2006). These reports are consistent with our research. Further clinical correlation analysis indicated that the expression level of CLDN10 was significantly correlated with clinical T stage, OS events, and HPV status. That is, patients with a heavier tumor burden and poorer prognosis showed lower CLDN10 expression. The receiver operating characteristic (ROC) curve analysis revealed that CLDN10 had good diagnostic discriminatory ability for HNSC (AUC = 0.800), suggesting its potential value as a prognostic and diagnostic biomarker. However, its clinical translation still requires clarification of the diagnostic threshold through prospective cohort studies and its evaluation together with existing markers (such as PD-L1, HPV status) to determine its practical value in the early screening or stratified management of HNSC. The expression of CLDNs in epithelial cells is a dynamic equilibrium pattern (Gunzel and Yu, 2013). Based on these findings, we hypothesize that the progression of HNSC may be accompanied by the downregulation of CLDN10. This change may disrupt the structural integrity and barrier function of tight junctions, thereby promoting local invasion and progression of the tumor. This is consistent with the previous research reports that the expression of Claudin family members is dysregulated in various epithelial-derived malignant tumors and affects prognosis. These findings imply that CLDN10 may act as an inhibitor of HNSC progression and could potentially serve as an independent prognostic marker for HNSC patients. Hence, it is essential to further clarify the role of CLDN10 in the pathogenesis of HNSC.

To explore the potential biological functions of CLDN10 in HNSC, we conducted GO and GSEA enrichment analyses. The results showed that the expression of CLDN10 was significantly correlated with B-cell-related immune pathways The rationale behind the metastatic and recurrence of HNSC is likely due to the interactions of the surrounding tissue matrix and immune cells that make up the tumor microenvironment (TME) (Chen et al., 2020). As a subgroup of cells in TME, B cells play an increasingly important role in tumor immunity. B cells have been shown to contribute to tumor immune escape in a variety of TME, including HNSC (Jeske et al., 2020), BRCA (Ragonnaud et al., 2019), Lung cancer (Tousif et al., 2021) and stomach cancer (Murakami et al., 2019). It has been found that B cells play an immune escape role through classical intercellular contact and secretion of cytokines, immunoglobulins, and small molecule metabolites (Li et al., 2022). Regulatory B cells (Breg) secretes TGF-β1, mediating immune escape from colorectal cancer (Wang et al., 2022). Immunoglobulin A (IgA) can mediate tumor immune escape (Zhong et al., 2021). In addition, it has been found that IgG has a pro-tumor effect in hepatocellular carcinoma (Wu et al., 2023). Adenosine (ADO) has been found in tumor tissue and peripheral blood of HNSC patients, and it is speculated that ADO binds to regulatory Breg to inhibit BCR signaling pathway (Lechner et al., 2019). Based on this, we further evaluated the correlation between CLDN10 expression and the level of immune cell infiltration in the tumor microenvironment of HNSC. We found that CLDN10 was only significantly positively correlated with the abundance of B cell infiltration. To verify this finding, we conducted immunohistochemical staining in an independent HPV-negative HNSC cohort. The results showed that the infiltration density of CD20⁺ B cells and CD38⁺ plasma cells in the CLDN10 high-expression group was significantly higher than that in the CLDN10 low-expression group. These multiple pieces of evidence indicate that there is a stable and reproducible association between CLDN10 and the infiltration of B cells in HPV-negative HNSC. However, it is important to note that the current research conclusions are limited to the level of correlation. Tumor-infiltrating B cells form a highly heterogeneous group, and their functions exhibit significant duality: they include effector B cells and plasma cells that exert anti-tumor effects through antigen presentation, antibody secretion, and germinal center reactions, as well as regulatory B cells that mediate immunosuppression by secreting inhibitory cytokines such as IL-10 and TGF-β. This study is unable to precisely distinguish the functionally distinct B cell subpopulations based on the available data. Therefore, the increase in B cell infiltration associated with high CLDN10 expression may represent an active anti-tumor immune response, or it may reflect the enrichment of immunosuppressive B cell subpopulations. It's reported that infiltration by intratumoural B cells was significantly increased in samples from patients with HPV-positive HNSCC compared with samples from patients with HPV-negative disease, and high densities of CD20 + B cells were associated with favourable outcomes in HNSC (Russell et al., 2013; Li Q. et al., 2020; Pretscher et al., 2009). Anthony R. Cillo et al. discovered that germinal center B cells are present across various stages of progression through germinal center reactions in HPV-positive HNSC, while B cells are found in fewer numbers and non-germinal center states in HPV-negative HNSC (Cillo et al., 2020). These reports are in line with our research results, suggesting that CLDN10 may be one of the key molecules that reshape the tumor immune microenvironment and regulate B-cell homeostasis. However, the tumor microenvironment consists of various components, such as T cells, macrophages, fibroblasts, and other subsets, which collectively promote tumor development. Only by comprehensively examining the tumor microenvironment can personalized treatment plans be provided for HNSC patients.

This study distinctly identifies CLDN10 as a differentially expressed gene in HNSC that strongly correlates with immune B cells. Additionally, immunohistochemical validation was performed on an independent cohort of 112 patients. Nevertheless, our study has certain limitations: firstly, although immunohistochemical analysis confirmed the consistency of CLDN10 with CD20 and CD38 expression in the independent cohort, it did not employ detection methods such as multiple immunofluorescence or flow cytometry that can simultaneously label both phenotypic and functional markers, which limited our precise definition of B cell subpopulations. Second, this study was a correlational study and failed to establish a causal relationship between CLDN10 and B cell function regulation. Third, the tumor microenvironment is a complex ecosystem consisting of various components such as T cells, macrophages, and fibroblasts. This study focused only on B cells and insufficiently explored the potential interactions of other components in this network. Finally, in this study cohort, the proportion of HPV-positive patients was relatively low, resulting in a much smaller sample size for the HPV-positive subgroup compared to the HPV-negative group. This distribution pattern objectively reflects the prevalence of HPV infection in non-oropharyngeal head and neck squamous cell carcinomas in the real world. However, the small sample size of the positive subgroup limits the test efficacy of statistical analysis within this subgroup. Therefore, we did not observe a significant association between CLDN10 and prognosis in the HPV-positive group, which may be partly attributed to the risk of Type II error (false negative) due to insufficient sample size, rather than a complete absence of the biological effect. In the future, it is necessary to expand the HPV-positive cohort or conduct multi-center studies to further verify the prognostic value of CLDN10 in this subgroup.

In summary, this study systematically described the expression characteristics and clinical significance of CLDN10 in head and neck squamous cell carcinoma for the first time. The main findings are as follows: CLDN10 was significantly downregulated in HNSC tissues, especially in the HPV-negative subgroup. Its low expression was significantly associated with advanced clinical pathological features and poor prognosis, and was an independent prognostic factor for HNSC, with potential diagnostic biomarker value. Bioinformatics enrichment analysis and immunohistochemical verification both indicated that the expression level of CLDN10 was significantly positively correlated with the infiltration abundance of B cells in the tumor microenvironment. This association was particularly stable in HPV-negative HNSC. However, B cell populations have high functional heterogeneity, and this study did not precisely distinguish B cell subgroups.

This study revealed the potential prognostic marker role of CLDN10 in HPV-negative HNSC and the new phenomenon of its association with B cell immunity. It provided preliminary evidence for further exploration of the interaction between tight junction proteins and immune cells in the tumor microenvironment. Whether CLDN10 affects the distribution of B cell subgroups and their anti-tumor/pro-tumor functions by regulating the function of tight junctions still needs to be verified through further functional experiments.