Background: N6-methyladenosine (m6A) RNA modification plays a critical role in various physiological and pathological conditions. However, the role of m6A modification in head and neck squamous cell carcinoma (HNSCC) remains elusive. Methods: In this study, the expression of m6A demethylases was detected by HNSCC tissue microarray. m6A-RNA immunoprecipitation (MeRIP) sequencing and RNA sequencing were used to identify downstream targets of ALKBH5. Comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) was used to explore the m6A “readers”. Tumor-infiltrating lymphocytes were analyzed in SCC7-bearing xenografts in C3H mice. Results: Here, we demonstrate the downregulation of m6A status and upregulation of two demethylases in HNSCC. Silencing the m6A demethylase alkB homolog 5, RNA demethylase (ALKBH5) suppresses tumor progression in vitro and in vivo. m6A-RNA immunoprecipitation sequencing reveals that ALKBH5 downregulates the m6A modification of DDX58 mRNA. Moreover, RIG-I, encoded by the DDX58 mRNA, reverses the protumorigenic characteristics of ALKBH5. ChIRP-MS demonstrates that HNRNPC binds to the m6A sites of DDX58 mRNA to promote its maturation. ALKBH5 overexpression inhibits RIG-I-mediated IFNα secretion through the IKKε/TBK1/IRF3 pathway. The number of tumor-infiltrating lymphocytes in C3H immunocompetent mice is reduced by ALKBH5 overexpression and restored by IFNα administration. Upregulation of AKLBH5 negatively correlates with RIG-I and IFNα expression in HNSCC patients. Conclusions: These findings unveil a novel mechanism of immune microenvironment regulation mediated by m6A modification through the ALKBH5/RIG-I/IFNα axis, providing a rationale for therapeutically targeting epitranscriptomic modulators in HNSCC.

Head and neck squamous cell carcinoma is the sixth most common cancer worldwide and has high heterogeneity and unsatisfactory outcomes. To better characterize the tumor progression trajectory, we perform single-cell RNA sequencing of normal tissue, precancerous tissue, early-stage, advanced-stage cancer tissue, lymph node, and recurrent tumors tissue samples. We identify the transcriptional development trajectory of malignant epithelial cells and a tumorigenic epithelial subcluster regulated by TFDP1. Furthermore, we find that the infiltration of POSTN⁺ fibroblasts and SPP1⁺ macrophages gradually increases with tumor progression; their interaction or interaction with malignant cells also gradually increase to shape the desmoplastic microenvironment and reprogram malignant cells to promote tumor progression. Additionally, we demonstrate that during lymph node metastasis, exhausted CD8⁺ T cells with high CXCL13 expression strongly interact with tumor cells to acquire more aggressive phenotypes of extranodal expansion. Finally, we delineate the distinct features of malignant epithelial cells in primary and recurrent tumors, providing a theoretical foundation for the precise selection of targeted therapy for tumors at different stages. In summary, the current study offers a comprehensive landscape and deep insight into epithelial and microenvironmental reprogramming throughout initiation, progression, lymph node metastasis and recurrence of head and neck squamous cell carcinoma.

Tertiary lymphoid structures (TLSs) hold the potential role in the prediction of immunotherapy response in several clinical trials. TLSs in head neck squamous cell carcinoma (HNSCC) have been investigated through IHC analysis, whereas there is no TLS gene signature to evaluate the level of TLS neogenesis. We here proposed a TLS signature containing 13 chemokines and determined TLS-hi and TLS-low groups in HNSCC samples from The Cancer Genome Atlas. TLS-hi condition signified a better overall survival. A more inflamed immune infiltrative landscape was identified in the TLS-hi tumors characterized by higher proportion of T cells, TCR/BCR activation and antigen processing. High level of TLSs has a determined role in the clinical significance of T cells. Interesting discovery was that innate lymphoid cells and cancer-associated fibroblasts were positively associated with TLS neogenesis in TME of HNSCC. Furthermore, by integrated TLSs with stromal cells and score, immune cells and score, TMB and malignant cells, we proposed a novel HNSCC TME classifications (HNSCC-TCs 1–5), unravelling the counteracted role of stromal cells and score in inflamed immune landscape, which may provide a novel stromal targeted modality in HNSCC therapy. Finally, we verified that TLS statue is an ideal predictor for immune checkpoint blockade immunotherapy. Current study indicated that the TLSs serve as a novel prognostic biomarker and predictor for immunotherapy, which may provide directions to the current investigations on immunotherapeutic strategies for HNSCC.

Immunotherapy is a regimen that is especially utilized in many advanced cancers. Tumor antigens include tumor-specific antigens and tumor-associated antigens, and they function as targets for immunotherapy, such as cancer vaccines and autologous T cells. Cancer/testis antigens (CTAs), which is a group of genes that are restrictedly expressed in malignant cells as well as some germline cells, are tumor-associated antigens. These expression characteristics make CTAs promising candidates for vaccine or T cell therapy targets. Cancer vaccines utilize cancer antigens to induce specific cellular and humoral immune responses to strengthen the body's immune system. T cell transfer therapy refers to genetically modifying T cells to express antigen-specific T cell receptors or chimeric antigen receptors, both of which can be directly activated by tumor antigens. Moreover, combined therapies are being investigated based on CTAs. Current studies have mainly focused on MAGE-A, NY-ESO-1, and IL-13Rα. And we will review clinical trials of CTA-based immunotherapies related to these three antigens. We will summarize completed trials and results and examine the future trends in immunotherapy.

Tumor microenvironment (TME) plays an important role in the tumor progression. Among TME components, cancer‐associated fibroblasts (CAFs) show multiple tumor‐promoting effects and can induce tumor immune evasion and drug‐resistance. Regulating CAFs can be a potential strategy to augment systemic anti‐tumor immunity. Here, the study observes that hydrogen treatment can alleviate intracellular reactive oxygen species of CAFs and reshape CAFs’ tumor‐promoting and immune‐suppressive phenotypes. Accordingly, a controllable and TME‐responsive hydrogen therapy based on a CaCO₃ nanoparticles‐coated magnesium system (Mg‐CaCO₃) is developed. The hydrogen therapy by Mg‐CaCO₃ can not only directly kill tumor cells, but also inhibit pro‐tumor and immune suppressive factors in CAFs, and thus augment immune activities of CD4⁺ T cells. As implanted in situ, Mg‐CaCO₃ can significantly suppress tumor growth, turn the “cold” primary tumor into “hot”, and stimulate systematic anti‐tumor immunity, which is confirmed by the bilateral tumor transplantation models of “cold tumor” (4T1 cells) and “hot tumor” (MC38 cells). This hydrogen therapy system reverses immune suppressive phenotypes of CAFs, thus providing a systematic anti‐tumor immune stimulating strategy by remodeling tumor stromal microenvironment.