Multi-omics investigation of perineural invasion in head and neck squamous cell carcinoma: neuroimmune mechanisms and clinical implications

Background Perineural invasion (PNI) is an aggressive feature in head and neck squamous cell carcinoma (HNSCC), but its molecular basis and neuroimmune implications, including potential links to immunotherapy response, remain unclear. Methods We performed an integrative multi-omics analysis using public datasets and an independent clinical cohort. Transcriptomic, proteomic, single-cell, and spatial transcriptomic data were jointly analyzed to identify PNI-associated molecular patterns, construct prognostic signatures, and characterize immune infiltration and cell-cell communication. Regulatory elements were further explored by super-enhancer mapping and target-gene prediction. Pharmacologic inhibition experiments using the TLR2 inhibitor C29 were performed in FaDu cells for functional validation. Results PNI was associated with extracellular matrix and neuroactive signaling changes. A protein-based signature (ADIPOQ, MB, PLIN1, ADH1B) stratified survival risk. PNI-positive/high-risk tumors showed an immune-suppressed phenotype with lower predicted immunotherapy sensitivity and reduced CD8+ T-cell, B-cell, and Tfh-cell infiltration. Spatial analysis showed higher PNI scores at the invasive front, positive correlations with neural programs, and enrichment of TLR2-related signaling. TLR2 expression was associated with the PNI score, neural markers, and immune exclusion. In FaDu cells, C29 suppressed proliferation, migration, and invasion. Super-enhancer analysis identified candidate SE-target genes, including MYL4, CMYA5, and TNNT3, linked to PNI-associated biology. Conclusions PNI in HNSCC is associated with coordinated extracellular-matrix, neuroimmune, and immune-suppressive remodeling. These findings support PNI-related molecular signatures for risk stratification and identify TLR2-related signaling and SE-associated programs as candidate mechanisms for further study.