Integrated Single-Cell and Spatial Multi-Omics Reveal S100A12+ Neutrophil Reprogramming Across Oral Inflammatory States and Immunotherapy-Associated Perturbation.

Oral squamous cell carcinoma (OSCC) develops through a continuum of tissue states from chronic inflammation and potentially malignant lesions to invasive cancer, yet the dynamic remodeling of the oral immune microenvironment and its relevance to immunotherapy-associated inflammation remain poorly understood. Here, we integrated single-cell RNA sequencing, spatial transcriptomics, murine immunotherapy datasets, and experimental validation to characterize immune and stromal alterations across healthy mucosa, periodontitis, leukoplakia, and OSCC. This integrated analysis revealed distinct oral immune microenvironment states and identified condition-associated neutrophil programs, including an S100A12-enriched inflammatory state, an S100A14-associated metabolic state in leukoplakia, and tumor-associated neutrophil states in OSCC. Spatial analyses and cell-cell communication modeling further nominated immune-stromal interaction networks involving S100A12+ neutrophils and fibroblast-associated inflammatory signaling. A machine-learning framework identified a nine-gene Neu_S100A12-associated signature that discriminated periodontitis-associated inflammatory states across two retrospective cohorts, with seven concordantly upregulated genes prioritized for biological validation. Multiplex immunofluorescence and an IFN-γ-primed OSCC inflammatory model provided experimental support for a TNF-associated inflammatory phenotype linked to the S100A12 program. Together, these findings establish a cross-sectional multi-omics framework for defining oral immune states and identify S100A12-associated neutrophil programs as candidate mediators of oral inflammation and immunotherapy-related immune perturbation, providing a foundation for future prospective and mechanistic studies.