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Head and neck squamous cell carcinomas (HNSCCs) are the seventh most common cancer and represent a global health burden. Immune checkpoint inhibitors (ICIs) have shown promise in treating recurrent/metastatic disease with durable benefit in ∼30% of patients. Current biomarkers for HNSCC are limited in their dynamic ability to capture tumor microenvironment (TME) features with an increasing need for deeper tissue characterization. Therefore, new biomarkers are needed to accurately stratify patients and predict responses to therapy. Here, we have optimized and applied an ultra-high plex, single-cell spatial protein analysis in HNSCC. Tissues were analyzed with a panel of 101 antibodies that targeted biomarkers related to tumor immune, metabolic and stress microenvironments. Our data uncovered a high degree of intra-tumoral heterogeneity intrinsic to HNSCC and provided unique insights into the biology of the disease. In particular, a cellular neighborhood analysis revealed the presence of six unique spatial neighborhoods enriched in functionally specialized immune subsets. In addition, functional phenotyping based on key metabolic and stress markers identified four distinct tumor regions with differential protein signatures. One region was marked by infiltration of CD8+ cytotoxic T cells and overexpression of BAK, a proapoptotic regulator, suggesting strong immune activation and stress. Another adjacent region within the same tumor had high expression of G6PD and MMP9, known drivers of tumor resistance and invasion, respectively. This dichotomy of immune activation-induced death and tumor progression in the same sample demonstrates the heterogenous niches and competing microenvironments that may underpin variable clinical responses. Our data integrate single-cell ultra-high plex spatial information with the functional state of the TME to provide insights into HNSCC biology and differential responses to ICI therapy. We believe that the approach outlined in this study will pave the way toward a new understanding of TME features associated with response and sensitivity to ICI therapies. [ABSTRACT FROM AUTHOR]