The development of a high-plex spatial proteomic methodology for the characterisation of the head and neck tumour microenvironment.

Head and neck squamous cell carcinoma (HNSCC) is a debilitating disease that accounts for an estimated 890,000 new cases per year. Despite advancements in chemotherapy, radiotherapy, surgery and immunotherapy, the prognosis of HNSCC has remained relatively unchanged for more than a decade. Insight into the tumour microenvironment (TME) using spatially resolved approaches, and its association with clinical endpoints, may provide useful prognostic tools and refine current treatment outcomes. Here, we profiled 84 mucosal HNSCC tissue samples using next-generation ultra-high plex spatial protein profiling (580-proteins, Immuno-Oncology Proteome Atlas (IPA)) and spatial transcriptome mapping (18,000 mRNA, Whole Transcriptome Atlas (WTA)) from Bruker Spatial Biology. Samples were collected during tumour resection, after which patients went on to receive either chemotherapy and/or radiotherapy. Each sample was subdivided into tumour and stromal regions prior to digital spatial profiling. We found that patient survival outcomes were associated with anatomical subsite and tumour stage. Independent validation of key proteomic findings (including CD34 and CD44) was performed using single-cell protein profiling (PhenoCycler-Fusion, Akoya Biosciences). Harnessing the breadth of the 580-plex protein panel and WTA, we identified region-specific proteins and RNA that associate with patient survival. These findings include the expression of immune-specific proteins, CD3e and CXCR5, differentially expressed in the tumour compartment and indicate that the location of these immune signals is important for understanding disease progression. Taken together, this study provides a systematic workflow for the discovery and validation of high-plex protein and transcriptomic profiling in mucosal HNSCC.