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 the seventh most common cancer globally. Despite advancements in treatment modalities, patient prognoses remain unimproved. 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. We profiled 84 mucosal HNSCC tissue samples using next-generation ultra-high plex spatial protein and transcriptome mapping (580-proteins, 18,000 mRNA targets). We found patient survival outcomes (both disease-free and overall survival) were associated with anatomical subsite and tumour stage. We identified proteomic and transcriptomic functional T cell markers within discrete TME regions that were associated with survival. GSEA revealed elevated glycolysis and hypoxia pathways in patients with worse outcomes. Key proteomic findings (including CD34 and CD44) were independently validated using single-cell protein profiling. This study provides a systematic workflow for high-plex protein and transcriptomic profiling in mucosal HNSCC. GeoMX technology [GeoMx Immuno-Oncology Proteome Assay (IPA, 580-plex); Nanostring, USA] was applied to tumour cores collected from 68 chemotherapy and/or radiotherapy treated patients with tissues acquired from a range of sites across the mucosal head and neck regions (i.e. tongue, oral cavity, pharynx and lip). Each core were segmented into tumour and stroma regions based on PanCK staining, resulting in 136 fields of view (FoVs) across 68 cores. The following cell surface markers were used for morphology visualization: SYTO-13, CD45, and PanCk