Spatial mapping of B7-H3 mediated cellular plasticity in treatment naive pancreatic cancer

Pancreatic Ductal Adenocarcinoma (PDAC) remains one of the most lethal cancers, with only 20% of patients surviving 5 years post pancreatectomy. Despite rationale for targeting immune pathways, existing immunotherapies have provided minimal benefit to date. The immune checkpoint molecule, B7-H3, has emerged as a promising therapeutic target across solid tumours. However, its cellular origins, spatial context and clinical relevance in PDAC is poorly defined. This study aims to address this unmet need. An integrated spatial multi-omics strategy was applied across two treatment naïve PDAC cohorts. These FFPE tissue microarrays were divided into a test cohort (n=27), and a validation cohort (n=74). GeoMx regional proteomics (Bruker) was applied on section of the test cohort, while GeoMx regional whole transcriptome (Bruker), CosMx single cell 64-plex protein (Bruker) and CosMx 6K RNA (Bruker) assays were applied to serial sections of the validation cohort. Additional CosMx single cell whole transcriptome (WTx) data was generated from an Intraductal Papillary Mucinous Neoplasm (IPMN) whole section cohort (n=6). GeoMx samples were fluorescently stained with Syto13, PanCk, CD45 and alphaSMA, and masks were selected for PanCk+ve epithelium and tumour microenvironment (TME). CosMx samples were fluorescently stained for DAPI, PanCk, CD298/B2M, CD45 and CD68. Extensive clinicopathological data was available for all samples. Across regional protein and transcriptome profiling, B7-H3 expression was consistently elevated in TME and fibroblast enriched compartments compared to epithelium. Reduced epithelial expression significantly correlated with improved disease specific survival. B7-H3 ranked transcriptomic regional signatures revealed suppression of T-cell activation and antigen presentation pathways, alongside enrichment of fibro-inflammatory, myeloid and stromal signalling pathways. Single-cell spatial proteomics and RNA validated and resolved these signatures to discrete cellular niches. B7-H3 high tumours were characterised by dense clustering of fibroblasts, antigen-presenting macrophages and exhausted T-cell phenotypes, whereas B7-H3 low tumours demonstrated epithelial dominant clustering and increased spatial separation between B7-H3 enriched cell types and cytotoxic lymphocytes. WTx data further demonstrated graded B7-H3 expression variation from normal to dysplasia to IPMN cancer, highlighting spatial-temporal remodelling throughout oncogenesis. Across all spatial platforms, B7-H3 consistently mapped within fibro-inflammatory and immunosuppressive niches, features strongly associated with aggressive disease and poor prognosis. Collectively, these findings establish B7-H3 as a robust spatial biomarker in PDAC and provide compelling biological rationale for therapeutic targeting of B7-H3 in a malignancy historically resistant to immune-based treatments.