Development of functional spatial mapping of the tumour immune microenvironment (FuncO:TiME) in advanced melanomas

Abstract Introduction Current spatial proteomic approaches quantify immune checkpoint expression but do not directly measure functional receptor-ligand (PD-1/PD-L1) interactions within the tumour-immune microenvironment (TiME). Therapeutic antibodies disrupt receptor-ligand interactions and do not target protein abundance; therefore, methods capable of resolving functional checkpoint interactions may provide biologically distinct insight beyond expression-based assays. Methods We developed FuncO:TiME by integrating functional oncology mapping (FuncOmap), histopathology and multiplex spatial immune profiling to map PD-1/PD-L1 interaction states within clinically annotated melanoma tissues collected before and after neoadjuvant immune checkpoint blockade (ICB). Results FuncOmap quantified millions of per-pixel PD-1/PD-L1 interactions and revealed marked spatial heterogeneity in checkpoint interactions that were not reflected by PD-1 or PD-L1 expression alone. Post-treatment tissues exhibited increased PD-1/PD-L1 interaction states despite no corresponding increase in expression, highlighting the divergence between functional checkpoint interactions and expression-based measurements. Integrating with spatial immune profiling further demonstrated the feasibility of contextualising regional differences in interaction states within distinct immune populations and patterns of PD-1/PD-L1 expression in melanoma TiMEs. Conclusion We establish the feasibility of FuncO:TiME spatially resolved mapping of functional checkpoint interactions in human melanoma tissues. Our findings demonstrate that receptor-ligand interactions diverge from checkpoint protein expression patterns and support further development of integrated approaches that contextualise functional checkpoint interactions within cellular features of the TiME. Such approaches may enable deeper investigation of checkpoint biology and inform the future development of functionally informed biomarkers for cancer immunotherapy.