Functional Spatial Mapping of the Tumour Immune Microenvironment In Advanced Melanoma Patients

Abstract

IntroductionCurrent spatial proteomic approaches quantify immune checkpoint expression but do not directly measure functional receptor/ligand (PD-1/PD-L1) interactions within the tumor immune microenvironment (TiME). Therapeutic antibodies disrupt receptor-ligand interactions and do not target protein abundance. Methods that resolve functional checkpoint interactions provide biologically distinct insight beyond expression-based assays MethodsWe combined computation and quantitative spatial imaging, FuncO:TiME, [Functional Oncology Mapping (FuncOmap)], to map PD-1/PD-L1 interaction states to spatially defined regions of the TiME, in clinically annotated melanoma specimens, collected before and after neoadjuvant immune checkpoint blockade (ICB), ResultsFuncOmap spatially quantified millions of per-pixel PD-1/PD-L1 interactions demonstrated spatial heterogeneity in checkpoint interaction, not reflected by PD-1 expression levels alone. Post-treatment tissues exhibited increased PD-1/PD-L1 interaction states despite no corresponding increase in expression, indicating persistent or augmented functional checkpoint interaction despite therapy. Integration with spatial immune profiling further demonstrated that checkpoint interaction intensity can be contextualized within distinct immune cell populations. ConclusionWe have established the feasibility of spatially resolved functional checkpoint mapping in human melanoma tissues. We demonstrate that receptor-ligand interactions diverge from protein expression patterns. By enabling direct interrogation of functional checkpoint interaction dynamics within intact tissue architecture, FuncO:TiME advances a functional paradigm for studying immune regulation in cancer.