Design and Validation of Ultrahigh-plex Discovery Panels for Immune Checkpoint Targets

Abstract Ultrahigh-plex single-cell spatial phenotyping is revolutionizing immuno-oncology research. To enable in-depth characterization of the tumor microenvironment (TME), we have developed and validated pre-optimized antibody panels on cancer tissues to reveal key cell types and cellular architecture using the PhenoCycler™-Fusion system. We designed multiple panels that answer specific biological questions about the TME, and once multiplexed together create a comprehensive view of the structure and function of the TME. The resulting PhenoCode™ Discovery panels include markers that identify immune cell types, activation states, immune checkpoints, and mediators of proliferation. Each antibody in the panel was conjugated to an oligo barcode and stained on human FFPE tissue. Staining specificity of each conjugated antibody was assessed by comparing immunofluorescent results from PhenoCycler™ with a DAB chromogenic immunohistochemistry assay. The slide stained with a PhenoCode™ Discovery panel was imaged on a PhenoCycler™-Fusion platform, where dye-labeled oligo reporters (complementary to the barcodes) are hybridized to the barcode to visualize the antibody. Antibody concentration, exposure time, and corresponding dye were further optimized, verified, and validated as a whole module using tonsil and cancer tissues based on image analysis and intensity analysis. In this study, we showcased the rigorous process used for development and validation of PhenoCode™ Discovery panels and demonstrated how they can work together to collect comprehensive data from one tissue sample. The introduction of these pre-optimized antibody panels marks a pivotal point in the evolution of ultrahigh-plex spatial phenotyping.