Spatially Resolved High-Plex Protein Profiling of Immune States in FFPE Tissue 2334779

Abstract Introduction Understanding immune cell states and signaling within intact tissue architecture remains a central challenge in immunology, particularly for formalin-fixed paraffin-embedded (FFPE) specimens that dominate clinical pathology and retrospective studies. While spatial transcriptomics has enabled high-plex mRNA profiling in FFPE tissue, comparable capabilities in protein profiling remain far behind. Methods Here, we describe a high-plex spatial protein profiling approach that integrates DNA-barcoded antibodies with a high-resolution spatial transcriptomics platform (Visium™ HD FFPE), enabling multiplexed protein detection directly within FFPE tissue sections. Tissues are stained with a panel of 200 DNA-barcoded antibodies targeting diverse proteins, including lineage markers, cytokines, phospho-epitopes, and transcription factors. Antibody-derived tags are captured, spatially barcoded, and sequenced, generating integrated spatial maps of protein expression across intact tissue architecture. Results Using human pancreatic cancer FFPE samples, we demonstrate spatially resolved detection of epithelial, immune, and innate immune compartments, including localized expression of T cell markers (CD3, CD8), cytokines (IFNG), chemokine receptors (CXCR4), and lineage-defining transcription factors (T-bet). Spatial patterns of immune activation and exclusion are observed alongside pathway-level protein signals, including Wnt/β-catenin activation, illustrating how immune contexture can be interrogated within its native microenvironment. This sequencing-based protein readout avoids optical spectral limitations, enables scalable plex expansion, and remains compatible with archived FFPE cohorts. Conclusion Together, this approach provides a robust framework for spatially mapping immune cell identity, functional states, and signaling networks at protein resolution in FFPE tissue, enabling retrospective immune profiling across disease states, therapeutic contexts, and experimental systems. Funding Source n/a Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)