Abstract 2081: Tissue-niche-based and cell-type-selective proteomics

Abstract Background: The spatial organization of cell populations within tissues is crucial for maintaining physiological function and understanding the mechanisms underlying various diseases. Proteins are the functional components that drive essential processes such as tissue homeostasis, disease progression, and therapeutic response. Spatial proteomics integrates molecular profiling with cellular location data, offering a powerful approach to decode these mechanisms. Here, we present an innovative spatial proteomics (SP) tool that combines widely adopted methodologies in biological research, enabling cell type-specific and comprehensive proteomic insights within tissue niches. Method: Our approach utilizes thick tissue macrosections (≥300 μm) to precisely select regions of interest (ROIs) based on 3D multiplexed microscopy images. We perform microscopic photobleaching on the immunofluorescence-stained cells in ROIs to create a distinguishable fluorescent label for capturing their spatial context. These labeled cells are subsequently dissociated and collected via flow sorting. Lastly, we conducted LC-MS/MS analysis for a comprehensive spatial proteomic analysis of the collected cells. Result: We confirmed that our SP technology does not significantly alter the protein expression in the samples. No significant changes in protein expression were observed in the heatmap and the volcano plot compared to the control samples. Using this technology, we investigated dendritic cell (DC) subsets in mouse spleen during inflammation. The result reveals significant proteome differences among three splenic DC subsets, categorized by their locations (“inside” or “outside” spleen T-cell zones) and control DCs. Our results align with published proteome data on inflammatory splenic DCs, demonstrating the feasibility and reproducibility of our technology. Importantly, we identified 50 proteins, including cytoskeletal proteins FSCN1 and SH3BP1, with differential expression between the “outside” and “inside” DC subsets in the inflamed spleen. These findings suggest varying migration capabilities between these subsets, providing potential therapeutic targets for inflammatory diseases. Additionally, we adapted this technology to study T cell-macrophage interaction in mouse peripheral T cell lymphoma and explore tumor cell heterogeneity in human tumors. Conclusion: Our SP technology provides extensive protein profiling tailored to cell-type-specific spatial proteomics. It also demonstrates a broad applicability across diverse tissue specimens in various diseases. These findings hold promise for identifying novel cell types and potential drug targets to enhance treatment strategies for inflammatory diseases and cancers. Citation Format: Yi-Chien Wu, Elie Abi Khalil, Aditi Upadhye, Jalees Rehman, Nermin Kady, Ryan Wilcox, Steve Lee. Tissue-niche-based and cell-type-selective proteomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2081.