Background/Purpose: Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent inflammation, fibroblast proliferation and progressive joint damage. Understanding the heterogeneity of RA and identifying therapeutic targets remain challenging using traditional bulk transcriptomics alone, as it lacks the spatial and protein-level resolution needed to fully capture disease and tissue complexities. While scRNA-seq provides a high-resolution of dynamics of gene expression at the single-cell level, it lacks the topographical or spatial context.

Methods: In this study, we applied Laser Capture Microdissection (LCM) coupled with mass spectrometry-based proteomics to analyze histopathological niches of the RA synovium, enabling the identification of protein expression profiles of the diseased synovial lining and sublining microenvironments compared to their healthy counterparts. To enhance our understanding of cellular dynamics within the dissected regions, we further integrated the proteomic dataset with single-cell RNA sequencing (scRNA-seq) and deduced cell type enrichment including T cells, fibroblasts, NK cells, myeloid cells, B cells and synovial endothelial cells.

Results: Using the in-house spatial proteomics workflow, we generated a protein expression abundance dataset containing 3,818 quantified proteins. protein expression changes were more pronounced between disease conditions than between different histopathological compartments. Key pathogenetic RA proteins like membrane proteins (TYROBP, AOC3, SLC16A3, TCIRG1 and NCEH1), and extracellular matrix (ECM) proteins (PLOD2, OGN and LUM) showed different expression patterns in diseased synovium compartments. Integration of scRNAseq and spatial proteomics provide cellular and molecular landscapes of cell type/cluster enrichments in distinct histopathological regions, highlighting fibroblasts enrichment in RA fibrous sublining regions and immune cells enrichment in RA immune sublining regions.

Conclusion: By combining high-resolution spatial proteomics and transcriptomic analyses, we provide novel insights into the molecular mechanisms driving RA and highlight potential protein targets for therapeutic intervention. This integrative approach offers a more comprehensive view of RA synovial pathology and mitigates the limitations of traditional bulk transcriptomics in target discovery.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/integrative-spatial-proteomics-and-single-cell-rna-sequencing-unveil-molecular-complexity-in-rheumatoid-arthritis-for-novel-therapeutic-targeting/