Targeting Modulated Vascular Smooth Muscle Cells in Atherosclerosis via FAP-Directed Immunotherapy [Visium]

Vascular smooth muscle cell (VSMCs) cell diversification drives atherosclerotic coronary artery disease (CAD). Mechanisms governing these cell state transitions remain unclear. We applied multi-omic single-cell profiling, epitope mapping, and spatial transcriptomics across 27 human coronary arteries, identifying fibroblast activation protein (FAP) as a marker of modulated VSMCs. Lineage tracing in mice indicated that FAP? cells originate from Myh11? VSMCs, and FAP PET imaging in CAD patients showed plaque uptake. FAP? cells states resided in the macrophage-rich neo-intima. Therapeutically, we developed an anti-FAP bispecific T-cell engager, which reduced plaque burden and remodeled the stromal–immune microenvironment through T-cell clonal expansion. Our study delivers a single-cell and spatial atlas of human CAD, establishes FAP as a marker of modulated VSMCs, and highlights immunotherapy for lipid-independent targets. Overall design: Visium FFPE: Fresh coronary tissue was fixed in 4% PFA overnight, washed with PBS, and embedded in FFPE blocks. FFPE blocks were sectioned onto Visium slides and underwent H&E staining before imaging. Libraries were generated using the Visium FFPE v1 kit (#1000188; #1000362; #1000364) and sequenced on a NextSeq2000 according to manufacturer's recommendations. Libraries were sequenced on a NovaSeq 6000. Xenium in situ: A custom gene panel was designed (Supplementary Table 4) based on the CITE-seq reference map and combined with the Xenium Human Multi-Tissue and Cancer Panel (#1000626, 10x Genomics). FFPE blocks and sectioned and the 10x Genomics Xenium In Situ Gene Expression with Cell Segmentation Staining User Guide protocol (10x Genomics, Protocol number CG000749) used for sample (#1000661; #1000460; #1000487. 10x Genomics,).