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

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: Human primary coronary artery smooth muscle cells (SMC) (Lot# 20TL266549, Cat# CC-2583, Lonza) were cultured in completed SmGM-2 growth medium (#CC-3182, Lonza). Foam cell conditioned medium was collected from cultures of THP-1-derived foam cells as previously described, with supernatant retrieval following centrifugation at 150 g for 5 minutes and filtration through a 0.22 µm membrane filter (#GSWP02500, Millipore). On day 0, the medium swap group was plated in 6-well plates (#140675, Thermo Fisher Scientific) at a density of 1 x 105 cells per well in SmGM basal medium containing 5% FBS (BM-FBS medium). On day 4 and day 8, the medium was replaced with fresh BM-FBS medium containing 50% foam cell conditioned medium. The control group was plated at the same density in completed SmGM-2 growth medium three days before harvest. On day 12, SMCs were detached by adding 1 mL of 0.05% trypsin-EDTA (#25300-054, Gibco) per well and incubating at 37°C for 3 minutes, then neutralized with 10 mL of BM-FBS medium. Viable cells were counted using trypan blue solution (#15250061, Gibco). The cell suspension was subsequently filtered through a 100 µm nylon cell strainer (#352360, Falcon) to remove clumps, resulting in a uniform single-cell suspension for further analysis.