Single-cell fixed RNA profiling uncovers SEMA4D and LMCD1 as therapeutic targets in a liver fibrosis model

Background & Aims Single-cell fixed RNA profiling (FLEX) is a novel technique that captures RNA expression in frozen tissues at a single-cell resolution. We applied FLEX to mouse model of liver fibrosis progression and regression to identify novel anti-fibrotic targets. Methods Mice were administered intraperitoneal thioacetamide (TAA) for 10 weeks to induce fibrosis, regression was assessed 2 weeks after cessation. The livers were fixed, dissociated, and analysed using FLEX. Molecular validation included immunoblotting, immunohistochemistry, gene silencing/overexpression, cytokine/phosphokinase arrays, and human liver samples. Results Approximately 40,000 liver cells were profiled, integrated, and annotated into 10 major cell types using lineage-specific markers. Pericentral signature restoration in hepatocytes, scar-resolving genes ( Mmp14 and Ctsl ), fenestrae restoration in liver sinusoidal endothelial cells, anti-inflammatory Kupffer cells, reduced fibrogenic cholangiocytes, and recovery-associated immune cell subsets were observed. During fibrosis, monocyte-derived macrophages secrete semaphorin-4D (SEMA4D), which binds to Plexin B2 on hepatic stellate cells (HSCs). SEMA4D + cells were upregulated in mouse fibrotic livers (n = 6, p