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

<h2>Abstract</h2><h3>Background & Aims</h3> 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. <h3>Methods</h3> 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. <h3>Results</h3> 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 (<i>Mmp14</i> and <i>Ctsl</i>), 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 <0.05). Recombinant SEMA4D induces type I collagen in HSCs, whereas humanised monoclonal IgG4 SEMA4D blockade (VX15/2503) attenuates fibrosis in vivo (n = 5, p <0.05). LIM and cysteine-rich domains 1 (LMCD1) was enriched in fibrotic HSCs and suppressed during regression. LMCD1 knockdown reduced the expression of fibrotic protein, while LMCD1 overexpression promoted the expression of fibrotic protein via AKT/mTOR signalling. LMCD1 and SEMA4D are localised in the fibrotic septa and are correlated with the fibrotic stage of MASLD (n = 34, p <0.05) and HCV (n=76, p < 0.05) in humans. <h3>Conclusions</h3> This FLEX-based single-cell atlas revealed critical transcriptional programs and cell-cell interactions, identifying SEMA4D and LMCD1 as promising therapeutic targets for liver fibrosis. <h3>Impact and Implication</h3> In this study, we applied a novel technique, single-cell fixed RNA profiling (FLEX), to profile 38,136 cells obtained from control, thioacetamide-induced liver fibrosis, and regression-phase mouse livers, generating a high-resolution atlas of liver fibrosis progression and regression. We identified the transcriptomic patterns of regressed cell subpopulations and uncovered two key therapeutic targets: the macrophage-derived factor semaphorin-4D (SEMA4D) and the hepatic stellate cell-specific transcription factor LIM and cysteine-rich domains 1 (LMCD1). Treatment with a humanized monoclonal IgG4 antibody against SEMA4D significantly alleviated liver fibrosis in the TAA-induced mouse model. SEMA4D and LMCD1 expression correlated with the metabolic dysfunction-associated steatotic liver disease-related fibrosis stage, and the attenuation of SEMA4D and LMCD1 after hepatitis C virus-sustained virologic response reduced the risk of progression to hepatocellular carcinoma.