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 antifibrotic targets. METHODS: Mice were administered intraperitoneal thioacetamide 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: 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. CONCLUSIONS: 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. IMPACT AND IMPLICATIONS: In this study, we applied a novel technique, single-cell fixed RNA profiling, 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 humanised monoclonal IgG4 antibody against SEMA4D significantly alleviated liver fibrosis in the thioacetamide-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 HCV-sustained virologic response reduced the risk of progression to hepatocellular carcinoma.