ScRNA-seq reveals dynamic macrophage heterogeneity in chronic liver disease progression and prognostic biomarkers KLF2/SPP1 in HCC

Qi Pan¹Xinru Wang²Borui Li²Zhenzhen Cai¹Shuwen Chen¹Jiahong Hu¹Xuenan Wang²Jie Yang²An-Yuan Guo³Zhihong Zhang^1,2*

• ¹Hainan University, Haikou, China
• ²Huazhong University of Science and Technology, Wuhan, China
• ³Sichuan University, Chengdu, China

Background: Metabolic dysfunction-associated steatohepatitis (MASH)-induced chronic liver diseases (CLDs) were worldwide prevalence and incidence. The stage-resolved cellular and molecular programs remained incompletely defined. This study aimed to resolve stage-specific immune and transcriptional features across CLDs processes and to identify prognostic biomarkers. Methods: We integrated single-cell RNA sequencing datasets from healthy liver, MASH, cirrhosis and HCC to construct a stage-resolved cellular atlas. We performed cell-state scoring, diffusion pseudotime, gene regulatory network inference, and cell–cell interaction to decipher various macrophages and T cells transcriptional profiles. We established a method of gene sets enrichment score to detect prognostic markers and employed RNA fluorescence in situ hybridization (FISH) to validate macrophage subtype abundances and spatial interactions. Results: The integrated atlas revealed the heterogeneity cell-subtype composition and transcriptional features across CLD stages. In MASH, CXCL3⁺ macrophage and CXCL10⁺ macrophage were enriched and characterized by ETS2- and IRF1-driven inflammatory programs that might potentially contribute to the transition from MASH to HCC. SPP1⁺ macrophage was exclusive to HCC and might contribute to cytotoxic T-cell (Tc) dysfunction but do not directly demonstrate functional suppression or exhaustion. Subsequently, we sought to validate the robustness of these signature genes. We integrated clinical datasets from the TCGA-LIHC to validate signature genes in HCC derived from the scRNA-seq results and identify prognostic biomarker. Survival-linked analyses uncovered SPP1 and KLF2 as prognostic biomarkers. FISH confirmed stage-specific shifts in macrophage abundances and close spatial interactions between SPP1⁺ macrophages and Tc in HCC specimens. Conclusion: We provided a stage-resolved framework to delineated macrophage heterogeneity during CLDs progression and identified SPP1 and KLF2 as candidate prognostic biomarkers and potential therapeutic targets in HCC.