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

## TL;DR

This study uses single-cell RNA sequencing to track changes in liver immune cells during chronic liver disease progression and identifies KLF2 and SPP1 as potential biomarkers for liver cancer prognosis.

## Contribution

The study introduces a stage-resolved cellular atlas of chronic liver diseases and identifies SPP1 and KLF2 as novel prognostic biomarkers for hepatocellular carcinoma.

## Key findings

- CXCL3+ and CXCL10+ macrophages are enriched in MASH and linked to inflammatory programs driving progression to HCC.
- SPP1+ macrophages are exclusive to HCC and associated with T-cell dysfunction but not direct suppression.
- KLF2 and SPP1 are validated as prognostic biomarkers for HCC using clinical datasets and RNA FISH.

## Abstract

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.

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.

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.

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.