Hepatic macrophages play a vital role in the defense mechanisms and maintaining the internal environment stability of body, and are also major cellular components involved in liver injury and repair. Macrophages derived from hematopoietic stem cells exhibit distinct gene regulation patterns compared to resident macrophages in the liver. More than 90% of primary liver cancer occurs on the basis of cirrhosis, and the dynamic changes of macrophages in the progression of cirrhosis to hepatocellular carcinoma are worth exploring.

To analyze the transcriptomic differences of hepatic macrophages originating from diverse sources, analyze the dynamic pattern of macrophage changes in liver cirrhosis and liver cancer progression, and explore potential strategies for preventing the progression of liver cancer.

In this study, single-cell transcriptomics data of healthy, cirrhotic and hepatocellular carcinoma (HCC) tissues were obtained from the Gene Expression Omnibus (GEO) database. The healthy and liver fibrosis data were obtained from the GSE136103 dataset of the GEO database, which included samples from five healthy liver tissues and five liver cirrhosis tissues. The HCC data were obtained from the GSE149614 dataset of the GEO database, which consisted of 21 samples from ten HCC patients. Utilizing the Seurat package, a clustering analysis was conducted on the transcriptomic data derived from liver fibrosis and HCC samples to identify distinct cell types. Notably, three distinctive clusters of macrophage subtypes were identified within the fibrosis samples, from which the top 200 marker genes were extracted. Metascape online analysis software was applied to functionally analyze each subcluster-specific expressed gene. Subgroup-specific expressed genes in liver fibrosis were extracted, and the function of macrophages in cirrhosis was explored by KEGG functional analysis. The CellChat software package was utilized to analyze intercellular interactions within liver fibrosis and HCC single-cell transcriptome data, differences in macrophage communication between cirrhosis and HCC samples were compared. Additionally, normal, fibrotic and cancerous macrophages were extracted, and batch effect correction was performed using the Harmony package. Subsequently, the Monocle package was employed for pseudo-time analysis to construct the developmental trajectory of macrophages spanning from a healthy state to fibrosis and eventually to the HCC microenvironment. The limma package was utilized to find genes that are continuously up-regulated and down-regulated during the evolution of macrophages from healthy state to cirrhotic state and finally to HCC, and functional enrichment analysis was performed.

Unsupervised clustering was performed, and a total of three macrophage subclusters (designated as Mac1, Mac2, Mac3) were identified based on the expression patterns of marker genes. Mac1 originates from tissue-resident macrophages (Kupffer cells). Mac2 and Mac3 derived from blood monocytes and their numbers were significantly increased in cirrhotic tissue. Mac1 in cirrhotic tissue showed up-regulation of adaptive immune system-related functions. Mac2 and Mac3 subgroups show down-regulation of phagosome-related functions and antigen presentation functions. There were significant differences in communication between macrophages and other cell types in cirrhotic tissue and HCC tissue. Certain intercellular communication occurs only in cirrhotic macrophages, including cell communication of signaling pathways such as IFN-Ⅱ and CD40. After batch effect correction, pseudo-time series analysis was performed on macrophages from healthy liver, liver cirrhosis and HCC, the results suggest that there is a specific temporal relationship between the three groups of macrophages. This study identified 81 genes that were continuously down-regulated during the process, however, no genes were identified that were continuously up-regulated during the evolution of healthy-cirrhotic-HCC macrophage. Functional analysis suggested that the continuously down-regulated genes are functionally enriched for immune responses to bacteria.

Cirrhotic macrophages can be divided into three subgroups, of which Mac1 derived from liver-resident Kupffer cells and Mac2 and Mac3 derive from blood monocytes. Many immune-related cell communications in liver cirrhosis, such as IFN-Ⅱ and CD40 pathways, disappear in HCC. There is a continuous down-regulation of immune responses to bacteria in the evolution of healthy -cirrhotic-HCC macrophages, which may exacerbate the destructive effect of portal hypertension-induced gut microbiota displacement. For patients with liver cirrhosis, early treatment of portal hypertension-induced intestinal leakage (leaky gut) may be an important treatment strategy.