Differential metabolites facilitate metabolic dysfunction-associated fatty liver disease malignancy via immune evasion and M2-polarized macrophages

Figure 1 Dynamic pathology and differentially expressed genes in the liver during metabolic dysfunction-associated fatty liver disease malignancy. A: Livers of Sprague-Dawley rats fed a high-fat diet plus 2-fluorenylacetamide; B: Liver grouping according to hematoxylin and eosin staining; C: Lipid analysis via Oil Red O staining; D: Enrichment analysis of differentially expressed genes associated with metabolic dysfunction-associated fatty liver disease malignancy; E: Lipid metabolism differentially expressed genes network. NC: Normal control; MAFLD: Metabolic dysfunction-associated fatty liver disease; MASH: Metabolic dysfunction-associated steatohepatitis; LC: Liver cirrhosis; HCC: Hepatocellular carcinoma; H&E: Hematoxylin and eosin.

Figure 2 Dynamic alterations in hepatic differential metabolites during metabolic dysfunction-associated fatty liver disease malignancy. A and B: Differential metabolite (DM) volcano maps (P value vs fold change). Metabolic dysfunction-associated fatty liver disease (MAFLD) vs normal control (NC) (A). Hepatocellular carcinoma (HCC) vs metabolic dysfunction-associated steatohepatitis (MASH) (B); C and D: Principal least squares-discriminant analysis of DM. MAFLD vs NC (C). HCC vs MASH (D); E and F: S-shaped orthogonal principal least squares-discriminant analysis plot analysis of DM. MAFLD vs NC (E) and HCC vs MASH (F). NC: Normal control; MAFLD: Metabolic dysfunction-associated fatty liver disease; FC: Fold change; PLS-DA: Principal least squares-discriminant analysis; Splot-OPLS-DA: S-shaped orthogonal principal least squares-discriminant analysis plot analysis.

Figure 3 Correlation analysis of differential metabolites during metabolic dysfunction-associated fatty liver disease malignancy. A: Hierarchical clustering of differential metabolites significantly different between the hepatocellular carcinoma (n = 10) and metabolic dysfunction-associated steatohepatitis (n = 10) groups. Horizontal, samples; vertical, differential metabolite (DM). Blue, low-abundance DM; red, high-abundance DM; B: Differential metabolites (hepatocellular carcinoma vs metabolic dysfunction-associated steatohepatitis) were analyzed with Pearson’s correlation coefficient. Blue, negative correlation; red, positive correlation. rHCC: Rat hepatocellular carcinoma; H-deg: Hepatocyte degeneration.

Figure 4 Hepatic cell clustering under lipid aggregation. A: Cell clustering of hepatic cells in healthy liver; B: Number of hepatic cell in each subpopulation; C: Dynamic liver cell subpopulations during metabolic dysfunction-associated fatty liver disease malignancy. Identifiable cells: Hepatocytes (0, 20, 23, 24, 27); B cells (2, 25, 28); endothelial cells (3, 17); natural killer cells (6, 10, 13); T cells/monocytes/macrophages (7, 9, 2, 15); dendritic cells (8); neutrophils (11); bile duct cells (19); red blood cells (21); plasma cells (26); hepatic stellate cells (29); dendritic cells (33). Unidentified cells (1, 4, 5, 14, 16, 18, 22, 30, 31, 32); D: The 16 most abundant subpopulations of immune cells in the healthy liver: Hepatocytes (0); macrophages (1, 4, 5, 14, 16); B cells (2); endothelial cells (3); natural killer cells (6, 10, 13); T cells/monocytes (7, 9, 12, 15); dendritic cells (8); and neutrophils (11); E: The percentage of immune/inflammatory cells during metabolic dysfunction-associated fatty liver disease malignancy progression. NC: Normal control; MAFLD: Metabolic dysfunction-associated fatty liver disease; MASH: Metabolic dysfunction-associated steatohepatitis; LC: Liver cirrhosis; HCC: Hepatocellular carcinoma; NK: Natural killer; DC: Dendritic cell; HSC: Hepatic stellate cell.

Figure 5 Gene transcripts of immune cells during metabolic dysfunction-associated fatty liver disease malignancy progression. A: Gene transcripts of hepatic immune cells; B: Transcript numbers of genes whose expression was upregulated or downregulated from healthy liver to metabolic dysfunction-associated fatty liver disease; C: Transcript numbers of genes whose expression increased or decreased from metabolic dysfunction-associated fatty liver disease to hepatocellular carcinoma; D: Interaction analysis of hepatic immune cells; E: Macrophages with immune cells in the programmed death ligand 1-activating network. MAFLD: Metabolic dysfunction-associated fatty liver disease; MASH: Metabolic dysfunction-associated steatohepatitis; HCC: Hepatocellular carcinoma.

Figure 6 M2 macrophages and signaling pathways during metabolic dysfunction-associated fatty liver disease malignancy. A: Dynamic alterations in the expression of the main genes in hepatic macrophages during metabolic dysfunction-associated fatty liver disease malignancy; B: M2-polarized macrophages (CD68+ or CD163+); C: Gene signal intensity of M1- or M2- macrophages; D: Inhibition of the steroid biosynthesis pathway; E: Activation of the cell cycle signaling pathway; F: Schematic diagram for interactions among differential metabolites, inhibited T cells and M2 macrophages promoting metabolic dysfunction-associated fatty liver disease malignancy. PD-L1: Programmed death ligand 1; HFD: High-fat diet; CPT-II: Carnitine palmitoyl transferase II; DMs: Differential metabolites; NC: Normal control; MAFLD: Metabolic dysfunction-associated fatty liver disease; MASH: Metabolic dysfunction-associated steatohepatitis; LC: Liver cirrhosis; HCC: Hepatocellular carcinoma.