Fig. 3: Prevalence of metallothioneins enriched TAMs in HBV-mediated HCC microenvironment.

A UMAP plot of subclustered myeloid cells labelled in different colours. Cluster number and cell type annotations are provided in the Figure. B The UMAP plot, showing patient IDs by colour (left panel); the bar graph represents the cell counts of each individual (right panel). C UMAP plot, showing tumor type by colour, HBV(blue), or non-HBV-HCC(green) myeloid. D UMAP coloured by viral status, HBV infected (green), and non-infected cells (red). E Expression of marker genes correlated with myeloid subtypes (macrophages, monocytes, and DCs). F Scatterplots showing M1 and M2 polarization scores (top panel) and pro- and anti-inflammatory scores (bottom panel), colour-coded according to patient ID. G Boxplot showing the fraction of myeloid cell subtypes in HBV (purple) and non-HBV (green) (left panel), violin plot indicating the fraction of myeloid cell subtypes in HBV (purple) and non-HBV (green) samples from the Validation cohort 2 (right panel). Error bars represent mean ± standard deviation (SD). H Representative IHC staining, indicating CD68⁺ cells, in paired HBV-HCC and non-HBV samples. Scale bar, 20 μm. I The volcano plot shows differentially expressed genes between HBV (red dots) and non-HBV myeloid (blue dots). The names of the most significant genes are indicated in the plot. J Violin plots indicating the expression of metallothionein genes (MT1G and MT1H) from macrophage clusters between HBV(blue) and non-HBV tumors (green) (top panel) violin plots showing the MT1G and MT1H genes expression in HBV (blue) and non-HBV (green) samples from the Validation cohort 2 (bottom panel). K Violin plots indicate the average metallothionein gene expression score from HBV (blue) and non-HBV (green) macrophage clusters (left panel) or infected and non-infected macrophages (middle panel); violin plots depicting the average metallothionein gene expression score in HBV (blue) and non-HBV (green) samples from the Validation cohort 2(right panel). L Representative IHC staining, indicating CD68⁺MT1G⁺ macrophages, in paired HBV and non-HBV HCC samples. Scale bar, 20 μm. The p-values in (J) and (K) were calculated using Student’s t-test; in (G), using the unpaired Wilcoxon test. All statistical comparisons were performed on biologically independent samples (n = 5 HBV-HCC and n = 3 non-HBV-HCC) from both the discovery and validation cohort 2. See also Supplementary Figs. 6 and 7.