Fig. 3

Single-cell sequencing analysis revealed that PARD3 maintained stemness of TICs in the early tumour ecosystems of HCC. (A) Uniform manifold projection and approximation (UMAP) plot showing the main cell lineages in early tumour ecosystems. (B) Proportions of various cell lineages identified in PARD3-overexpressing and wild-type early tumour tissues. (C) Cancer cells were additionally classified into 3 subclusters. (D) Trajectories of the 3 subclusters of cancer cells. Left panel: The expression patterns of PARD3 and Prom1 along the phylogenetic tree are visualized. Right panel: Pard3 and Prom1 are positively correlated in cluster 0 and 1 with lower differentiation potency. (E) CytoTRACE analysis of cancer cells. The CytoTRACE scores are visualized in a violin plot. (F) GSEA revealed the activated pathway in PARD3-overexpressing tumour cells in the early tumour ecosystem. (G) Immunoblotting showing the overexpression of PARD3 in CD133⁺ Hepa1-6 cells. H&I. PARD3 overexpression increased the CD133⁺ cell percentage and stemness-associated gene expression in Hepa1-6 cells. J&K. PARD3-overexpressing CD133⁺ Hepa1-6 cells showed stronger tumour sphere formation ability than their wild-type counterpart, as measured by an in vitro limiting dilution assay. L&M. An in vivo limiting dilution assay showed that the tumorigenic ability of CD133 + Hepa1-6 cells was enhanced by PARD3 overexpression. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not statistically significant