Abstract
Hepatocellular carcinomas (HCC) with multinodular morphology were typically polyclonal and exhibited extensive heterogeneity. We explored the multiple-layer heterogeneity of HCC utilizing single-cell multiomics sequencing together with multiregional sampling. We found that Confluent Multi-Nodular samples exhibit more heterogeneous immune landscapes characterized by increased transcriptome heterogeneity and more complex immune-related interactions compared to Single Nodular (SN) samples. We identified differential DNA methylation patterns among distinct tumor foci in a subset of liver cancer patients. Global DNA hypomethylation in cancer cells predominantly occurs in partially methylated domains. We predicted and validated two genes, GADD45A and SNHG6, as potential drivers of DNA hypomethylation. Lastly, we demonstrated that DNA methylation distance serves as a more robust metric than gene expression distance for reconstructing tumor evolutionary trajectories.