Abstract

Background Current liver cancer research lacks reliable in vitro models that replicate tumor pathophysiology. This study establishes primary liver cancer (PLC) organoids from three major subtypes—hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and combined hepatocellular-cholangiocarcinoma (CHC)—to enable precise diagnostics and personalized therapies through comprehensive genomic profiling. Methods Organoid cultures were generated from 11 PLC patients (5 HCC, 3 ICC, 3 CHC). Whole exome sequencing (WES), RNA-seq, and single-cell RNA-seq (scRNA-seq) were performed to analyze molecular differences. Drug screening targeting subtype-specific pathways was conducted to validate sequencing findings. Results WES and RNA-seq confirmed that organoids retained parental tumor genetics and heterogeneity, distinct from paracancerous tissues. scRNA-seq revealed distinct cell populations in HCC, ICC, and CHC organoids. Lipid metabolism was enriched in HCC organoids; tumor migration pathways were upregulated in ICC organoids; and mitochondrial function was enhanced in CHC organoids. Rosuvastatin inhibited HCC growth by targeting lipid metabolism, while pemigatinib reduced ICC malignancy by suppressing epithelial-mesenchymal transition. Regorafenib impaired mitochondrial function in CHC organoids, slowing progression. Conclusions PLC-derived organoids serve as robust tools for biomarker discovery and drug screening. scRNA-seq elucidates inter- and intra-tumoral heterogeneity, offering insights for precision therapy in liver cancer. This model advances personalized treatment strategies for diverse PLC subtypes.