High-Ammonia Microenvironment Promotes Stemness and Metastatic Potential in Hepatocellular Carcinoma Through Metabolic Reprogramming

Background Hepatocellular carcinoma (HCC) is a prevalent and aggressive form of liver cancer, characterized by frequent recurrence and metastasis, which remain significant obstacles to effective treatment. Ammonia accumulates in the tumor microenvironment of HCC due to dysfunction in the urea cycle, but the detailed impact of ammonia on HCC cells remains insufficiently understood. Methods We exposed HCC cell lines to high concentrations of ammonium chloride to evaluate alterations in proliferation, stemness, and migratory potential. After ammonia removal, changes in cellular behavior were assessed using colony formation, wound healing, and spheroid assays. Transcriptomic and metabolomic analyses were conducted to investigate ammonia-induced metabolic reprogramming and alterations in gene expression. Additionally, animal models were employed to validate the impact of ammonia on tumor growth and metastasis. Results Exposure to high-ammonia conditions transiently suppressed HCC cell proliferation without inducing apoptosis. However, following ammonia removal, cells demonstrated increased proliferation, enhanced spheroid formation, and elevated migratory capacity. Transcriptomic analysis revealed the upregulation of genes associated with cell adhesion, migration, and glycolysis. Concurrently, metabolomic profiling indicated increased lactate production, facilitating the aggressive behavior of HCC cells after ammonia withdrawal. Animal experiments confirmed that high-ammonia exposure accelerated tumor growth and metastasis. Conclusion Ammonia exerts a dual effect on HCC progression: it initially suppresses cell growth but later promotes stemness, proliferation, and metastasis through metabolic reprogramming. Targeting ammonia metabolism or glycolysis in the tumor microenvironment may represent a promising therapeutic strategy for mitigating HCC recurrence and metastasis. Future studies utilizing clinical samples are required to validate these findings and identify potential therapeutic strategies targeting ammonia metabolism.