Hepatic radiofrequency ablation induces widespread cellular activation throughout the liver

Objective: We investigated the extent of cellular, transcriptional, and translational activation throughout the liver following radiofrequency ablation (RFA). Materials and methods: RFA of the healthy liver was performed in two 8–10-week-old male C57/Bl6 mice, no/sham procedure in one. One and 7 days after, single-cell RNA sequencing (scRNAseq) was performed on distant, untreated liver to examine > 6,000 genes from normalized datasets of > 6,000 cells/sample, enabling identification of ten major cell populations. We defined cell-to-cell interactions by CellphoneDB and identified active pathways via STRING-db analysis with Markov clustering. Twelve distant liver lobe samples were homogenized on day 3 or day 6 after RFA/sham procedure for SomaLogic proteomic analysis (> 1,300 genes), subsequent STRING-db analysis, and assessment of cellular origin (PanglaoDB-2021). Results: CellphoneDB identified crosstalk among all ten populations with 4,658 and 4,218 receptor/ligand pairs, identified on day 1 and day 7 post-RFA, respectively. On day 1, 360 differentially expressed genes were identified; on day 7, 430. Activated genes distributed into 16 clusters, including 66 chemokines/cytokines, including Ccl2 and Ccl7; 57 immunomodulators, including Il6, Ctla4 and Pdcd1; and 54 growth factors, including Vegf, Hgf, Pdgf, and Fgf. Angiogenesis pathway genes were observed in endothelial cells and hepatocytes. Pdcd1 and Ctla4 were notably increased transiently in T cells. Proteomic analysis included 228/443 genes (51%) identified by scRNAseq; 73/228 proteins (32%) demonstrated 25% elevation over controls. Overall, 427 proteins were elevated, with 9/10 cell populations contributing to increased protein expression (odds ratio 4.9‒7.0). Conclusion: RFA diffusely activates cellular processes remotely from the ablation zone on both transcriptional and translational levels, altering tumorigenic and immunologic pathways simultaneously. Relevance statement: This study offers insights into liver tissue biology after RFA and provides a comprehensive picture of the molecular mechanisms put into motion by this procedure. A better understanding of these processes could provide a potential basis to develop specific biomarkers and effective adjuvant therapies following local tumor ablation. Key Points: RFA activates a multiplicity of hepatic cellular processes remotely from the ablation zone on a transcriptional and translational level. Single-cell RNA sequencing provides insights into widespread cellular origins of activated pro-immunogenic, pro-tumorigenic, and other pathways detected post-ablation. Consideration of the nature of this response may help achieve the clinical goals of adjuvant therapies and predictive biomarkers.