Hepatic radiofrequency ablation induces widespread cellular activation throughout the liver.

objectiveWe investigated the extent of cellular, transcriptional, and translational activation throughout the liver following radiofrequency ablation (RFA). MATERIALS AND

methodsRFA 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).

resultsCellphoneDB 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).

conclusionRFA 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.