Journal: JHEP Reports

Article Title: Elevation of liver elasticity following radiofrequency ablation reflects neutrophil-mediated abscopal effect in liver cancer

doi: 10.1016/j.jhepr.2026.101824

Figure Lengend Snippet: Increasing liver SWE values post RFA correlate with intrahepatic neutrophil infiltration. (A) Left: representative plots showing SWE measurements under different conditions. Right: mean ± SEM liver SWE measurements across groups (n = 5; ∗∗ p <0.01; two-way ANOVA). (B) Schematic of the treatment plan. (C) DEGs were identified from the bulk RNA-seq results of murine livers (n = 4), visualized in a heatmap. GO enrichment analysis based on these genes revealed pathways that were specifically enriched in the PBS- and RFA-treated groups. (D) Murine liver immune cells were evaluated through FACS, and visualized through t-SNE analysis performed with FlowJo. The bar plots show the proportional changes. The gating strategy used is demonstrated below the graphs (n = 5; ∗∗∗ p <0.001; two-way ANOVA). (E) Schematic of the treatment process. (F) Left: representative plots of the change in SWE values after neutrophil depletion. Right: mean ± SEM liver SWE values in RFA, RFA + neutrophil deletion, and control groups in C57BL/6 mice (n = 5; ∗ p <0.05; two-way ANOVA). (G) Routine blood test results of patients illustrated in C in the main text. The proportional change in peripheral neutrophils before and 1 month after RFA was compared among different groups showing increased SWE (∗∗ p <0.01; two-sided paired t test). (H) KM survival curve of patients with HCC receiving RFA treatment in our center from 2013 to 2022. Patients were spilt based on their pre-RFA peripheral neutrophil levels, post-RFA peripheral neutrophil levels, and the post-RFA peripheral neutrophil:pre-RFA peripheral neutrophil ratio. DEG, differentially expressed gene; FACS, fluorescence-activated cell sorting; HCC, hepatocellular carcinoma; KM, Kaplan-Meier; RFA, radiofrequency ablation; RNA-seq, RNA sequencing; SWE, shear wave elastography.

Article Snippet: Blue = DAPI; Yellow = Ly6G; Green = iNOS. (K) Mean ± SEM mRNA expression of iNOS in TANs (n = 3; ∗∗ p <0.01; two-sided unpaired t test). (L) Western blot showing iNOS expression in TANs. (M) Mean ± SEM relative tumor cell viability when co-cultured with neutrophils isolated from different groups, with the addition of an iNOS inhibitor (20 μM; HY-18731, MCE, China; n = 3; ∗ p <0.05, ∗∗ p <0.01; two-way ANOVA).

Techniques: RNA Sequencing, Control, Fluorescence, FACS, Shear

Journal: JHEP Reports

Article Title: Elevation of liver elasticity following radiofrequency ablation reflects neutrophil-mediated abscopal effect in liver cancer

doi: 10.1016/j.jhepr.2026.101824

Figure Lengend Snippet: Neutrophils suppress tumor progression in an iNOS-dependent manner post RFA. (A) Schematic of the treatment process. (B) Representative FACS plots and mean ± SEM tumor neutrophil infiltration levels (n = 5; ∗∗∗ p <0.001; two-sided unpaired t test). (C) Schematic of the treatment process. (D) Mean ± SEM tumor volume across groups before RFA (n = 5; two-sided unpaired t test). (E) Representative plots of tumors and mean ± SEM tumor volume across groups at Day 14 (n = 5; ∗∗ p <0.01; two-sided unpaired t test). (F) t-SNE visualization of tumor-infiltrating neutrophil single-cell transcriptomes in mice in the control group (n = 2) and RFA group (n = 2). (G) Top: slingshot trajectories for tumor-infiltrating neutrophils in mice from control and RFA groups; bottom: mRNA expression of related markers of neutrophils in different clusters. (H) Neutrophil function scores evaluated by the AUCell algorithm. (I) Representative FACS plots of iNOS expression in TANs (n = 5). (J) Representative plots of IF staining. Blue = DAPI; Yellow = Ly6G; Green = iNOS. (K) Mean ± SEM mRNA expression of iNOS in TANs (n = 3; ∗∗ p <0.01; two-sided unpaired t test). (L) Western blot showing iNOS expression in TANs. (M) Mean ± SEM relative tumor cell viability when co-cultured with neutrophils isolated from different groups, with the addition of an iNOS inhibitor (20 μM; HY-18731, MCE, China; n = 3; ∗ p <0.05, ∗∗ p <0.01; two-way ANOVA). FACS, fluorescence-activated cell sorting; IF, immunofluorescence; RFA, radiofrequency ablation; TAN, tumor-associated neutrophil.

Article Snippet: Blue = DAPI; Yellow = Ly6G; Green = iNOS. (K) Mean ± SEM mRNA expression of iNOS in TANs (n = 3; ∗∗ p <0.01; two-sided unpaired t test). (L) Western blot showing iNOS expression in TANs. (M) Mean ± SEM relative tumor cell viability when co-cultured with neutrophils isolated from different groups, with the addition of an iNOS inhibitor (20 μM; HY-18731, MCE, China; n = 3; ∗ p <0.05, ∗∗ p <0.01; two-way ANOVA).

Techniques: Single Cell, Control, Expressing, Staining, Western Blot, Cell Culture, Isolation, Fluorescence, FACS, Immunofluorescence

Journal: JHEP Reports

Article Title: Elevation of liver elasticity following radiofrequency ablation reflects neutrophil-mediated abscopal effect in liver cancer

doi: 10.1016/j.jhepr.2026.101824

Figure Lengend Snippet: RFA activates neutrophils in an IFN-γ-dependent manner. (A) Bar plot of the relative information flow of signal pathways as assessed by CellChat. (B) GSEA of DEGs from TAN single-cell transcriptomes in RFA and control groups. (C) Left: Ifng mRNA expression in T/NK cell single-cell transcriptomes in RFA and control groups. Right: line graphs demonstrating the communication network for Ifng signaling, with the width of connecting lines indicating the probability of communication. (D) Representative FACS plots and mean ± SEM IFN-γ expression of tumor-infiltrating lymphocytes in double liver tumor-burden models, in which the large tumors received either acupuncture or RFA (n = 5; ∗ p <0.05; two-sided unpaired t test). (E) Representative plots of IF staining of tumors from mice in control and RFA groups. Blue, DAPI; red, Ly6G; green, CD8A. (F) Mean ± SEM iNOS + neutrophils when co-cultured with lymphocytes isolated from tumors, and with the addition of anti–IFN–γ (50 μg/ml; Clone XMG1.2, BioXCell; n = 3; ∗∗ p <0.01; two-way ANOVA). (G) Schematic of the treatment process. (H) Left: bar plots of IFN-γ expression in CD8+ T cells in tumors from control and anti–IFN–γ groups. Right: mean ± SEM iNOS expression in TANs in control and anti–IFN–γ groups (n = 5; ∗∗ p <0.01; two-sided unpaired t test). DEG, differentially expressed gene; FACS, fluorescence-associated cell sorting; GSEA, Gene Set Enrichment Analysis; RFA, radiofrequency ablation; TAN, tumor-associated neutrophil.

Article Snippet: Blue = DAPI; Yellow = Ly6G; Green = iNOS. (K) Mean ± SEM mRNA expression of iNOS in TANs (n = 3; ∗∗ p <0.01; two-sided unpaired t test). (L) Western blot showing iNOS expression in TANs. (M) Mean ± SEM relative tumor cell viability when co-cultured with neutrophils isolated from different groups, with the addition of an iNOS inhibitor (20 μM; HY-18731, MCE, China; n = 3; ∗ p <0.05, ∗∗ p <0.01; two-way ANOVA).

Techniques: Single Cell, Control, Expressing, Staining, Cell Culture, Isolation, Fluorescence, FACS

Journal: JHEP Reports

Article Title: Elevation of liver elasticity following radiofrequency ablation reflects neutrophil-mediated abscopal effect in liver cancer

doi: 10.1016/j.jhepr.2026.101824

Figure Lengend Snippet: Neutrophil-activating therapy enhances the abscopal effect of RFA. (A) Potential TFs of neutrophils in RFA groups from F in the main text, predicted through Scenic algorithms. (B) Relative TF activity of neutrophils from F in the main text. (C) Schematic of the treatment process. (D) Mean ± SEM tumor abundance among groups in (C) (n = 5; ∗ p <0.05; two-sided unpaired t test). (E) Mean ± SEM mRNA expression of TANs in mice in (C), detected by PCR (n = 3; ∗∗ p <0.01, ∗∗∗∗ p <0.0001; two-sided unpaired t test). (F) Schematic of the treatment process. (G) Representative FACS plots of the iNOS expression of TANs in mice from different groups in (F). (H) Mean ± SEM iNOS+ TANs in mice from different groups in (F) (n = 3; ∗∗ p <0.01, ∗∗∗∗ p <0.0001; two-way ANOVA). (I) Heatmap of the relative mRNA expression of TANs in mice in (F), detected by PCR (n = 3). Clusters 0, 1, and 2 represent the related cluster markers in scRNA-seq in F in the main text. (J) Schematic of the treatment process. (K) Plots of tumor abundance among groups in (J). (L) Mean ± SEM tumor abundance in D7 and D21 among groups in (J) (n = 6; ∗ p <0.05, ∗∗ p <0.01; two-sided paired t test). (M) Representative H&E images and IHC staining for Ly6G and iNOS among groups in (J). FACS, fluorescence-activated cell sorting; IHC, immunohistochemistry; RFA, radiofrequency ablation; scRNA-seq, single-cell RNA sequencing; TAN, tumor-associated neutrophil; TF, transcription factor.

Article Snippet: Blue = DAPI; Yellow = Ly6G; Green = iNOS. (K) Mean ± SEM mRNA expression of iNOS in TANs (n = 3; ∗∗ p <0.01; two-sided unpaired t test). (L) Western blot showing iNOS expression in TANs. (M) Mean ± SEM relative tumor cell viability when co-cultured with neutrophils isolated from different groups, with the addition of an iNOS inhibitor (20 μM; HY-18731, MCE, China; n = 3; ∗ p <0.05, ∗∗ p <0.01; two-way ANOVA).

Techniques: Activity Assay, Expressing, Immunohistochemistry, Fluorescence, FACS, Single Cell, RNA Sequencing