High-frequency irreversible electroporation synergizes with PD-1/PD-L1 inhibitor BMS-1 and TLR7/8 agonist R848 to potentiate liver cancer antitumor immunity

Figure 1 High-frequency irreversible electroporation induces immunogenic cell death in subcutaneous tumor cells. A and B: Immunofluorescence images of key immunogenic cell death markers (high mobility group box 1 protein/calreticulin) in tumors (DAPI for nuclear counterstaining); C and D: Quantitative analysis of their mean fluorescence intensity (n = 8). ^dP < 0.0001. H-FIRE: High-frequency irreversible electroporation; HMGB1: High mobility group box 1 protein; CRT: Calreticulin; MFI: Mean fluorescence intensity.

Figure 2 Combinatorial therapy with high-frequency irreversible electroporation, BMS-1, and R848 prolongs survival and inhibits tumor growth in hepatoma 22 subcutaneous tumor-bearing mice. A: Schematic of the hepatoma 22 subcutaneous tumor model and experimental treatment timeline; B: Kaplan-Meier survival curves across the different treatment groups (n = 8); C: Tumor growth kinetics presented as dynamic volume curves over the observation period (n = 8); D: Comparison of subcutaneous tumor volumes measured on day 21 post-treatment (n = 8). ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001. i.p.: Intraperitoneal; i.t.: Intratumoral; H-FIRE: High-frequency irreversible electroporation; H22: Hepatoma 22.

Figure 3 Representative coronal T2-weighted magnetic resonance scans of mouse tumors before treatment and 1 week post-treatment across all experimental groups. H-FIRE: High-frequency irreversible electroporation.

Figure 4 High-frequency irreversible electroporation combined with BMS-1 and R848 impairs tumor cell viability and suppresses proliferation in hepatoma 22 subcutaneous tumors. A: Schematic of the hepatoma 22 subcutaneous tumor-bearing mouse model and treatment regimen; B: Weight of excised subcutaneous tumors from the right flank at 1 week post-treatment; C: Hematoxylin-eosin staining of subcutaneous tumor sections across treatment groups (n = 8); D: Quantitative analysis of Ki67-positive rate (%) in right-sided subcutaneous tumors 1 week post-treatment (n = 8); E: Representative images of Ki67 immunohistochemical staining in right-flank subcutaneous tumors 1 week post-treatment. ^cP < 0.001, and ^dP < 0.0001. i.p.: Intraperitoneal; i.t.: Intratumoral; H-FIRE: High-frequency irreversible electroporation; H22: Hepatoma 22.

Figure 5 High-frequency irreversible electroporation combined with BMS-1 and R848 reprograms the tumor immune microenvironment in hepatoma 22 subcutaneous tumors. A-E: Representative flow cytometry dot plots of intratumoral immune cell subsets post-treatment across all groups: CD3+ CD4+ T cells, CD3+ CD8+ T cells, CD4+ CD25+ FOXP3+ Tregs, CD11c+ CD80+ CD86+ dendritic cells, F4/80+ CD11b+ CD86+ M1 macrophages, and F4/80+ CD11b+ CD206+ M2 macrophages; F-K: Quantitative analysis of the proportions of the above-listed intratumoral immune cell subsets in each group post-treatment (n = 5); L: Quantitative analysis of the intratumoral M1/M2 macrophage ratio across groups post-treatment (n = 5). ^aP < 0.05, ^cP < 0.001, and ^dP < 0.0001. H-FIRE: High-frequency irreversible electroporation.

Figure 6 Safety assessment of high-frequency irreversible electroporation combined with BMS-1 and R848 in hepatoma 22 tumor-bearing mice. A: Dynamic body weight changes of mice across all groups post-treatment (n = 8); B: Quantification of mouse body weights in each group at 21 days post-treatment (n = 8); C: Hematoxylin-eosin staining of major organs (heart, liver, spleen, lung, kidney) in mice from each group post-treatment, showing no obvious histopathological damage; D-H: Serum biochemical analysis shows no significant intergroup differences in liver function markers (aspartate aminotransferase, alanine aminotransferase), a cardiac injury marker (creatine kinase MB isoenzyme), and renal function parameters (creatinine, blood urea nitrogen) post-treatment (n = 8). H-FIRE: High-frequency irreversible electroporation; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; CK-MB: Creatine kinase MB isoenzyme; CREA: Creatinine; BUN: Blood urea nitrogen.

Figure 7 Therapeutic efficacy of high-frequency irreversible electroporation combined with BMS-1 and R848 in the hepatoma 22 bilateral tumor-bearing mice. A: Schematic of the hepatoma 22 bilateral tumor-bearing mice and treatment regimen; B: Dynamic changes in left tumor volume of hepatoma 22 bilateral tumor-bearing mice post-treatment (n = 8); C: Comparison of left tumor volumes on day 21 post-treatment (n = 8); D: Quantitative analysis of TUNEL-positive cell proportion in left tumors 1 week post-treatment (n = 8); E: Representative TUNEL staining images of left-sided tumors from each group 1 week post-treatment. ^dP < 0.0001. i.p.: Intraperitoneal; i.t.: Intratumoral; H-FIRE: High-frequency irreversible electroporation; H22: Hepatoma 22.

Figure 8 High-frequency irreversible electroporation combined with BMS-1 and R848 elicits systemic antitumor immunity by modulating splenic immune cell subsets. A and B: Proportion of splenic CD3+ CD4+ T cells; C and D: Proportion of splenic CD3+ CD8+ T cells; E and F: Proportion of splenic CD4+ CD25+ FOXP3+ Tregs; G and H: Proportion of splenic CD11c+ CD80+ CD86+ dendritic cells; I-L: Polarization of splenic macrophages: Proportion of F4/80+ CD11b+ CD86+ M1 and F4/80+ CD11b+ CD206+ M2 macrophages and the M1/M2 macrophage ratio. Each panel shows representative flow cytometry plots alongside quantitative data (n = 5). ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, and ^dP < 0.0001. H-FIRE: High-frequency irreversible electroporation.