Irreversible electroporation enhances immunotherapeutic effect in the off-target tumor in a murine model of orthotopic HCC

Abstract

Irreversible electroporation (IRE) has been postulated to have an off-target effect on lesions not in the tumor-ablative field, possibly through heightened immunologic response. In this study, we evaluated whether combination IRE and immunotherapy would lead to increased tumor necrosis and T cell recruitment to both the treated tumors and tumors outside the local ablative field. An in vitro cell-IRE model was established to evaluate the ability of T lymphocytes (EL4 cell and HH cells) migration in response to Hepatocellular carcinoma (HCC) cells (Hepa1-6 and HepG2) with IRE treatment. An orthotopic HCC mouse model was established by implantation of 1mm^3 sections of Hepa1-6 tumor tissues into the right and left lobes of the liver. The Hepa1-6 cells and HepG2 cells with IRE treatment increased the migration ability of EL4 cell and HH cells, specifically when they were pretreated with immunotherapeutic agents in vitro. In the orthotopic HCC mouse model, IRE+immunotherapy treatment enhanced the necrosis and subpopulation of infiltrated CD8 positive cells, but attenuated the tumor associated inflammatory cells in both IRE target tumor tissues and IRE off-target tumor tissues from the mice with 4 weeks of immunotherapy following IRE. This study provided the evidence that combination of IRE and immunotherapy enhances tumor necrosis and immune responses, not only in the IRE-treated tumor but also in the off-target tumor.

Keywords: Irreversible electroporation; hepatocellular carcinoma; immunotherapy; tumor microenvironment; tumor-associated macrophages; tumor-associated neutrophils.

Figure 1

A: Schematic diagram for the IRE off-target tumor and IRE target tumor. B: Surgical operation for IRE on the tumor in right liver lobe. Arrow: tumor.

Figure 2

Representative images of cell migration capacity by a trans-well assay with crystal violet staining. The numbers of crystal violet stained cells were analyzed in all 4 treatment groups as well as the controls. A: Cell migration capacity of EL4 cells co-cultured with Hepa1-6 cells. B: Cell migration capacity of HH cells co-cultured with HepG2 cells. HPF: high power field. Scale bar = 100 µm. *, P<0.05; **, P<0.01.

Figure 3

Representative histological images by H&E staining. A: Successfully established HCC orthotopic model showing an abnormal hepatic architecture occupied by tumor nodules, compared to normal hepatic architecture. B: Twenty-four hours Post-IRE treatment showing massive haemorrhage and extensive tumor tissue damage as well as hepatic architecture damages. C: Necrosis and inflammatory cells infiltrated were detected in all the tissues of treatment groups (IRE, IT treatment, direct IRE with IT treatment, and IRE-off target with IT treatment). Necrotic index and index of inflammatory cells infiltrated were calculated based on the histology. LPF: low power field. Scale bar = 200 µm. Dash circle, necrotic area; Arrow, area of inflammatory infiltration. *, P<0.05.

Figure 4

Representative histological images of CD8 staining by IHC staining. CD8 positive cells extensively distributed into the direct-IRE tumor tissues and distributed peritumorally into the IRE off-target tumor tissues in the mice with 4 weeks of IT following IRE. HPF: high power field. Scale bar = 200 µm. UT: untreated control. *, P<0.05; **, P<0.01.

Figure 5

A: Representative images of CAE staining for neutrophil detection in the IRE target tumor tissues and IRE off-target tumor tissues from the animals with 4 weeks of IT following IRE, in comparison with the tumor tissues without treatment (no-IRE, no-IT). B: Dual immunofluorescent staining using the antibodies of anti-F4/80 and anti-CD206 to detect TAMs in the IRE target tumor tissues and IRE off-target tumor tissues from the animals with 4 weeks of IT following IRE, in comparison with the tumor tissues without treatment (no-IRE, no-IT). LPF: Low power field. Scale bar = 100 µm. *, P<0.05; ***, P<0.001.

Figure 6

Dual immunofluorescent staining using the antibodies of anti-CD11b and anti-Ly6C to detect the M-MDSC derived TAMs in the IRE target tumor tissues and IRE off-target tumor tissues from the animals with 4 weeks of IT following IRE, in comparison with the tumor tissues without treatment (no-IRE, no-IT). LPF: low power field. Scale bar = 100 µm. **, P<0.01; ***, P<0.001.

Figure 7

Flow Cytometry to detect F4/80⁺CD11b⁺ cells and F4/80⁺CD11b⁺CD206⁺ cells for potential M-MDSC derived TAMs in the isolated peritoneal inflammatory cells from the HCC mice with 4 weeks of IT following IRE and the untreated HCC controls. Ctrl, control. n.s, no statistical significance. *, P<0.05.