OX40 agonist combined with irreversible electroporation synergistically eradicates established tumors and drives systemic antitumor immune response in a syngeneic pancreatic cancer model

Abstract

In this study, we intended to explore a novel combination treatment scheme for pancreatic cancer, using irreversible electroporation (IRE) and OX40 agonist. We further aimed to investigate the capacity and mechanism of this combination treatment using an in vivo mouse aggressive pancreatic cancer model. To this end, mice subcutaneously injected with KPC1199 pancreatic tumor cells were treated with IRE, followed by intraperitoneal injection of OX40 agonist. Tumor growth and animal survival were observed. Flow cytometry analysis, immunohistochemistry, and immunofluorescence were used to evaluate the immune cell populations within the tumors. The tumor-specific immunity was assessed using ELISpot assay. Besides, the cytokine patterns both in serum and tumors were identified using Luminex assay. After combination therapy with IRE and OX40 agonist, 80% of the mice completely eradicated the established subcutaneous tumors, during the 120 days observation period. Rechallenging these tumor-free mice at day 120 with KPC1199 tumor cells leads to complete resistance to tumor growth, suggesting that the combination therapy generated long-term-specific antitumor immune memory. Moreover, combination therapy significantly delayed the growth of contralateral untreated tumors, and significantly prolonged animal survival, suggesting that a potent systematic anti-tumor immunity was induced by combination therapy. Mechanically, combination therapy amplified antitumor immune response induced by IRE, as manifested by the increased quality and quantity of CD8⁺ T cells trigged by IRE. Together, these results provide strong evidence for the clinical assessment of the combination of IRE and OX40 agonist in patients with pancreatic cancer.

Keywords: OX40; Pancreatic cancer; combination treatment; immunotherapy; irreversible electroporation.

Figure 5

The anti-tumor activity of combination therapy is CD8⁺ T-cell dependent. A. Tumor cell implantation, immune cell depletion, and treatment strategy in unilateral subcutaneous mouse model. B. Successful depletion of CD4⁺ T and CD8⁺ T cells is confirmed using flow cytometry, and no cells are depleted after control IgG antibody injection. C and D. Tumor-free rates and survival rates after depletion of CD4⁺ or CD8⁺ T cells in unilateral subcutaneous tumor mice treated with combination therapy. N = 6 per group. E. Tumor cell implantation, immune cell depletion, and treatment strategy in bilateral subcutaneous tumor mouse model. F and G. Growth curves of the secondary untreated tumors and survival rates after depletion of CD4⁺ or CD8⁺ T cells in bilateral subcutaneous tumor mice treated with combination therapy. N = 6 per group. **P < 0.01, ***P < 0.001.

Figure 1

Efficacy of IRE and anti-OX40 mAb treatment of mouse subcutaneous pancreatic tumors. A. The schema for animal experiments using unilateral tumor mouse model. N = 10 per group. B. Volume changes in tumors in unilateral tumor mouse model following the indicated treatments. C. Tumor-free rates in IRE vs. IRE+Anti-OX40 groups after treatment initiation. D. Survival rates in unilateral tumor-bearing mice in the indicated treatment groups up to 120 days after initiating treatment. E. The schema for animal experiments using bilateral tumor mouse model. N = 10 per group. F. Volume changes of the untreated secondary tumors in bilateral tumor-bearing mice following the indicated treatments. G. Survival rates in bilateral tumor-bearing mice in the indicated treatment groups. N = 10 per group. ***P < 0.001. IRE, irreversible electroporation.

Figure 2

Immune cell infiltration in the primary tumor 10 days after initiation of treatments. (A-C) Representative images of immunohistochemistry and corresponding quantifications for CD3 (A), CD4 (B), and CD8 (C). (D, E) Representative images of immunofluorescence for MDSCs (D), and T-regs (E) and corresponding quantifications. MDSCs: CD11b⁺Gr1⁺; T-regs: CD4⁺Foxp3⁺. Scale bars = 50 µm. N = 5 per group. *P < 0.05 vs. control group, **P < 0.01 vs. control group; #P < 0.05 vs. IRE group. IRE, irreversible electroporation.

Figure 3

Immune cell infiltration and levels of cytokines or chemokines in the secondary tumor 10 days after initiation of treatments. A. Proportions of tumor-infiltrating CD3⁺CD8⁺ T cells, CD3⁺CD4⁺ T cells, ki-67⁺CD8⁺ cells, ki-67⁺CD4⁺ cells, IFN-γ⁺CD8⁺ cells, IFN-γ⁺Foxp3^-CD4⁺ cells, T-regs (CD4⁺CD25⁺Foxp3⁺), and MDSCs (Gr-1⁺CD11b⁺) assessed using flow cytometry, n = 5 per group. B and C. Levels of cytokines or chemokines in the secondary tumor were measured using a Mouse Cytokine 23-plex Luminex Assay. Data values (pg/mL) are normalized to total protein content and are presented in pg/mg protein. Cytokines or chemokines that did not show any significant difference in all groups were not included in this figure. n = 5 per group. *P < 0.05 vs. control group.

Figure 4

Levels of cytokines in serum and numbers of IFN-γ secreting CD8⁺ T cells in spleen at 10 days after initiation of treatments. (A) The concentration of serum cytokines in each group, assessed using Luminex assay. Cytokines that did not show any significant difference in all groups were not included in this figure. N = 5 per group. (B, C) IFN-γ secreting CD8⁺ T cells are analyzed using ELISpot. Representative ELISpot images from each group for (B), and the ELISpot count in each group for (C). N = 5 per group. *P < 0.05 vs. control group, **P < 0.01 vs. control group, ***P < 0.001 vs. control group, #P < 0.05 vs. IRE group, ##P < 0.01 vs. IRE group, ΔΔP < 0.01 vs. Anti-OX40 group. IRE, irreversible electroporation.

Figure 6

Immunological memory induced by combination therapy. A. Representative flow cytometry plots of splenic CD8⁺ central memory T cells (CD8⁺CD44⁺CD62L⁺) and CD8⁺ effector memory T cells (CD8⁺CD44⁺CD62L^-) from cured mice isolated before rechallenge. B. Percentage of splenic CD8⁺ central memory T cells and CD8⁺ effector memory T cells from cured mice isolated before rechallenge. N = 3 per group. C. Growth curves of rechallenging tumors in the cured mice treated with IRE+anti-OX40 mAb. Age-matched healthy mice are used as controls (n = 5 per group). D. Representative images of IFN-γ spots from the ELISpot assay. E. Quantification of IFN-γ spots from the ELISpot assay. N = 5 per group. F. Cytokine levels in serum from mice isolated 18 days after cured mice were rechallenged with KPC1199 tumor cells. Cytokines that did not show any significant difference in all groups were not included in this figure. n = 5 per group. *P < 0.05, **P < 0.01, ***P < 0.001. IRE, irreversible electroporation.

Figure 7

Percentages of OX40⁺ T lymphocytes in spleen and TDLN in mice in the IRE and control groups 24 h, 48 h, 7 days, and 10 days post-IRE, analyzed using flow cytometry. A. Representative flow cytometry plots showing OX40⁺CD8⁺ T cells in spleen. B and C. Percentages of OX40⁺CD8⁺ and OX40⁺CD4⁺ T cells in spleen. D. Representative flow cytometry plots showing OX40⁺CD8⁺ T cells in TDLN. E and F. Percentages of OX40⁺CD8⁺ and OX40⁺CD4⁺ T cells in TDLN. n = 5 per group. *P < 0.05, **P < 0.01. IRE, irreversible electroporation; TDLN, tumor-draining lymph nodes.