Irreversible electroporation combined with anti-programmed cell death protein 1 therapy promotes tumor antigen-specific CD8+ T cell response

Abstract

BACKGROUND

Irreversible electroporation (IRE) is a novel local tumor ablation approach with the potential to activate the host’s immune system. However, this approach is insufficient to prevent cancer progression, and complementary approaches are required for effective immunotherapy.

AIM

To assess the immunomodulatory effects and mechanism of IRE combined anti-programmed cell death protein 1 (PD-1) treatment in subcutaneous pancreatic cancer models.

METHODS

C57BL-6 tumor-bearing mice were randomly divided into four groups: Control group; IRE group; anti-PD-1 group; and IRE + anti-PD-1 group. Tumor-infiltrating T, B, and natural killer cell levels and plasma concentrations of T helper type 1 cytokines (interleukin-2, interferon-γ, and tumor necrosis factor-α) were evaluated. Real-time PCR was used to determine the expression of CD8 (marker of CD8⁺ T cells) in tumor tissues of the mice of all groups at different points of time. The growth curves of tumors were drawn.

RESULTS

The results demonstrated that the IRE + anti-PD-1 group exhibited significantly higher percentages of T lymphocyte infiltration, including CD4⁺ and CD8⁺ T cells compared with the control group. Additionally, the IRE + anti-PD-1 group showed increased infiltration of natural killer and B cells, elevated cytokine levels, and higher CD8 mRNA expression. Tumor volume was significantly reduced in the IRE + anti-PD-1 group, indicating a more pronounced therapeutic effect.

CONCLUSION

The combination of IRE and anti-PD-1 therapy promotes CD8⁺ T cell immunity responses, leading to a more effective reduction in tumor volume and improved therapeutic outcomes, which provides a new direction for ablation and immunotherapy of pancreatic cancer.

Key Words: Irreversible electroporation; Pancreatic cancer; Programmed cell death protein 1 blockade; CD8⁺ T cell; Anticancer immunity

Core Tip: This study highlighted the synergistic effect of combining irreversible electroporation with anti-programmed cell death protein 1 therapy in the treatment of subcutaneous pancreatic cancer. The combination significantly enhanced T lymphocyte infiltration, elevated key cytokine levels, and promoted CD8⁺ T cell-mediated immune responses, resulting in a marked reduction in tumor volume. These findings suggest a promising avenue for improving immunotherapy strategies in pancreatic cancer through enhanced local tumor ablation methods.

INTRODUCTION

Pancreatic cancer is recognized as a notably aggressive type of cancer and is characterized by an alarmingly unfavorable prognosis. The 5-year survival rate for individuals diagnosed with this disease is persistently below 8%[1]. Although surgical resection remains the sole therapeutic option that may provide a potential cure, the majority of patients are diagnosed with locally advanced pancreatic cancer, and fewer than 20% of newly diagnosed individuals qualify for surgical intervention[2,3]. Additionally, locally advanced pancreatic cancer demonstrates a limited response to standard chemoradiotherapy, leading to only minimal enhancements in survival rates[4].

Irreversible electroporation (IRE) represents an innovative, non-thermal approach to localized tissue ablation. This technique employs high-voltage electrical pulses to induce disruption of cellular membranes, leading to the formation of irreversible nanoscale pores and ultimately precipitating apoptotic cell death[5,6]. As an effective local treatment, the safety and effectiveness of IRE have been explored in various types of cancer[7], with the most promising overall survival and recurrence results in pancreas[8], liver[9], and prostate cancer[10]. Boosting IRE-induced immune activation was also proposed[11,12]. Moreover, Babikr et al[13] and Scheffer et al[14] demonstrated that IRE for locally advanced pancreatic cancer temporarily reduced immune suppression, thereby establishing an opportunity for the activation of antitumor T cells. Meanwhile, recent studies have shown that an immune response can be elicited after IRE ablation, but this immune response is not sufficient to restrain tumor progression and recurrence[15-18].

Immune checkpoint inhibitors play a pivotal role in enhancing the ability of the immune system to target and combat tumors, thereby transforming the landscape of cancer treatment. Notably, therapies that target programmed cell death protein 1 (PD-1) have demonstrated significant effectiveness across a range of malignancies, such as melanoma, non-small cell lung carcinoma, and renal cell carcinoma[19-21]. PD-1 serves as an inhibitory receptor found on activated T cells. The interaction of PD-1 with its ligands, PD-L1 and PD-L2, which are often found in elevated levels in tumor cells, results in T cell exhaustion and a weakened immune response against tumors. By blocking this interaction, anti-PD-1 therapies reinvigorate T cells by enhancing their proliferation and cytotoxic activity against tumor cells. Moreover, T helper type 1 (Th1) cytokines, primarily produced by CD4⁺ Th1 cells, play a crucial role in driving robust immune responses[22]. Crucial cytokines, including interferon (IFN)-γ and tumor necrosis factor (TNF)-α, play a significant role in bolstering antitumor immunity. They achieve this by facilitating enhanced antigen presentation, activating macrophages, and supporting the maturation of cytotoxic CD8⁺ T cells[23,24]. Elevated levels of Th1 cytokines create a proinflammatory tumor microenvironment that can combat tumor growth. Despite the promising mechanisms of immune checkpoint inhibitors, their use in pancreatic cancer has been disappointing due to the immunosuppressive microenvironment and low tumor immunogenicity. Therefore, combining IRE with anti-PD-1 therapy could potentially augment the immunogenicity of pancreatic tumors and facilitate a more robust antitumor immune response, thus overcoming existing limitations.

In this study, we investigated the therapeutic efficacy and immune response of combining IRE with anti-PD-1 therapy in a C57BL-6 mouse model of pancreatic cancer using Panc02 cells. We hypothesized that the combination of IRE and anti-PD-1 would potentially augment the immunogenicity of pancreatic tumors and facilitate a more robust antitumor immune response compared to either treatment alone. To test this hypothesis, we established a pancreatic tumor model in C57BL-6 mice and evaluated tumor growth and immune cell populations following treatment.

Our findings provide critical insights into the potential synergistic effects of IRE and anti-PD-1 therapy in pancreatic cancer. By elucidating the mechanisms underlying the enhanced antitumor immune response, this study aimed to pave the way for novel combination therapies that could improve outcomes for patients with this devastating disease.

MATERIALS AND METHODS

Cell lines and cell culture

Panc02 cells were purchased from BeNa Culture Collection (BNCC, Beijing, China). Panc02 cells were cultured in 90% DMEM (Gibco) and 10% FBS (Hyclone). The cells were then maintained in an incubator at 37 °C and 5% CO₂.

Animal models

To establish a mouse model with a normal immune system, female C57BL/6 mice were subcutaneously inoculated with 1 × 10⁶ Panc02 cells in 100 µL of Hanks balanced salt solution into the right flank of 6-week-old C57BL/6 mice. This animal experiment was examined and approved by the laboratory animal welfare ethics committee of the Jinan University.

Study design and treatment

Forty-eight female C57BL-6 mice were randomly assigned to four groups: Control group without any treatment (n = 12); IRE group (n = 12); anti-PD-1 group (n = 12); and IRE + anti-PD-1 group (n = 12). Panc02 cells (1 × 10⁶/mL) were injected subcutaneously into the right flank subcutaneous area of mice, and the tumor growth was detected every 2 days. The maximum diameter (a) and shortest diameter (b) of the tumor were measured and recorded with vernier calipers, and the volume of the tumor was calculated according to the formula a × b²/2. When the maximum diameter of the tumor reached 10 mm, IRE was performed, an anti–PD-L1 antibody (200 μg/mouse) was administered intraperitoneally immediately after the IRE procedure, and tumor tissues and peripheral blood were taken from the mice at different time points (1, 3, 7, and 14 days) after the IRE procedure. The tumor size was recorded every 3 days by calculating the length and the width of the tumor. The flowchart for model establishment and IRE ablation parameters are shown in Figure 1.

Figure 1 Establishment of pancreatic cancer model and ablation parameters. A: Schematic drawing of the study design. C57BL/6 mice were enrolled for treatment once the tumor size reached about 10 mm; B: Tumor growth on day 20; C: Irreversible electroporation procedure was performed with two electrodes; D: Ablation parameters (1200 kV/cm, 70 pulses, 70 μs). IRE: Irreversible electroporation; PD-1: Programmed cell death protein 1.

IRE procedure and anti-PD-1 administration

Mice were fixed on the operating table, anesthetized with tribromoethanol intraperitoneally according to their body weight, and the surgical area was prepared, disinfected, and toweled. Two electrode needles were inserted along the long axis of the tumors (the parameters were set at 1200 V/cm, 50 A, 70 pulses, 70 μs wavelength, and 100 ms intervals, and the distance of the needles was adjusted according to the size of the tumors). The PD-1/PD-L1 inhibitor was purchased from Aladdi. Anti-PD-1 (200 μg) was injected intraperitoneally once every 3 days for a total of 4 times. The needle distance was adjusted according to the tumor size.

Flow cytometry analysis

Mouse peripheral blood samples (1 mL) were drawn at different time points. Flow cytometry techniques (FACS caliber, four-color system, BD Bioscience, CA, United States) were used to analyze the absolute number of CD3⁺ T cells (CD3⁺), CD4⁺ T cells (CD3⁺ CD4⁺), CD8⁺ T cells (CD3⁺ CD8⁺), the ratio of CD4⁺ T cells (CD3⁺ CD4⁺) and CD8⁺ T cells, natural killer (NK) cells (CD16⁺ CD56⁺), and B cells (CD19). A similar method was used to measure serum cytokines, including interleukin-2 (IL-2), TNF-β, and IFN-γ. A record of the data was made.

Real-time PCR

The expression of CD8 in mouse tumor tissues was detected by SYBR Green real-time PCR (RT-PCR) with glyceraldehyde 3-phosphate dehydrogenase as the internal reference gene. Tumor tissues were collected and cut into pieces of 0.3 cm × 0.3 cm × 0.3 cm. Total RNA was extracted by adding TRIzol reagent, and cDNA was synthesized according to the instructions. The reaction system (20 μL) was prepared according to the instructions of the reagents and then subjected to RT-PCR amplification and detection. The RT-PCR primer sequences were: β-actin (forward) GGGAAATCGTGCGTGAC, (reverse) AGGCTGGAA AAG -AGCCT; CD8 (forward) CAAACACGCTTTCGG CTCCTG, (reverse) CGGATTGGACTTCGCCTGTGA. The results were analyzed using the 2^-ΔΔCt method.

Statistical analysis

All statistical analyses were performed and compiled using GraphPad Prism (version 8.01, GraphPad Software, San Diego, CA, United States) and SPSS software (version 20.0, SPSS Inc., Chicago, IL, United States). The measured data were expressed as mean ± standard deviation. The t test was used to compare the data between two groups, and one-way analysis of variance was used to compare the data of multiple groups. P < 0.05 was considered statistically significant.

RESULTS

IRE combined with anti-PD-1 treatment increased T lymphocyte infiltration in peripheral blood cells

Flow cytometric evaluation revealed that the proportion of T lymphocytes was significantly elevated in the IRE group (P < 0.01), the anti-PD-1 group (P < 0.01), and the IRE + anti-PD-1 group (P < 0.001) compared with the control group, demonstrating a time-dependent effect. Additionally, the percentage of CD4⁺ T cells was markedly higher in all three treatment groups relative to the control group (P < 0.001). Notably, the IRE + anti-PD-1 group exhibited an increased proportion of infiltrating CD8⁺ T cells (P < 0.001, Figure 2). These findings indicated that the immune response induced by IRE ablation is primarily driven by the infiltration of CD8⁺ T cells, while the combination of IRE with anti-PD-1 therapy elicits a more robust CD8⁺ T cell response.

Figure 2 Irreversible electroporation combined with anti-programmed cell death protein 1 treatment increased T lymphocyte infiltration in peripheral blood cells. A: Percentages of CD3⁺ T cells within a single-cell suspension of tumor tissues; B: Percentages of CD4⁺ T cells; C: Percentages of CD8⁺ T cells; D: CD4⁺: CD8⁺ ratio (values represent mean ± standard deviation, ^aP < 0.05, ^bP < 0.01, ^cP < 0.001). IRE: Irreversible electroporation; PD-1: Programmed cell death protein 1.

IRE combined with anti-PD-1 treatment increased peripheral blood cell infiltration of NK and B cells

To explore whether humoral and innate immune responses were involved in the therapeutic response, we also analyzed the B cell and NK cell populations in the tumor tissues. The percentages of NK cells and B cells were significantly higher in the IRE group, the anti-PD-1 group, and the IRE + anti-PD-1 group compared to the control group. Notably, the extent of infiltration was significantly greater in the IRE + anti-PD-1 group relative to the anti-PD-1 group (P < 0.001, Figure 3). These findings indicated that all three therapeutic strategies bolstered both humoral and innate immune responses, with the combination of IRE and anti-PD-1 treatment demonstrating superior efficacy.

Figure 3 Irreversible electroporation combined with anti-programmed cell death protein 1 treatment increased peripheral blood cell infiltration of natural killer and B cells. A: Percentages of natural killer cells; B: Percentages of B cells (values represent mean ± standard deviation, ^aP < 0.05, ^bP < 0.01, ^cP < 0.001). IRE: Irreversible electroporation; NK: Natural killer; PD-1: Programmed cell death protein 1.

IRE combined with anti-PD-1 treatment altered peripheral blood cytokine concentrations

We conducted a quantitative analysis of cytokine levels, specifically IL-2, TNF-β, and IFN-γ, in the plasma of mice with tumors using flow cytometry techniques. Our findings revealed that the levels of IL-2, TNF-β, and IFN-γ were markedly elevated in the IRE, anti-PD-1, and IRE + anti-PD-1 treatment groups compared with the control group (P < 0.01 for both IRE and anti-PD-1 groups; P < 0.001 for the IRE + anti-PD-1 group). Notably, within the IRE + anti-PD-1 groups, the concentration of IL-2 exhibited a significant variation (P < 0.001, Figure 4). These observations indicated that all three therapeutic strategies successfully instigated a systemic immune response through the modulation of various cytokine levels; however, the combination of IRE ablation with anti-PD-1 therapy produced a more pronounced systemic immune response.

Figure 4 Irreversible electroporation combined programmed cell death protein 1 treatment modulated the concentrations of T helper type 1 cytokines in the peripheral blood. A: Plasma concentration of interleukin-2; B: Plasma concentration of tumor necrosis factor-β; C: Plasma concentration of interferon-γ (values represent mean ± standard deviation, ^aP < 0.05, ^bP < 0.01, ^cP < 0.001). IL-2: Interleukin-2; INF-γ: Interferon-γ; IRE: Irreversible electroporation; PD-1: Programmed cell death protein 1; TNF-β: Tumor necrosis factor-β.

IRE combined with anti-PD-1 treatment increased CD8 expression at the mRNA level

We examined the expression of CD8 mRNA by RT-PCR. It was found that the expression of CD8 mRNA in the IRE group, the anti-PD-1 group, and the IRE + anti-PD-1 group exhibited a markedly elevated response compared to the control group (P < 0.001). The expression of CD8 mRNA in the IRE + anti-PD-1 group was stronger in a time-dependent manner (P < 0.001, Figure 5). These results suggested that all three interventions can enhance CD8 mRNA. However, IRE ablation combined with anti-PD-1 treatment induced stronger expression of CD8 mRNA.

Figure 5 Irreversible electroporation combined programmed cell death protein 1 treatment increased the mRNA expression of CD8 in the tumor tissue. Values represent mean ± standard deviation. ^aP < 0.01, ^bP < 0.001. IRE: Irreversible electroporation; PD-1: Programmed cell death protein 1. IRE: Irreversible electroporation; PD-1: Programmed cell death protein 1.

IRE combined with anti-PD-1 treatment reduced tumor volume

We detected the tumor volume every 3 days after IRE treatment. The results revealed that the tumor volumes in the IRE group, the anti-PD-1 group, and the IRE + anti-PD-1 group were markedly reduced compared with those in the control group (P < 0.01, P < 0.001). However, the change in tumor volume in the IRE + anti-PD-1 group was significantly greater in a time-dependent manner (Figure 6). These results suggested that all three interventions can reduce tumor volume. However, IRE ablation combined with anti-PD-1 treatment induced a more significant reduction in tumor volume.

Figure 6 Irreversible electroporation combined programmed cell death protein 1 treatment increased the tumor regression. A: Tumor morphologic changes; B: Tumor size changes (values represent mean ± standard deviation, ^aP < 0.01, ^bP < 0.001). IRE: Irreversible electroporation; PD-1: Programmed cell death protein 1.

DISCUSSION

In this study, we investigated the therapeutic effects and immune responses of IRE and anti-PD-1 therapy in a C57BL-6 mouse model of pancreatic cancer. Our findings indicated that the combination of IRE and anti-PD-1 therapy significantly inhibited tumor growth compared with the control group (P < 0.01). Additionally, flow cytometry analysis revealed an increase in the absolute number and proportion of CD4⁺ T cells, CD8⁺ T cells, NK cells, and B cells in the peripheral blood of treated mice. RT-PCR further demonstrated elevated CD8 expression in tumor tissues, suggesting enhanced cytotoxic T cell activity. These results support our hypothesis that the synergistic effect of IRE and anti-PD-1 therapy can potentiate antitumor immunity and improve therapeutic outcomes in pancreatic cancer.

The primary novelty of this study lies in its comprehensive evaluation of the combined effects of IRE + anti-PD-1 therapy in a pancreatic cancer mouse model. This research fills a significant gap in the current understanding of how these two treatments interact to modulate the immune response and tumor dynamics. Previous studies have primarily focused on the individual effects of IRE or anti-PD-1 therapy. For instance, IRE has been shown to induce immunogenic cell death and enhance the infiltration of immune cells into the tumor microenvironment[25]. Similarly, anti-PD-1 therapy has been well-documented for its ability to reinvigorate exhausted T cells and promote antitumor immunity. However, the synergistic effects of combining these two modalities have not been thoroughly investigated. Our study demonstrated that the combination of IRE and anti-PD-1 therapy significantly enhanced CD8⁺ T cell infiltration, increased the levels of cytokines such as IL-2, TNF-β, and IFN-γ, and led to a more substantial reduction in tumor volume compared with either treatment alone. These findings suggested that IRE may prime the tumor microenvironment, making it more susceptible to immune checkpoint blockade. This synergistic effect could potentially translate into more effective therapeutic strategies for pancreatic cancer, a malignancy known for its poor prognosis and resistance to conventional therapies[26].

The main mechanism of IRE in treating pancreatic cancer is the induction of tumor cell apoptosis, which can lead to immune tolerance. Consequently, traditional theories suggest that combining IRE with immunotherapy may struggle to achieve synergistic effects. However, Shao et al[27] discovered that IRE is superior to thermal and cryoablation in inducing T cell immunity, indicating that IRE combined with immunotherapy may have synergistic effects. Zhao et al[15] utilized IRE in conjunction with PD-1 antibodies in a mouse model of orthotopic pancreatic cancer, and the results illustrated that the combined treatment significantly increased the survival time of mice compared with those receiving only PD-1 or IRE treatment. Narayanan et al[28] reported that the combination of IRE with PD-1 and TLR7 could enhance the survival of pancreatic cancer mice, along with increased infiltration of CD8⁺ T lymphocytes and dendritic cells. Furthermore, a recent study showed that IRE boosts specific T cell infiltration and immune memory by increasing the synthesis and secretion of damage-associated molecular patterns from tumor cells[29]. Our research aligns with these findings, confirming that the local innate immune system is activated after IRE ablation, leading to enhanced antigen presentation and the induction of adaptive immune responses, thereby strengthening the overall antitumor activity of the immune system.

The clinical implications of our study are significant, particularly in the context of pancreatic cancer, which remains one of the most lethal malignancies with limited treatment options. The combination of IRE and anti-PD-1 therapy demonstrated a synergistic effect in enhancing the immune response and reducing tumor burden in the mouse model. This combination therapy not only increased the infiltration of CD8⁺ T cells, NK cells, and B cells into the tumor microenvironment but also elevated systemic cytokine levels, suggesting a robust activation of both adaptive and innate immunity. These findings align with previous studies that have shown the potential of combining IRE with immunotherapy to overcome the immunosuppressive tumor microenvironment and improve therapeutic outcomes[15,30,31]. Clinically, this combination therapy could offer a novel and effective treatment strategy for patients with pancreatic cancer, potentially improving survival rates and quality of life. However, it is important to acknowledge the limitations of our study, including the use of a single animal model and the need for further validation in clinical trials. Future research should focus on optimizing the treatment parameters (both timing and dose) and exploring the long-term effects of this combination therapy to fully understand its clinical potential.

CONCLUSION

Our study demonstrated that the combination of IRE and anti-PD-1 therapy significantly enhanced the therapeutic efficacy against pancreatic cancer mouse model. Furthermore, CD8⁺ T cell infiltration and activation, suggesting that the combination therapy not only directly targets tumor cells but also modulates the immune microenvironment to favor antitumor responses.

ACKNOWLEDGEMENTS

We wish to thank the staff from the Department of Surgery and Anesthesia of Guangzhou Fuda Cancer Hospital for their technical support.