Letter to the Editor: High-frequency irreversible electroporation as an immune primer in hepatocellular carcinoma

Abstract

We read with great interest the recent study by Huang et al published in the World Journal of Gastroenterology. High-frequency irreversible electroporation (H-FIRE) is an emerging non-thermal ablation method that has been shown to induce features consistent with immunogenic cell death in hepatocellular carcinoma (HCC). This process releases damage-associated molecular patterns that prime adaptive antitumor immunity. A recent preclinical study demonstrated that the triple combination of H-FIRE, the small-molecule programmed death-1 (PD-1)/PD-ligand 1 (PD-L1) interaction inhibitor BMS-1, and the TLR7/8 agonist resiquimod synergistically reprograms the immunosuppressive tumor microenvironment, suppresses primary and distant tumor growth, and produces a strong abscopal effect in a murine HCC model. While this work provides an important proof-of-concept, several methodological and translational limitations merit discussion. These include using a subcutaneous ectopic tumor model that does not replicate the native hepatic immune microenvironment, relying on a single, highly immunogenic cell line, conducting immune profiling at a single time point without longitudinal kinetic analysis, lacking tumor rechallenge data to confirm immune memory, and using a non-clinically approved small-molecule PD-1/PD-L1 inhibitor (BMS-1) rather than a clinically validated checkpoint inhibitor. This letter addresses the limitations, outlines the most effective methodological approaches to address them, and presents a prioritized translational research plan and advances toward a Phase I investigator-initiated clinical trial in unresectable HCC.

Key Words: High-frequency irreversible electroporation; Hepatocellular carcinoma; Immunogenic cell death; Tumor microenvironment; Programmed death-1; Programmed death-ligand 1; Abscopal effect; Tumor immunotherapy; Liver ablation; Translational oncology

Core Tip: High-frequency irreversible electroporation induces features consistent with immunogenic cell death in hepatocellular carcinoma tumors and, when combined with the small-molecule programmed death-1/programmed death-ligand 1 interaction inhibitor BMS-1 and TLR7/8 agonist resiquimod, reverses immunosuppression both locally and systemically. While this triple combination shows strong preclinical efficacy, including a robust abscopal effect, important limitations, such as dependence on a subcutaneous ectopic model, single-timepoint immune profiling, and lack of immune memory validation, need to be addressed before moving to clinical trials. Future research should focus on validating orthotopic models, conducting longitudinal immune studies, performing tumor rechallenge experiments, and optimizing nanoparticle drug delivery as important steps toward a Phase I trial in unresectable hepatocellular carcinoma.

TO THE EDITOR

We read with great interest the recent study by Huang et al[1] published in the World Journal of Gastroenterology, which presents compelling preclinical evidence that a triple therapeutic strategy-comprising high-frequency irreversible electroporation (H-FIRE), the small-molecule programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) interaction inhibitor BMS-1, and the TLR7/8 agonist resiquimod (R848)-synergistically reverses both local and systemic immune tolerance in a murine model of hepatocellular carcinoma (HCC). Unlike thermal ablation modalities such as radiofrequency and microwave ablation, H-FIRE preserves the extracellular matrix scaffold and avoids heat sink effects, conditions that may better support immune cell infiltration and antigen presentation. The study showed that this approach suppressed primary tumor growth and induced a significant abscopal effect, inhibiting distant untreated tumors through systemic immune activation. That said, this effect may be amplified in synchronized dual-tumor murine models and likely does not fully capture the immunological heterogeneity of metastatic HCC in clinical settings, where tumor burden, prior treatment history, and host immune competence vary substantially. This work provides an important proof-of-concept in the growing field of combining ablation with immunotherapy for HCC. However, it has several methodological and translational limitations that merit careful attention. We aimed to discuss these limitations, suggest effective strategies to address them, and outline future research directions to strengthen and broaden the clinical relevance of these findings.

First, the main limitation of this study is its reliance on a subcutaneous H22 HCC model in BALB/c mice. While this model is reproducible and allows for accurate tumor measurements, it does not fully replicate the anatomical, immunological, and biomechanical features of the native liver tumor environment[2]. The liver naturally has an immunotolerant microenvironment, shaped by various cell types, including Kupffer cells, liver sinusoidal endothelial cells, and hepatic stellate cells, as well as immunosuppressive signals from the portal circulation[3]. These cellular components work together to suppress antigen-specific T-cell responses, promote the differentiation of regulatory T-cells (Tregs), and encourage M2 macrophage polarization through mechanisms such as secreting interleukin-10, transforming growth factor-β, and prostaglandin E2-all of which are absent in the subcutaneous model. Additionally, most cases of HCC in humans develop in a setting of hepatic fibrosis or cirrhosis caused by chronic viral hepatitis, alcohol use disorder, or metabolic-associated steatotic liver disease (MASLD)[4]. This environment significantly alters the hepatic immune landscape, creating a highly immunosuppressive stroma that the subcutaneous model does not accurately mimic[5]. Therefore, while the results here are important, they may overestimate the strength and longevity of immunostimulatory responses achievable in a cirrhotic liver. For future research, it is important to focus on orthotopic intrahepatic tumor models, as these can be established by ultrasound-guided injections of H22 or Hepa1-6 cells directly into the liver of immunocompetent mice, which better mimic the anatomical and immunological features of HCC[5]. Furthermore, developing a fibrosis-based model-using methods like repeated CCl₄ administration or a methionine-choline-deficient diet before orthotopic tumor implantation-will be crucial for assessing the triple combination therapy in a cirrhotic environment that closely resembles human HCC[5]. Adding microcomputed tomography or small-animal magnetic resonance imaging guidance to intrahepatic H-FIRE delivery will also improve simulation of image-guided percutaneous procedures commonly used in clinical practice.

Second, the H22 murine hepatoma cell line is known for its high immunogenicity in BALB/c mice and a relatively favorable immune environment at baseline. However, relying on a single cell line limits the applicability of the findings, as HCC exhibits significant molecular diversity, including at least 6 distinct subclasses, each with notably different immune characteristics[6], which range from the immunologically active “inflamed” subclass to the severely immunosuppressive “transforming growth factor-beta high” subclass. As a result, outcomes from a highly immunogenic model may not accurately predict therapeutic responses in cold tumors, which comprise a significant proportion of patients resistant to checkpoint inhibitor monotherapy[7]. To overcome this limitation, it is recommended to perform parallel validation using the Hepa1-6 syngeneic HCC model in C57BL/6 mice. This model has a less immunogenic profile and a more suppressive tumor microenvironment (TME). Such an approach would provide complementary and vital insights into the effectiveness of the H-FIRE + BMS-1 + R848 strategy. Additionally, longer-term translational efforts should include patient-derived organoid models derived from resected or biopsied human HCC tissue, which would enable testing the triple combination therapy in a patient-specific manner that retains the molecular and immune diversity seen clinically[6,7].

Third, the immune microenvironment was characterized at a single post-treatment time point, 1 week after the intervention[1]. While this provides a useful snapshot of early immune activation, it does not capture the temporal dynamics of the immune response following H-FIRE-based combination therapy. The immunostimulatory phase triggered by H-FIRE-mediated features consistent with immunogenic cell death is short-lived, typically leading to a compensatory immunosuppressive rebound. This rebound includes the reaccumulation of Tregs, upregulation of PD-L1 on surviving tumor cells, and the resurgence of M2-polarized tumor-associated macrophages over the next few weeks[8]. Because this study lacks longitudinal profiling, it is challenging to determine the durability of the observed immune activation, the optimal timing for immunotherapy dosing relative to ablation, and the mechanisms underlying long-term tumor control vs relapse. Moving forward, future studies should include immune profiling at predetermined time points, such as day 7, day 14, and day 28 post-H-FIRE, to capture the course of the immune response. This should include phases of activation, peak effector function, contraction, and potential memory formation. Additionally, performing concurrent cytokine multiplex profiling to measure factors such as interferon-γ, tumor necrosis factor-α, interleukin-12, and interleukin-10 in both peripheral blood and the TME at these matched time points will provide mechanistic insight into the cellular changes observed by flow cytometry. Collecting this longitudinal data will be essential for guiding the development of optimal treatment schedules to improve clinical outcomes.

Fourth, the authors highlight an abscopal effect and demonstrate systemic immune activation within splenic immune cell subsets. Based on these observations, the authors suggested that a durable antitumor immune memory may be induced. While these findings are biologically plausible and supported by underlying mechanisms, the claim of immunological memory induction lacks strong evidence without a formal tumor rechallenge experiment. Without such rechallenge data, it is difficult to distinguish between long-lasting T-cell memory and a temporary expansion of effector T cells that dissipates without forming a persistent memory pool[9]. To verify this, a rechallenge experiment, in which mice that have achieved complete tumor regression following H-FIRE + BMS-1 + R848 treatment are re-implanted with H22 cells at a different anatomical site 30-60 days later, is considered the gold standard for demonstrating immune memory in preclinical studies. Including this in a revised or follow-up study is highly recommended[10].

Fifth, the statistical methods included one-way analysis of variance, paired with Student’s t-test for pairwise comparisons, and Kaplan-Meier analysis with the log-rank test for survival, which are suitable given the study’s design. However, analyzing eight experimental groups across multiple outcome variables without adjusting for multiple comparisons increases the risk of type I errors. Moreover, although tumor volume measurements were taken at several time points during the observation period, the primary statistical analysis considers only the endpoint at day 21, thereby missing potential statistical power and deeper insights from a longitudinal repeated-measures analysis. To improve this, all multi-group analysis of variance comparisons should be followed by Tukey’s honest significant difference post-hoc test, and adjusted P values should be reported for each pairwise comparison. Longitudinal tumor volume data ought to be analyzed using a linear mixed-effects model, with treatment group as a fixed effect and individual animals as random effects. This approach accounts for within-subject correlations across repeated measurements and provides a more robust statistical assessment of growth differences between treatment groups[11].

Finally, all experiments in this study were conducted with male BALB/c mice, and the potential influence of sex as a biological variable was not assessed[1]. Studies have consistently shown sex-based differences in innate and adaptive immune responses, including variations in macrophage polarization, natural killer cell activity, CD8+ T-cell cytotoxicity, and baseline Treg frequency. These differences can significantly affect the response to both ablative and immunotherapeutic interventions[12]. Given that HCC affects both genders, with a prevalence ratio of approximately 2-4:1 favoring males, the exclusive use of male animals may introduce bias into the immune response parameters observed. To address this, future studies should include equal numbers of male and female animals and use sex as a stratification variable in the randomization process, thereby enabling a thorough assessment of whether treatment-induced immune responses vary by sex.

Future directions

The proof-of-concept data provided by Huang et al[1] laid a strong scientific foundation for an organized translational research initiative. We propose the following prioritized research agenda: (1) In the short term, the most urgent next step is to validate the H-FIRE + BMS-1 + R848 triple combination in an orthotopic intrahepatic tumor model, both with and without a fibrotic background[5]. In addition, conducting a tumor rechallenge experiment and performing TCR repertoire sequencing will allow us to directly evaluate the induction of immune memory and the expansion of clonal T cells[9,10]. A thorough immune profiling and cytokine analysis over a 60-day post-treatment period should be implemented to better understand the durability and dynamics of immune activation[8]; (2) In the medium term, replacing BMS-1 with an approved murine surrogate anti-PD-1 antibody will enhance the translational validity of our findings. Developing a nanoparticle-based formulation of R848 will help address the delivery challenges in orthotopic models and clinical applications. Furthermore, combining this triple therapy with transarterial chemoembolization (TACE), the current standard treatment for intermediate-stage HCC, presents a clinically relevant medium-term strategy, as TACE is known to promote tumor antigen release and has an established safety record in patients with cirrhosis[13,14]. This TACE integration should follow, not precede, the core orthotopic mechanistic validation, and should be pursued only after the primary H-FIRE + BMS-1 + R848 strategy has been validated. It bears noting that CCl₄-induced fibrosis models provide a useful approximation, they incompletely replicate the full immunosuppressive milieu of human cirrhosis, particularly the distinct immune alterations seen in MASLD-associated and viral hepatitis-driven fibrosis; and (3) Looking ahead to the long term, one of the most significant translational steps will be designing a Phase I investigator-initiated clinical trial to evaluate H-FIRE in conjunction with an approved anti-PD-1 checkpoint inhibitor and intratumoral R848 in patients with unresectable or locally advanced HCC[1,4]. The preclinical data presented in this study provide the crucial mechanistic rationale needed to support such a trial design, assuming the aforementioned validations are completed. Importantly, the regulatory pathway for this trial will involve coordinating the Investigational New Drug application for intratumoral R848 with the Investigational Device Exemption for the H-FIRE generator, necessitating early engagement with regulatory bodies to outline a clear development pathway.

CONCLUSION

The study conducted by Huang et al[1] makes a significant contribution to the preclinical literature on the combination of ablation and immunotherapy for HCC. It highlights how H-FIRE, the small-molecule PD-1/PD-L1 inhibitor BMS-1, and R848 work in concert to reprogram the TME, enhance systemic antitumor immunity, and produce a strong abscopal effect while maintaining a favorable safety profile. To turn these promising findings into a viable clinical treatment strategy, addressing the methodological limitations outlined in this letter will be crucial, including validating results in orthotopic models, conducting longitudinal immune profiling, performing rechallenge experiments, and optimizing dosing studies[13]. We applaud the authors for this original work and eagerly anticipate the continued progression of this research program.