Oncolytic peptide LTX-315 targets PD-L1 to improve antitumor immune response of nanosecond pulse electric field in liver cancer.

Introduction
Hepatocellular carcinoma (HCC) is a prevalent malignant tumor globally.1 2 For unresectable HCC, current guidelines recommend local ablation therapies, such as radiofrequency ablation, microwave ablation and irreversible electroporation (IRE), as potential treatment options.3 4 However, the efficacy of thermal ablation may be compromised in perivascular liver tumors because of the “thermal sink effect”. Furthermore, thermal ablation may damage surrounding structures, potentially leading to severe complications.5
Nanosecond pulsed electric field (nsPEF), an emerging non-thermal ablation derived from IRE, delivers ultra-short, high-voltage electric pulses. nsPEF induces tumor cell apoptosis by generating electroporation of the plasma membrane and intracellular organelles.6 Importantly, nsPEF functions independently of thermal effects, making it suitable for the treatment of tumor lesions adjacent to major blood vessels, bile ducts and diaphragms.7 8 Clinical trials have demonstrated that nsPEF functioned as a safe and effective treatment for liver cancer with minimal damage to the adjacent blood vessels and bile ducts.9 10 Furthermore, nsPEF could trigger immunogenic cell death (ICD) to activate dendritic cells (DCs) and promote the infiltration of CD4+ and CD8+ T cells, macrophages and natural killer (NK) T cells within the tumor microenvironment (TME).1113 Nevertheless, the antitumor immunity induced by nsPEF is often inadequate to avert tumor recurrence, and some patients in clinical trials have developed needle-tract seeding, local recurrence or distant metastasis after nsPEF treatment.14 It has been reported that nsPEF upregulates the expression of programmed cell death ligand 1 (PD-L1) in HCC cells, leading to the exhaustion of tumor-infiltrating CD8+ T cells and subsequent tumor progression.15 Moreover, the efficacy of nsPEF depends heavily on the electric field distribution generated by parallel electrode configurations, which is challenging to achieve in clinical practice.16 Therefore, integrating nsPEF with other therapeutic strategies is essential to prevent tumor recurrence and metastasis.
Cationic antimicrobial peptides (CAPs) are naturally occurring molecules that protect against bacteria and other pathogens, and they also exhibit potential cytotoxic activity against a variety of cancer cell types. As a novel CAP, LTX-315 exerts potent oncolytic effects by targeting anionic phospholipids on tumor cell membranes, while sparing normal cells.17 Importantly, LTX-315 initiates the antitumor immunity through promoting ICD,18 increasing tumor-infiltrating lymphocytes (TILs) and reducing the tumor-infiltrating immunosuppressive regulatory T cells (Tregs).19 In a recent clinical trial, intratumoral injection of LTX-315 demonstrated significant efficacy and an acceptable safety profile in patients with advanced solid tumors.20 Notably, LTX-315 reduces PD-L1 expression in pancreatic cancer cells via ATP11B-CMTM6 axis-mediated lysosomal degradation pathway.21 However, the therapeutic potential of combining nsPEF with LTX-315 for liver cancer remains largely unexplored.
This study aims to investigate the therapeutic efficacy and underlying immunological mechanisms of nsPEF combined with LTX-315 in the treatment of liver cancer. We found that LTX-315 acted synergistically with nsPEF to further activate DCs by promoting ICD of liver cancer cells. Furthermore, LTX-315 enhanced the cytotoxic activity of CD8+ T cells by counteracting the nsPEF-induced upregulation of PD-L1. In addition, LTX-315 potentiated antitumor immune response of nsPEF, thereby significantly suppressing tumor growth and prolonging survival of tumor-bearing mice. Overall, this work conceptually highlights the efficacy of the “interventional dual therapy” strategy based on the ablation and oncolytic peptide.

Materials and methods

Cell lines and cell culture
The human HCC cell line SMMC-7721 and mouse HCC cell line Hepa 1–6 were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). The methods of cell culture are presented in online supplemental materials.

Mouse tumor model and drug treatment
The establishment of mouse tumor model and drug treatment is described in detail in the online supplemental materials.

nsPEF treatment
The parameters of nsPEF, including pulse voltage, duration, frequency and number, were set up via the control panel of nsPEF producer (online supplemental figure 1A). The nsPEF producer is provided by Ruidi Pharmaceutical (Hangzhou, China). For the in vitro experiment, cells were trypsinized and resuspended in phosphate-buffered saline at a concentration of 5×105 cells/mL, and subsequently were added to an electroporation cuvette (165–2091, Bio-Rad, USA) embedded with two electrodes 4 mm apart (online supplemental figure 1B). The cell suspension was exposed to nsPEF treatment at room temperature under the following parameters: voltage of 25 kV/cm, pulse duration of 100 ns, repetition frequency of 1 Hz, 40, 60, 80 pulses and rise time of 15 ns. For the in vivo experiment, nsPEF was administered using a two-needle electrode with a 5 mm gap to penetrate into the subcutaneous tumor of mice after anesthesia (online supplemental figure 1B). The parameters were listed as follows: voltage of 20 kV/cm, pulse duration of 100 ns, repetition frequency of 1 Hz, 100 pulses and rise time of 15 ns.

Cell isolation
Bone marrow cells from the femurs of C57BL/6 mice were suspended and plated at 1.5×106/mL in RPMI 1640 with 10% fetal bovine serum, 10 ng/mL interleukin (IL)-4 and 20 ng/mL granulocyte-macrophage colony-stimulating factor (PeproTech). A half volume of fresh medium was added to the culture on day 3, and non-adherent cells were harvested and re-plated in fresh medium on day 5 and 7. On day 9, the non-adherent cells were collected for further use as naive DCs.
CD8+ T cells were isolated from spleen cells of C57BL/6 mice using an Easysep Mouse CD8+ T cell Isolation Kit (STEMCELL) following the manufacturer’s protocols, and were activated in RPMI 1640 in the presence of 5 ug/mL anti-mouse CD3 (100340, BioLegend), 5 ug/mL anti-mouse CD28 (102116, BioLegend) and 20 ng/mL IL-2 (PeproTech) for 72 hours.

Statistical analysis
Statistical analyses were performed by SPSS V.21.0 and GraphPad Prism V.9.0. Quantitative data were presented as mean±SD at least three independent experiments. The differences of two groups and multiple groups were compared using the Student’s t-test and one-way analysis of variance, respectively. Survival differences were estimated through the log-rank test. Statistical significance is considered as a two-tailed test with p<0.05.

Results

nsPEF treatment weakens PD-L1-mediated immune response and displays a temporal tumor control
nsPEF treatment in tumor-bearing mice resulted in a significant reduction in tumor growth shortly after treatment; however, tumor growth began to resume by day 4 post-treatment compared with the control group (figure 1A). The antitumor effect of nsPEF is reportedly closely linked to the function of tumor-infiltrating CD8+ T cells.22 To investigate the cytotoxic function of CD8+ T cells, tumors were harvested at a later stage after nsPEF for flow cytometry analysis. The percentage of Granzyme B+CD8+ TILs was significantly increased, whereas the proportions of Perforin+, tumor necrosis factor alpha (TNF-α)+ and interferon-gamma (IFN-γ)+ CD8+ TILs were decreased following nsPEF compared with those in the control group (figure 1B). This suggests a marked dysfunction of CD8+ TILs and tumor progression beginning on day 4 after nsPEF.
To assess the cytotoxic effect of nsPEF in vitro, apoptosis assays demonstrated that nsPEF induced apoptosis in liver cancer cells in pulse number-dependent manner (figure 1C). GSE76427 dataset from the Gene Expression Omnibus (GEO) database revealed that patients with liver cancer with high PD-L1 expression had significantly shorter overall survival than those with low PD-L1 expression, suggesting an association between elevated PD-L1 levels and poor prognosis (figure 1D). Given the critical role of the PD-L1/programmed cell death protein-1 (PD-1) axis in regulating CD8+ T cells function and mediating tumor immune escape, we further explored the potential involvement of nsPEF in modulating PD-L1 expression. nsPEF treatment significantly upregulated PD-L1 expression in tumor cells (figure 1E), and the increased membrane PD-L1 levels were further confirmed by flow cytometry, showing a positive correlation with increasing pulse numbers (figure 1F). Taken together, these findings suggest that nsPEF may attenuate the cytotoxic activity of CD8+ T cells by upregulating PD-L1 expression in liver tumor cells.

LTX-315 reduces the viability of liver cancer cells and attenuates nsPEF-induced PD-L1 expression
LTX-315, a novel cationic peptide, exerts potent oncolytic activity through selectively targeting tumor cell membranes. CCK-8 assays showed that increasing concentrations and prolonged exposure to LTX-315 significantly reduced the viability of Hepa 1–6 and SMMC-7721 cells, illustrating a concentration-dependent and time-dependent cytotoxic effect of LTX-315 (online supplemental figure 2). Furthermore, LTX-315 has been reported to downregulate PD-L1 level in pancreatic cancer cells by ATP11B-CMTM6-mediated lysosomal degradation pathway.21 As for liver cancer cells, western blot and flow cytometry revealed that LTX-315 decreased PD-L1 expression in a dose-dependent manner in both Hepa 1–6 and SMMC-7721 cells (figure 2A, online supplemental figure 3A). To investigate whether LTX-315 modulates PD-L1 expression in liver cancer cells via ATP11B-CMTM6-lysosomal pathway, western blot was conducted and indicated that LTX-315 decreased PD-L1, ATP11B and CMTM6 expression in liver cancer cells in a dose-dependent manner (figure 2B). Moreover, LTX-315-induced downregulation of PD-L1 was reversed by treatment with the lysosome inhibitors of aloxistatin and pepstatin A (figure 2C). When nsPEF was combined with LTX-315, a significant reduction in PD-L1 expression was observed compared with nsPEF treatment alone (figure 2D,E, online supplemental figure 3B). These findings revealed that LTX-315 reduced liver cancer cell viability and mitigated the nsPEF-induced upregulation of PD-L1, potentially through the ATP11B-CMTM6-lysosomal degradation pathway.

LTX-315 restores CD8+ T cell cytotoxicity against liver cancer cells after nsPEF treatment
To evaluate whether LTX-315-induced reduction in PD-L1 expression exerts an influence on CD8+ T cell cytotoxicity, a co-culture system was established using CD8+ T cells isolated from mouse spleens and Hepa 1–6 cells pretreated with LTX-315, nsPEF, or their combination (figure 3A). After 24 hours of co-culture, both CD8+ T cells and Hepa 1–6 cells were harvested for flow cytometry analysis. The results revealed a significant reduction in the proportions of IFN-γ+, TNF-α+ and IL-2+CD8+ T cells in the nsPEF group in comparison with the control group, whereas Granzyme B+ and Perforin+CD8+ T cells remained unchanged. In contrast, the proportions of IFN-γ+, TNF-α+ and IL-2+CD8+ T cells were significantly elevated after co-culture with Hepa 1–6 cells pretreated with nsPEF in combination with LTX-315 (figure 3B).
Next, the killing ability of CD8+ T cells was assessed based on the apoptosis rates of Hepa 1–6 cells co-cultured with or without CD8+ T cells. The relative killing ability of CD8+ T cells was calculated using the following formula: Relative T-cell killing ability=test group apoptosis % (with T cells − without T cells) / control group apoptosis % (with T cells − without T cells). The findings showed that the killing ability of CD8+T cells toward Hepa 1–6 cells subjected to nsPEF was markedly reduced compared with the control group; however, CD8+ T cells exhibited enhanced cytotoxicity against LTX-315-treated Hepa 1–6 cells. Furthermore, the combination treatment significantly improved CD8+ T cell-mediated cytotoxicity compared to nsPEF treatment alone (figure 3C). Collectively, nsPEF conferred resistance to CD8+ T cell-mediated killing in liver cancer cells and thereby diminished their cytotoxic efficacy. However, this effect was markedly reversed by LTX-315, which restored tumor-cell sensitivity to CD8+ T cell-mediated cytotoxicity.

LTX-315 promotes immunogenic cell death in liver cancer cells and activation of dendritic cells and CD8+ T cells after nsPEF treatment
Both LTX-315 and nsPEF were capable of inducing ICD in tumor cells, as evidenced by the release of ATP and HMGB1 and the exposure of CRT. To determine whether LTX-315 further augments nsPEF-induced ICD in liver cancer cells, luciferase-based ATP and ELISA assays showed significantly increased levels of ATP and HMGB1 in the supernatants of cells treated with either LTX-315 or nsPEF compared with the control group. Notably, the combination treatment group exhibited a further increase in ATP and HMGB1 release (figure 4A), as well as enhanced CRT exposure as observed by confocal microscopy (figure 4B). To investigate the downstream impact of ICD on DCs and CD8+ T cells, a triple co-culture system was established comprising Hepa 1–6 cells, DCs, and CD8+ T cells (figure 4C). Regarding DC activation, a significant increase in CD86+ DCs was noted in the LTX-315 group compared with the control group. The percentages of CD80+ and CD86+ DCs were significantly increased when co-cultured with nsPEF-treated Hepa 1–6 cells. Furthermore, the combination treatment group exhibited significantly higher proportions of CD80+ and CD86+ DCs than the nsPEF group (figure 4D).
CD80 and CD86 molecules on activated DCs interact with CD28 on T cells to induce T cell activation. To assess the impact of LTX-315/nsPEF-induced DC activation on CD8+ T cells cytotoxicity, CD8+ T cells were collected after co-culture for flow cytometry. Significantly higher proportions of Granzyme B+, IFN-γ+, TNF-α+, IL-2+and Perforin+CD8+ T cells were noted when co-cultured with DCs exposed to Hepa 1–6 cells pretreated with LTX-315 or nsPEF compared with control group. In the combination treatment group, the proportions of IFN-γ+, Granzyme B+, Perforin+, and IL-2+CD8+ T cells were significantly higher than those in the nsPEF group, except for TNF-α+ CD8+ T cells (figure 4E). These findings suggested that LTX-315 enhanced nsPEF-induced ICD in liver cancer cells, thereby promoting the activation of DCs and CD8+ T cells.

LTX-315 enhances endogenous antigen processing and presentation in liver cancer cells following nsPEF treatment
Transcriptomic analysis was performed on nsPEF-treated Hepa 1–6 cells with or without subsequent LTX-315 exposure, to explore the underlying immunomodulatory effects of LTX-315 on nsPEF treatment. The results revealed that LTX-315 substantially altered the gene expression profile of nsPEF-treated cells (online supplemental figure 4A). Gene set enrichment analysis indicated significant enrichment of multiple immune-related pathways on LTX-315 treatment, including antigen presentation (folding, assembly and peptide loading of class I major histocompatibility complex (MHC)), endoplasmic reticulum (ER)-phagosome pathway, inflammatory response, peptide antigen binding, protein processing in ER, T cell receptor binding, immunoregulatory interactions between a lymphoid and a non-lymphoid cell, and antigen processing and presentation of endogenous peptide antigen via MHC class Ib (online supplemental figure 4B). To validate the enhancement of antigen presentation induced by the combination therapy, flow cytometry was employed to assess MHC-I molecule expression on tumor cells. The combination therapy led to an even greater upregulation of H-2 (murine MHC-I) expression than either LTX-315 or nsPEF treatment alone (online supplemental figure 4C). In summary, LTX-315 enhanced nsPEF-induced processing and presentation of endogenous antigens in liver cancer cells.

LTX-315 augments the cytotoxic effects of nsPEF against liver cancer cells
In addition to the immunomodulatory effects, the cytotoxic effect of LTX-315 on liver cancer cells following nsPEF treatment was evaluated. Transmission electron microscopy revealed more pronounced cell damage in the combination treatment group compared with either treatment alone, including nuclear dissolution, mitochondrial swelling, membrane disruption and cytoplasmic matrix dissolution (figure 5A). Besides, the combination treatment induced a significantly higher apoptosis rate in Hepa 1–6 and SMMC-7721 cells than either single treatment (figure 5B). Furthermore, a marked reduction in the number of colony-forming cell clusters was observed in both LTX-315 and nsPEF groups compared with the control group, with an even greater decrease in the combination treatment group (figure 5C). Transwell assays demonstrated that the number of liver cancer cells migrating through the polycarbonate membrane, with or without Matrigel coating, was significantly reduced in both LTX-315 and nsPEF groups compared with the control group, and further decreased in the combination group (figure 5D). Collectively, the addition of LTX-315 enhanced nsPEF-induced apoptosis and weakened the colony-forming, invasion and migration abilities of liver cancer cells.

The combination of nsPEF and LTX-315 inhibits tumor growth by remodeling the tumor immune microenvironment
In this study, nsPEF was combined with LTX-315 for the first time to evaluate their potential synergistic therapeutic efficacy against liver cancer in vivo, and a microneedle and microsyringe system was used to LTX-315 delivery (online supplemental figure 5A,B). Tumor response to varying numbers of nsPEF pulses indicated that tumor growth inhibition intensified with increasing pulse numbers (online supplemental figure 6A). Considering the clinical reports of tumor residue and recurrence following nsPEF, a mild nsPEF regimen (20 kV/cm, 100 ns, 1 Hz, 100 pulses) was applied to mimic these conditions in vivo. In addition, a previous study reported that intratumoral administration of LTX-315 significantly inhibited tumor growth and PD-L1 expression at a dose of 0.5 mg/mouse two times a week.21 In this study, 1 mg LTX-315 per mouse once was adopted to reduce injection frequency and minimize animal discomfort, due to comparable tumor growth between the regimens of 0.5 mg, two times a week and 1 mg once (online supplemental figure 6A). Furthermore, a significant reduction was observed in CD31+ vascular density in LTX-315-treated tumors, while nsPEF-treated tumors remained largely unchanged (online supplemental figure 6B), suggesting a rapid vasculature disruption effect of LTX-315 in liver cancer. Therefore, performing nsPEF prior to LTX-315 injection may preserve transient vascular integrity, facilitating the infiltration of activated immune cells into the tumor. Besides, the sequential administration of LTX-315 1 day after nsPEF resulted in optimal antitumor efficacy (online supplemental figure 6C), which was adopted for further treatment regimen.
The combination treatments elicited a more potent and sustained suppression of tumor growth with over 50% reduction in tumor volume and weight compared with the control, LTX-315 and nsPEF groups (figure 6A). Furthermore, tumor-bearing mice subjected to combination treatment demonstrated significantly prolonged survival in comparison to the nsPEF group (33 days vs 24 days, p<0.05) (figure 6B). To evaluate the impact of combination treatment on tumor-infiltrating immune cells, tumor tissues were harvested for immunofluorescence staining and confocal imaging. Notably, the number of CD8+ TILs was markedly nearly doubled in the combination group compared with the nsPEF group, while the number of PD-L1+ cells was markedly reduced (figure 6C). Tumors treated with either LTX-315 or nsPEF alone showed significant decreases in Ki67+ cells, CD11b+ myeloid-derived suppressor cells and Tregs (Foxp3+) compared with controls, with an even more pronounced reduction in the combination group. In contrast, there was a markedly increased number of macrophages (F4/80+) in both LTX-315 and nsPEF groups without further elevation in the combination group. In addition, NCR1+ NK cells were increased in the LTX-315 group, but not in the nsPEF or combination groups (online supplemental figure 7).
It is reported that the antitumor immunity of both LTX-315 and nsPEF largely depends on CD8+ T cells.22 CD8+ T cell depletion study showed the depletion of CD8+ T cells abolished the antitumor effects of the combination treatment (online supplemental figure 8A,B), suggesting that CD8+ T cells are indispensable for the therapeutic efficacy of nsPEF plus LTX-315 treatment. In tumor-draining lymph nodes (TdLNs), type 1 conventional DCs (cDC1s) are regarded as one of the most important antigen-presenting cells for CD8+ T cell activation in tumor immunity. The validation of tumor-infiltrating immune cells illustrated that the combination therapy boosted the infiltration of cDC1s (CD45+MHC-II+CD11c+CD11b-XCR1+) within tumor compared with nsPEF alone (online supplemental figure 9A,B). In parallel, the frequencies of Granzyme B+, IFN-γ+, TNF-α+, IL-2+ and Perforin+CD8+ TILs were significantly elevated compared with nsPEF-treated tumors (figure 6D). In addition, we observed that the combination of nsPEF and LTX-315 promoted the accumulation of migratory DCs (MHC-IIhiCD11cint) and XCR1+ cDC1 in TdLNs compared with nsPEF or LTX-315 alone (online supplemental figure 10). Collectively, LTX-315 cooperated with nsPEF to enhance the migration of DCs to TdLNs and cDC1s infiltration within TdLNs and tumor to restore CD8+ TILs cytotoxicity within the TME.

Immune memory against tumor metastasis elicited by the combination treatment
Immunological memory plays a pivotal role in protecting against tumor recurrence, characterized by the generation of central memory T cells (Tcm, CD44+CD62L+) and effector memory T cells (Tem, CD44+CD62L-).23 To evaluate the immune memory response induced by the combination treatment, the peripheral blood was collected from mice 16 days post-treatment for flow cytometric analysis (figure 6E). As illustrated in figure 6F, the proportions of CD4+ Tem and CD8+ Tcm cells in both LTX-315 and nsPEF groups were significantly higher than those in the control group, whereas the combination group exhibited the most pronounced increase. It is noteworthy that nsPEF alone did not increase CD4+ Tcm cells; however, the combination treatment significantly elevated CD4+ Tcm cells compared with nsPEF alone. Subsequently, the inhibitory effects of various treatments on pulmonary tumor metastasis were assessed. As shown in online supplemental figure 11, the control group developed numerous large metastatic nodules, whereas markedly fewer metastases were observed in both LTX-315 and nsPEF groups, with the combination treatment displaying the lowest metastatic burden.
These findings suggested that the administration of LTX-315 elicited a robust immune memory-like response capable of preventing tumor metastasis, which contributed to the prolonged survival in nsPEF-treated mice.

Safety of the combination treatment of nsPEF and LTX-315
The safety profile of this therapeutic approach was evaluated, as the combination of nsPEF and LTX-315 was applied for the first time. No significant differences in body weight were observed among the treatment groups (figure 7A). H&E staining of the heart, liver, spleen, lungs, and kidneys revealed no pathological abnormality or structural damage (figure 7B). Furthermore, the peripheral blood analysis indicated normal levels of white blood cells, hemoglobin, platelets, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, and creatinine (figure 7C). Taken together, the combination treatment of nsPEF and LTX-315 was safe and well tolerated in mice.

Discussion
Recent studies have explored the effects of nsPEF on tumor growth and tumor immune microenvironment.13 24 Single-cell sequencing analysis has further elucidated that nsPEF promoted the infiltration of DCs and immunosuppressive monocytes/macrophages in pancreatic cancer.25 Nonetheless, Rossid et al failed to observe nsPEF-induced antitumor immune response in mouse melanoma model.26 Another study reported a significant increase in tumor-infiltrating TNF-α+ and IFN-γ+ CD8+ T cells in mouse subcutaneous liver cancer treated with nsPEF at 20 kV/cm, 100 ns, 1 Hz, and 300 pulses.12 However, when the pulse number was converted to 100 pulses, there were comparable populations of TNF-α+ and IFN-γ+ CD8+ T cells, accompanied by increased exhausted PD-1+ and LAG-3+CD8+ T cells.15 These inconsistent results may be attributed to the differences in tumor models and nsPEF pulse parameters. In light of the potential tumor residue associated with non-parallel electrode placement and recurrence and metastasis in patients with liver cancer after nsPEF,14 16 we employed a mild nsPEF regimen (20 kV/cm, 100 ns, 1 Hz, 100 pulses) to mimic these clinical conditions in vivo.
Our study demonstrated that mild nsPEF lacked long-term antitumor efficacy so that tumor growth began to resume during the mid-to-late stages post-treatment. We hypothesize that the transient inhibition of tumor growth observed in the early phase following nsPEF was primarily attributed to the apoptosis and necrosis of tumor cells. Subsequently, surviving cells upregulated PD-L1 expression and led to CD8+ TIL exhaustion through the PD-L1/PD-1 axis, ultimately promoting immune escape and tumor progression. To address this, LTX-315 was applied in our study to reverse the nsPEF-mediated increase in PD-L1 expression and restore the cytotoxic function of CD8+ T cells. Furthermore, previous studies have used nsPEF to promote the intracellular delivery of various molecules including drugs by generating membrane electroporation and increasing membrane permeability.27 28 Accordingly, the administration of LTX-315 after nsPEF may facilitate its intracellular uptake and potentiate its oncolytic activity in the present study. In addition, LTX-315 enhanced nsPEF-induced inhibition of cell proliferation, induction of ICD, activation of DCs and CD8+ T cells, reduced infiltration of immunosuppressing cells and generation of potential antitumor immune memory. Consequently, the combination of nsPEF and LTX-315 achieved durable tumor control and improved survival in mice by remodeling the tumor immune microenvironment and triggering systemic antitumor immune response.
The translocation of PD-L1 to the plasma membrane surface and its release into extracellular matrix in the form of extracellular vesicles may represent a major mechanism underlying the upregulation of PD-L1 levels following nsPEF treatment.15 It is reported that the lysosomal degradation pathway accounted for the LTX-315-induced reduction of PD-L1 expression in pancreatic cancer cells. Specifically, ATP11B serves as a critical regulator to maintain PD-L1 expression in cancer cells, and its downregulation induced by LTX-315 promotes CMTM6-mediated lysosomal degradation of PD-L1.21 Consistently, our results demonstrated that LTX-315 downregulated PD-L1 expression in liver cancer cells through the same axis. Notably, the administration of LTX-315 effectively reversed the nsPEF-induced PD-L1 upregulation, and consequently restored the cytotoxic activity of CD8+ T cell against tumor cells in co-culture systems.
A significant increase in tumor-infiltrating activated DCs has been observed in a mouse pancreatic cancer model treated with nsPEF,25 while the impact of LTX-315 on DCs remains unclear yet. In our study, the co-culture models comprising liver cancer cells, DCs and CD8+ T cells revealed that LTX-315 induced ICD in tumor cells, and cooperated with nsPEF to further enhance ICD-mediated activation of DCs and CD8+ T cells. It is noteworthy that LTX-315 selectively targets tumor cells characterized by elevated levels of anionic phospholipid, while sparing DCs and other immune cells.29
CD8+ T cells are pivotal for antitumor immunity owing to their potent cytotoxic capacity against malignant cells. A previous study has revealed that nsPEF accelerated tumor progression in CD8+ T cell-deficient rats but inhibited tumor growth in immunocompetent rats, highlighting the dependence of nsPEF-induced antitumor effects on CD8+ T cells.30 In our study, the depletion of CD8+ T cell abolished the therapeutic efficacy of nsPEF combined with LTX-315, which underscored the indispensable role of CD8+ T cells in combination treatment, consistent with previous observations.22 31 Moreover, the integration of nsPEF with LTX-315 boosted tumor antigen processing and presentation, CD8+ T cells recognition and infiltration, and production of Granzyme B, IFN-γ, TNF-α, IL-2 and Perforin. These changes collectively amplified the cytotoxic activity of CD8+ T cells against tumor cells compared with nsPEF monotherapy.
Beyond local tumor control, LTX-315 and nsPEF acted cooperatively to elicit a more robust and durable immune memory response. Notably, nsPEF has been shown to prevent tumor relapse for up to 8 months post-treatment by expanding memory T cell populations in the peripheral blood and spleen of rats.13 Similarly, in mouse breast cancer models, nsPEF treatment was associated with reduced distant metastases by increasing memory CD4+ and CD8+ T cells in spleen.31 However, the contribution of LTX-315 to the establishment of immune memory has not been previously explored. Our findings provided the evidence that LTX-315 may initiate the potential generation of immune memory, which was further potentiated when combined with nsPEF. Mechanistically, the enrichment of migratory DCs and cDC1s in TdLNs reflects enhanced antigen transport from the treated tumor and promotes a priming-competent LN microenvironment. It may be associated with improved T-cell priming quality and expansion of memory-phenotype T cells, due to enhanced antigen cross-presentation and costimulatory signals delivery to T cells.32 33 In this study, the increased migratory DCs and cDC1s in TdLNs after combination treatment, relative to nsPEF alone, provide potential stronger basis for LN remodeling and durable T-cell memory programming. This synergistic interaction of durable antitumor immunity holds promise for preventing liver cancer recurrence and metastasis (figure 8).
This study has several limitations. First, the subcutaneous liver tumor model was used in this study to facilitate the intratumoral injection, ablation needle placement and tumor size monitoring for the consistency across experiments. However, the orthotopic liver tumor models would more accurately replicate the hepatic TME; thus, we intend to further validate and extend our findings using orthotopic models in future research. Second, the precise molecular mechanism by which nsPEF modulates PD-L1 expression in liver cancer cells remains to be elucidated. Third, although the multisite intratumoral injection strategy improved local delivery, it could not ensure a homogeneous distribution of LTX-315 within the tumor. Several studies have proposed various nanocarrier systems for systemic delivery of LTX-315, including polymer-lipid hybrid nanoparticle and chimeric polymersomes, which enabled its serum stability and tumor accumulation. Therefore, future exploration of biomaterial-based intravenous delivery may represent a promising strategy for LTX-315 optimization. Fourth, because the efficacy of nsPEF is critically influenced by the distribution, intensity and electric field duration, further investigations are needed to refine electrode design and optimize both spatial and temporal dose distribution of nsPEF to maximize therapeutic outcomes.

Supplementary material
10.1136/jitc-2025-012438online supplemental file 1