Abstract

Background

   Irreversible Electroporation (IRE) offers a promising treatment for
   pancreatic cancer by using high-voltage pulses to kill tumor cells. But
   variations in tumor size and shape can lead to uneven electric fields,
   causing some cells to undergo only reversible electroporation (RE) and
   survive. However, RE can temporarily increase the permeability of the
   cell membrane, allowing small molecules to enter. H101 virus is an
   oncolytic adenovirus with deleted E1B-55kD and E3 regions that
   selectively targets and kills tumor cells. This study aimed to
   investigate whether the H101 oncolytic virus can serve as a
   supplementary therapeutic approach to kill tumors combined with RE.

Methods

   We first explored how RE and the H101 oncolytic virus, both
   individually and together, affected tumor cell proliferation and
   migration in cellular experiments. Subsequent in vitro studies further
   assessed the effects of different treatments on tumor growth. To
   understand the mechanisms of pathway changes in tumors from different
   treatment groups, we analyzed tumor samples from each group using bulk
   RNA sequencing (bulk RNA-seq) and single-cell RNA sequencing
   (scRNA-seq). Additional biochemical techniques were used to validate
   key molecular changes.

Results

   The combination of RE with the H101 oncolytic virus effectively
   inhibited pancreatic cancer cell proliferation and migration.
   Experiments using mouse subcutaneous tumor models confirmed that the
   combination therapy significantly reduced tumor growth. Further
   analysis bulk RNA-seq and scRNA-seq revealed that this combined
   approach activates the JNK-MAPK pathway, inducing apoptosis and
   enhancing therapeutic effects.

Conclusions

   This combination boosts therapeutic effectiveness by activating the
   JNK-MAPK pathway and promoting tumor cell apoptosis. These findings
   suggest that the H101 oncolytic virus could serve as a valuable adjunct
   to improve the efficacy of IRE treatment.

   Keywords: irreversible electroporation (IRE), reversible
   electroporation(RE), H101 oncolytic virus, JNK-MAPK, apoptosis

1. Introduction

   Pancreatic cancer (PC) is a highly aggressive gastrointestinal
   malignancy, ranking ninth in global incidence but fourth in mortality
   ([37]1). Surgery remains the only curative option for PC, yet only
   about 20% of patients are eligible for surgical intervention at the
   time of diagnosis ([38]2). Additionally, approximately 40% of cases are
   diagnosed as locally advanced pancreatic cancer (LAPC), characterized
   by vascular invasion that precludes immediate surgical resection
   ([39]3). For LAPC patients, the effectiveness of radiotherapy and
   chemotherapy is limited, and only a small subset qualify for conversion
   surgery. However, the success rates for such procedures are low, and
   postoperative complications are significant ([40]4, [41]5). This
   underscores the urgent need for innovative treatment strategies.

   Irreversible electroporation (IRE) is an emerging tumor ablation
   technique that differs from traditional thermal or cryoablation methods
   by not relying on temperature changes to destroy tumor cells. Instead,
   it uses high-voltage, short-duration electrical pulses to create
   irreversible nanopores in tumor cell membranes, altering their
   permeability and inducing cell death. IRE overcomes challenges such as
   heat sink effects and reduces thermal injury to adjacent structures,
   preserving vital blood vessels, bile ducts, and pancreatic ducts within
   the ablation zone ([42]6). This results in fewer postoperative
   complications, including pancreatic and biliary fistulas and
   infections, thereby improving outcomes for LAPC patients ([43]7,
   [44]8). However, the success of IRE depends on the precise application
   of electrical pulses, which can be influenced by tumor geometry,
   electrode positioning, and the electrical properties of surrounding
   tissues ([45]9). Given the aggressive and irregular nature of
   pancreatic cancer, areas of insufficient field intensity within the
   tumor can result in incomplete ablation. This phenomenon, termed
   reversible electroporation (RE), may paradoxically stimulate tumor
   growth ([46]10–[47]12). According to a previous research, electric
   field intensities below 1000 V/cm predominantly induced RE rather than
   IRE ([48]13). Consequently, combining IRE with supplementary therapies
   is essential to enhance its efficacy.

   The H101 oncolytic virus, a recombinant virus selectively targeting
   cancer cells while sparing normal ones ([49]14), has shown promise in
   treating advanced cancers such as nasopharyngeal carcinoma and melanoma
   ([50]15, [51]16). The virus exerts its anticancer effects through
   multiple mechanisms, including direct infection and lysis ([52]17),
   induction of immunogenic cell death ([53]18), and targeting of
   tumor-associated fibroblasts and other stromal cells, thereby
   disrupting the complex tumor structure and promoting the infiltration
   of cytotoxic immune cells ([54]19). Previous literature also indicated
   that IRE can reshape the tumor microenvironment by enhancing
   microvascular density and tumor vascular permeability ([55]20).
   Additionally, the membrane pores created by IRE or RE can facilitate
   the entry of drugs and genes ([56]21, [57]22), potentially increasing
   the local concentration of the H101 virus within tumors and eliminating
   residual cancer cells that survive after RE.

   In this study, we found that RE alone did not induce cytotoxicity but
   instead enhanced cellular migration. However, combining RE with the
   H101 oncolytic virus demonstrated significantly improved tumor-killing
   effects and reduced pancreatic tumor migration compared to H101 virus
   treatment alone. These findings suggested that the H101 oncolytic virus
   is a potent complementary therapy, capable of enhancing the efficacy of
   IRE for the treatment of pancreatic cancer.

2. Materials and methods

2.1. Cell culture and viruses

   The Bxpc-3 human pancreatic adenocarcinoma cell line and the Pan02
   murine pancreatic adenocarcinoma cell line were obtained from the Cell
   Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were
   maintained at 37°C in a 5% CO2 humidified incubator using DMEM
   (C11995500BT, Gibco) or RPMI 1640 (R8758, Gibco) media, both
   supplemented with 10% heat-inactivated fetal bovine serum (A5669701,
   Gibco) and 1% Penicillin-Streptomycin (15140122, ThermoFisher). The
   H101 oncolytic virus was generously donated by Shanghai Sunway Biotech
   (Shanghai, China).

2.2. Establishment of animal models

   In this study, 6-week-old C57BL/6 female mice were used. All procedures
   involving animals were authorized by the Animal Care and Use Committee
   of Sun Yat-sen University (ID, L102012021009A). 1×10^6 Pan02 cells were
   implanted into the right flank of C57BL/6 mice. After a period of 2
   weeks, palpable tumors with a diameter of approximately 7 to 8 mm were
   formed. The dimensions of the tumors, including length (A) and width
   (B), were assessed, and the tumor volume was determined using the
   formula: V = (A × B^2)/2. Upon reaching a tumor volume of 200-250 mm³,
   the mice were randomly assigned to four groups. The first group
   received intratumor injections of PBS three times a week, serving as a
   placebo. The second group received low-field electrical stimulation
   once a week. The third group received intratumor injections of H101
   virus (1×10^8 TCID50) three times a week. The fourth group received a
   combination of intratumor injections of H101 virus (three times a week)
   and low-field electrical stimulation (once a week). Tumors were treated
   with intratumoral virus injection immediately after exposure to
   low-field intensity stimulation. The dimensions of the tumors were
   recorded every 3 days until the sacrifice on day 35.

2.3. Electroporation

   The in vivo and in vitro electroporation experimental methods are as
   described in our previous article ([58]23). ECM 830 electroporator (BTX
   Harvard Apparatus, Holliston, MA) was used to generate electric pulses.
   For in vitro experiments, a 400-μL cell suspension of 1×10^6 cells,
   post-digestion and counting, was positioned in an electroporation
   cuvette (1652088; BTX, Holliston, MA, USA) between two 4-mm spaced
   aluminum electrodes. For in vivo experiments, electroporation was
   conducted using a 2-needle array electrode (BTX item #45–0168, BTX
   Harvard Apparatus, Holliston, MA) with a 5 mm gap. The electrode was
   inserted sequentially into the tumor along the X, Y, and Z axes to
   ensure the efficacy of electroporation. The parameters for
   electroporation were as follows: voltage: 150 V; pulse duration: 100
   ms; pulse frequency: 1 Hz; pulse number: 80.

2.4. Cell viability assay

   Cell Counting Kit-8 (CCK8) Assay Cell viability was analyzed using the
   cell counting kit-8 (CCK-8) assay (HYC500, HUAYUN). Triturated cells
   were seeded in 96-well flat-bottomed plates at a density of 2*103 in
   100 μL of conditioned medium per well, and 10 μL of CCK-8solution was
   added to each well. After 2 h of incubation, the absorbance of each
   well at a wavelength of 450 nm was quantified.

2.5. Flat plate clone formation

   Post-treatment, 500 cells were plated into each well of a 6-well plate
   and cultured for 14 days, with media changes occurring every 3 days.
   The cells were then fixed using a 4% formaldehyde solution and stained
   with crystal violet (E607309-0100, Sangon Biotech, Shanghai, China).
   Subsequently, the colonies were counted and subjected to analysis.

2.6. Cell scratch and transwell migration assay

   For the scratch wound assay, the tumor cells of different treatment
   groups were counted as 1×10^5 and then placed in 12 well plates.
   Subsequently, the cell layer was scratched with the tip of a 20 µL
   sterile pipette to create a wound gap. The migrated rate is quantified
   as the distance of wound closure at 0 and 24 hours relative to the
   initial wound length. For the transwell migration assay, 8 μm pore size
   transwell chambers (353097, Corning) were utilized. The lower chamber
   was supplemented with medium containing 20% FBS. After teatment, the
   cells were counted to 1×10^4 and placed separately in the upper
   chamber. After 24 hours, cancer cells that had penetrated and adhered
   to the underside of the filter were fixed with paraformaldehyde and
   stained with crystal violet. They were then imaged and counted under a
   20X objective lens. The statistical analysis of migrating cell counts
   was based on data from three replicate experiments, with the average
   determined from assessments of five microscopic fields per experiment.

2.7. Western blotting

   The protein lysates were generated using RIPA buffer, resolved by
   sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel, moved to
   polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA),
   and incubated with primary antibodies at 4°C overnight. The following
   antibodies were utilized: GAPDH (10494-1-AP, Proteintech), Snail
   (A11794, ABclonal), Vimentin (60330-1-Ig, Proteintech), E-cadherin
   (20874-1-AP, Proteintech), N-cadherin (22018-1-AP, Proteintech),
   Phospho-ERK1/2 (28733-1-AP, Proteintech), ERK (66192-1-Ig,
   Proteintech), Phospho -P38 (28796-1-AP, Proteintech), P38 (14064-1-AP,
   Proteintech), Phospho -JNK (80024-1-RR, Proteintech), JNK (24164-1-AP,
   Proteintech), Bc1-2 (60178-1-Ig, Proteintech), BAX (50599-2-Ig,
   Proteintech). After three washes with Tris-buffered saline containing
   0.05% Tween-20, the membranes were incubated with either secondary
   anti-rabbit (AS003, ABclonal) or anti-mouse antibodies (SA00001-1,
   Proteintech) for 1 hour at room temperature. Images were detected by
   Tanon-5200 chemiluminescent imaging system (Tanon, Shanghai, China).
   The signals were detected using the ECL Advance reagent ([59]P10300,
   New Cell & Molecular Biotech Co.,Ltd) and quantified with ImageLab
   software. The experiment was conducted in triplicate.

2.8. Immunohistochemistry assay

   4-μm-thick tumor sections were deparaffinized in xylene, rehydrated
   through a graded series of ethanol, and incubated in 0.3% H2O2 in
   methanol for 30 minutes. After washing with PBS, the sections were
   probed with a monoclonal antibody (ab15580, Abcam) at 4°C overnight.
   After washing, the sections were incubated with biotinylated goat
   anti-rabbit IgG for 2 hours at room temperature. Immunostaining was
   then detected using a streptavidin-horseradish peroxidase conjugate and
   diaminobenzidine. Subsequently, the sections underwent counterstaining
   with hematoxylin.

2.9. Flow cytometry

   Following the manufacturer’s guidelines, the Annexin V-FITC/PI
   apoptosis detection kit (GOONIE, Cat. No. 100-101) was employed to
   assess cellular apoptosis. Cells were collected at a concentration of
   1×10^5 cells per tube and stained in duplicate with 5 μl of APC-Annexin
   V conjugate and 5 μl of propidium iodide solution (10 μg/ml), with the
   staining process conducted in the dark for a duration of 30 minutes.
   Subsequently, the cells were analyzed using the CytoFLEX S system
   (Beckman Coulter, USA) via flow cytometry. The percentage of apoptotic
   cells was referred to as the apoptosis rate.

2.10. Tissue dissociation and cell purification

   The dissociation and purification process of the tissue was as
   previously described ([60]24). In short, the tissue were transported on
   ice in DMEM containing 1 mM protease inhibitor (CW2200S, CWBIO) to
   maintain viability, washed three times with PBS, then minced on ice. To
   digest the tissues, we employed a dissociation enzyme mixture
   containing 1 mg/ml of Type VIII Collagenase (C2139, Sigma), 2 mg/ml of
   Dispase II (4942078001, Sigma), 1 mg/ml of Trypsin Inhibitor (T6522,
   Sigma-Aldrich), and 1 unit/ml of DNase I (M0303S, NEB), all dissolved
   in PBS supplemented with 5% FBS. After filtration through a 40 μm nylon
   cell strainer (model 352340, Falcon), the cell suspension underwent
   erythrocyte removal using red blood cell lysis buffer (1966634,
   Invitrogen). Subsequently, cells were stained with 0.4% trypan blue
   solution ([61]T10282, Invitrogen) to discern cell viability. Finally,
   the cells were diluted in PBS containing 0.04% bovine serum albumin
   (BSA) to approximately 1 × 10^6 cells per milliliter for single-cell
   sequencing applications.

2.11. 10× library preparation and sequencing

   Cells were prepared in accordance with the standardized protocol of the
   Chromium single-cell 3′ reagent kit, aiming to capture a range of 5000
   to 10,000 cells per chip position (utilizing V2 chemistry). Subsequent
   steps, encompassing library preparation, were executed following the
   manufacturer’s established guidelines meticulously.

2.12. Single cell RNA-seq data processing

   Illumina HiSeqXTen platforms were utilized for the sequencing of
   single-cell libraries, employing a paired-end approach with 150
   nucleotide reads. The data obtained were then subjected to analysis via
   the Cell Ranger 2.1.0 software suite, adhering to the preset and
   advised settings. The resulting FASTQ files from the Illumina
   sequencing were mapped to the GRCm39 reference genome using the STAR
   alignment method ([62]25). The processed output was subsequently loaded
   into Seurat version 5.0.0 to conduct quality control and further
   analysis on our scRNA-seq dataset. Unless otherwise stated, all
   operations were performed using the default settings. Cells of inferior
   quality, characterized by having fewer than 300 genes per cell, fewer
   than 5 cells per gene, or a mitochondrial gene expression exceeding
   15%, were removed from the dataset.

2.13. Identification of cell types and subtypes by nonlinear dimensional
reduction (umap)

   The R-based Seurat package was utilized to discern primary cell types.
   A set of highly variable genes was derived and leveraged for conducting
   Principal Component Analysis (PCA). These clusters were then visualized
   through UMAP, utilizing the first ten principal components (PCs)
   previously computed via the RunUMAP function. To delineate the cellular
   identities within these clusters, we referenced the expression of
   established markers: Ptprc for immune cells, Krt8 and Krt19 for
   epicardial cells, Pecam1 for endothelial cells, and Acta2 for stromal
   cells ([63]26). Cluster-defining marker genes were pinpointed by
   executing the FindAllMarkers function within the Seurat package,
   applied to the normalized gene expression dataset.

2.14. Bulk RNA-seq

   In brief, we began with 500ng of purified RNA with a high integrity
   score (RIN >7), which was selectively targeted for polyadenylated mRNA.
   This was followed by a series of steps including fragmentation,
   synthesis of cDNA using random primers with the NEBNext protocol,
   indexing via PCR, and then selecting and measuring the cDNA fragments
   with KAPA reagents from Roche. The prepared cDNA libraries were then
   sequenced on the Illumina NovaSeq 6000 platform. The alignment to the
   GRCm39 reference genome and the counting of reads were executed using
   the previously outlined methods ([64]26), employing the STAR aligner
   (version 2.7.8), featureCounts (version 1.6.4), and Ensembl gene
   transcripts (version 104). The analysis of differential gene expression
   was conducted using the DESeq2 software package (version 1.30)
   ([65]27).

2.15. Biological signaling pathways analysis

   For the biological pathways of differential genes, we conducted Go
   (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), and
   GSEA (Gene Set Enrichment Analysis) analyses using the
   “clusterProfiler” (V.4.8.3) R package.

2.16. Statistical analysis

   Statistical analyses were conducted utilizing GraphPad Prism version
   8.00 software (GraphPad Software, La Jolla, CA, USA). Appropriate
   statistical tests, including Student’s t-test or analysis of variance
   (ANOVA), were selected to compare different groups in the in vitro
   study. Repeated-measures one-way ANOVA was employed to analyze tumor
   volume data. Standard deviations (SDs) were represented by error bars.
   Statistical significance was set at a P value of less than 0.05.

3. Results

3.1. Low-intensity electric fields enhance cell migration without causing
cytotoxicity

   We examined the effects of varying electric field intensities (100 V/cm
   to 1500 V/cm, including intermediate levels of 300 V/cm, 750 V/cm, and
   1000 V/cm) on tumor cell viability and migration. Cells were exposed to
   20 pulses, with each pulse lasting 100 microseconds at a frequency of 1
   Hz. Cytotoxicity was assessed immediately using CCK8 and plate colony
   formation assays. Results showed no significant cytotoxicity below 750
   V/cm, with most cells surviving intact ([66] Figures 1A, B ). Migration
   assays (scratch and transwell tests) demonstrated enhanced tumor cell
   migration at low-field intensities (<750 V/cm), while intensities above
   this threshold significantly inhibited migration ([67] Figures 1C, D ).
   Western blot analysis revealed that low-field stimulation activated
   epithelial-to-mesenchymal transition (EMT), as evidenced by increased
   expression of Snail, N-cadherin, and vimentin, along with decreased
   E-cadherin expression ([68] Figure 1E ).

Figure 1.

   [69]Figure 1
   [70]Open in a new tab

   Low-Intensity Electric Fields Enhance Cell Migration Without Causing
   Cytotoxicity. (A), The CCK-8 results for tumor cell survival rates
   after stimulation with different field strength groups, CCK-8 assay
   results showed that electric field intensities below 300 V/cm have no
   significant cytotoxic effect, while high-intensity fields (≥1000 V/cm)
   significantly reduce cell viability. (B), Colony formation assay of
   cell viability under different field strengths, no significant
   cytotoxicity was observed with electric field intensities below 750
   V/cm, while high-intensity fields (≥1000V/cm) significantly reduced
   colony formation. (C, D), Representative images of the transwell
   migration assay and cell scratch assay (at 0h and 24h), low-intensity
   electric fields (≤300 V/cm) enhance cell migration, while
   high-intensity fields (≥1000 V/cm) inhibit migration, scale bar, 100
   μm. (E), Western blot analysis of EMT markers in tumor cells treated
   with different electric field intensities for 24 hours. Low-intensity
   fields (100–750 V/cm) activate the EMT pathway, as indicated by
   increased N-cadherin, Vimentin, and Snail expression and decreased
   E-cadherin expression. High-intensity fields (≥1000 V/cm) inhibited
   EMT, as shown by the opposite expression pattern. Ns, not significant;
   *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001, significant
   difference compared with the control. Data are presented as mean ± SD.

3.2. Combined RE and H101 virus treatment suppresses tumor growth

   To evaluate the H101 virus’s cytotoxic effects, tumor cells were
   infected with an MOI of 100, and cell viability was monitored at 24,
   48, and 72 hours post-infection using CCK8 assays. The results
   demonstrated a time-dependent decline in cell viability following H101
   virus infection ([71] Figure 2A ). Colony formation tests showed that
   the combined treatment with low-field intensity and H101 virus produced
   fewer and smaller colonies compared to treatments with low-field
   intensity or H101 virus alone ([72] Figure 2B ). In vivo experiments
   using mouse models further confirmed these findings, as tumor growth
   was significantly suppressed in the combination therapy group compared
   to individual treatments ([73] Figures 2C-E ). Immunohistochemical
   analysis of tumor tissues revealed reduced proliferation markers, such
   as Ki-67, in the combined treatment group ([74] Figure 2F ). These
   results confirm that the combination therapy effectively inhibits tumor
   growth both in vitro and in vivo.

Figure 2.

   [75]Figure 2
   [76]Open in a new tab

   Combined RE and H101 Virus Treatment Suppresses Tumor Growth. (A), The
   CCK-8 results for tumor cells after infection with a multiplicity of
   infection (MOI) of 100 at 24, 48, and 72 hours, a significant decrease
   in cell viability was observed. (B), Colony formation assay of tumor
   cells after different treatment, the results showed that H101 virus
   alone reduces cell viability, and the combination of 300 V/cm electric
   field with H101 virus further enhanced the cytotoxic effect. (C), The
   timeline and scheme of the experimental setup for subcutaneous animal
   experiments, 1×10^6 Pan02 cells were implanted into the right flank of
   C57BL/6 mice. Treatments included weekly low-field electrical
   stimulation and thrice-weekly intratumor injections of H101 virus
   (1×10^8 TCID50) over 35 days. Yellow lightning bolts denote electrical
   stimulation, and red arrows indicate virus injections. (D),
   Representative images of tumors from each group at day 35. (E), Tumor
   growth curve diagram, data represent mean ± SD for each group at the
   indicated time points. (F), Representative Ki-67
   immunohistochemicalstaining in subcutaneous tumor sections. Scale bar,
   100 μm. Ns, not significant; **p < 0.01; ***p < 0.001, ****p <
   0.0001,significant difference compared with the control.

3.3. Combined RE and H101 virus treatment inhibits tumor cell migration

   To evaluate the impact of low-field intensity stimulation in
   combination with H101 virus on cellular migration, cell migration
   assays (scratch and transwell tests) were performed and showed that
   while low-field intensity stimulation initially enhanced tumor cell
   migration, subsequent treatment with H101 virus reversed this effect
   ([77] Figures 3A, B ). Western blot analysis indicated that the EMT
   process induced by low-field stimulation was mitigated by the H101
   virus, as demonstrated by downregulation of Snail, N-cadherin, and
   vimentin, along with upregulation of E-cadherin ([78] Figure 3C ).

Figure 3.

   [79]Figure 3
   [80]Open in a new tab

   Combined RE and H101 Virus Treatment Inhibits Tumor Cell Migration. (A,
   B),The low-intensity electric field (300 V/cm) enhanced tumor cell
   migration, while the addition of the H101 virus partially inhibited it,
   scale bar, 100 μm. Data are presented as mean ± SD. Ns, not
   significant; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001. (C)
   Western blot analysis of EMT-related protein levels.The low-intensity
   electric field stimulation promoted the EMT, while H101 virus partially
   reversed this effect.

3.4. Bioinformatics analysis reveals MAPK pathway activation

   Tumor samples from mouse models treated with either low-field intensity
   alone or the combination therapy underwent bulk RNA-seq and scRNA-seq.
   Differentially expressed gene analysis from bulk RNA-seq indicated that
   the MAPK signaling pathway was the most significantly activated ([81]
   Figure 4A ). GO and GSEA corroborated this finding ([82] Figures 4B, C
   ). ScRNA-seq further detailed the pathway enrichment, with KEGG
   analysis confirming that the MAPK pathway was the primary enriched
   pathway across treatment groups ([83] Figures 4D-H ).

Figure 4.

   [84]Figure 4
   [85]Open in a new tab

   Bioinformatics Analysis Reveals MAPK Pathway Activation. (A), The top
   10 pathways activated by differential genes of bulk RNA-seq, the MAPK
   signaling pathway is significantly activated. (B), GO enrichment
   analysis of MAPK-related biological processes. Key processes include
   the activation and regulation of the MAPK cascade, as well as
   apoptosis-related pathways. (C), GSEA enrichment plot for the MAPK
   signaling pathway. The enrichment score curve indicates significant
   activation of the MAPK pathway. (D-F), UMAP visualization of
   single-cell RNA sequencing data. (D) Cells are grouped by experimental
   conditions(Low means low-Intensity electric field. H-low means H101
   virus plus low-Intensity electric field.) (E) Clusters of cells
   identified, labeled with cluster numbers. (F) Major cell types
   annotated, including epithelial, immune, and stromal populations. (G),
   Dot plot of gene expression across different cell types. Expression of
   various marker genes (Krt18, Krt19, Ptprc, Pecam1, Acta2) is shown for
   stromal, immune, and epithelial cells. (H), KEGG pathway enrichment
   analysis of scRNA-sequencing data. The MAPK signaling pathway is
   prominently enriched, ranked third in terms of gene ratio.

3.5. JNK-MAPK pathway activation induces apoptosis in tumor cells

   Based on the bioinformatics analysis results, we initially performed
   Western blot analysis to assess the expression levels of the three
   major MAPK subfamilies—JNK, ERK, and p38. It was showed that the
   combination therapy specifically enhanced JNK phosphorylation, with no
   significant effect on ERK or p38 expression ([86] Figure 5A ). The JNK
   signaling pathway, a key MAPK subfamily, plays a pivotal role in
   apoptosis. Flow cytometry and Western blot analyses demonstrated
   increased BAX expression, decreased BCL-2 expression, and a
   significantly higher apoptosis rate in the combination therapy group
   ([87] Figures 5B, D ). To explore JNK’s role in apoptosis, cells were
   pretreated with the JNK inhibitor SP600125. The inhibition of JNK
   reduced apoptosis, as shown by decreased BAX expression, increased
   BCL-2 expression, and lower apoptotic cell percentages ([88]
   Figures 5B, E ). These findings identify JNK as a critical mediator in
   apoptosis induction and highlight its role in the enhanced therapeutic
   efficacy of the combined low-field intensity and H101 virus treatment.

Figure 5.

   [89]Figure 5
   [90]Open in a new tab

   JNK-MAPK Pathway Activation Induces Apoptosis in Tumor Cells. (A),
   Western blot analysis of MAPK-related protein levels. Low-intensity
   electric field (300 V/cm) combined with H101 virus treatment
   significantly activates the JNK-MAPK pathway, as indicated by increased
   p-JNK levels. No significant changes were observed in other MAPK
   pathway markers. (B), Western blot analysis of apoptosis-related
   protein levels. The combined treatment significantly increased the
   expression of apoptotic markers (Bax). (C), Western blot analysis of
   JNK-MAPK and apoptosis-related proteins. Treatment with JNK inhibitor
   (SP600125, 10 µmol/L) suppresses the JNK-MAPK and apoptosis pathways,
   as indicated by the reduced levels of p-JNK, and BAX. (D), Flow
   cytometric analysis of apoptosis in tumor cells. Low intensity electric
   fields did not significantly increase the apoptotic cell population.
   The treatment with H101 virus alone or combined with low-intensity
   electric field stimulation enhanced apoptosis, with the highest rate
   observed in the combination group. (E), Flow cytometric analysis of
   apoptosis after the addition of the JNK inhibitor.Treatment with the
   JNK inhibitor partially inhibits apoptosis in the combined treatment
   group. Data are presented as mean ± SD. Ns, not significant; ****p <
   0.0001.

4. Discussion

   In this study, we initially demonstrated that RE, while not
   significantly cytotoxic to tumor cells, unexpectedly enhanced their
   migratory ability. Conversely, the H101 oncolytic virus alone exhibited
   strong tumoricidal effects and reduced cell migration. When combined,
   the two approaches significantly enhanced tumor cell killing and
   migration inhibition. Bulk RNA-seq and scRNA-seq analyses revealed that
   the combination of RE and H101 virus activates the JNK-MAPK pathway,
   promoting tumor cell apoptosis and improving therapeutic efficacy
   compared to low-field stimulation alone.

   Due to the invasion of adjacent structures or distant metastasis, only
   about 20% of patients with pancreatic cancer have the opportunity for
   radical resection at initial diagnosis ([91]28). For patients with
   LAPC, which accounts for nearly 40% of all pancreatic cancer cases, the
   opportunity for radical surgical resection is also forfeited ([92]29).
   IRE, as an emerging and effective treatment modality, has been
   demonstrated to significantly prolong the survival of patients with
   LAPC ([93]8, [94]30, [95]31). However, due to tumor heterogeneity and
   variable electric field distribution within tumors, some areas may only
   experience RE, allowing tumor cells to survive and potentially leading
   to recurrence. As a new localized treatment, IRE can induce short and
   high-voltage current pulses to disrupt the integrity of cell membrane,
   causing cell apoptosis and death ([96]32). In this study, electric
   fields below 750 V/cm were shown to cause RE without cytotoxicity.
   Furthermore, cell migration assays revealed enhanced tumor cell
   motility after low-field stimulation, likely due to EMT. These findings
   suggested that RE might contribute to recurrence after IRE treatment,
   highlighting the need for complementary therapies.

   While RE does not directly kill tumor cells, it temporarily increases
   membrane permeability, allowing the delivery of therapeutic agents such
   as drugs or genes—a process known as electrochemotherapy (ECT). This
   technique has shown promising results in various cancer types,
   particularly in the treatment of cutaneous and subcutaneous metastases
   ([97]33–[98]35). Furthermore, the integration of ECT with other
   treatment modalities, such as immunotherapy and gene therapy, is being
   explored to enhance its efficacy and broaden its application in
   oncology ([99]36, [100]37). This also provides a theoretical
   possibility for the combination of RE with oncolytic viruses.

   Oncolytic virus is an emerging therapeutic regimen able to selectively
   kill tumor cells and has achieved remarkable results in clinical
   applications and basic research for various types of cancers ([101]16,
   [102]38–[103]40). It is noteworthy that the oncolytic virus H101 has
   also achieved significant efficacy in inhibiting or treating tumor
   recurrence. A case report by Wang et al. demonstrated pathological
   complete response in a patient with postoperative lymph node metastasis
   of colorectal cancer following intratumoral injection of oncolytic
   virus H101 in combination with capecitabine therapy ([104]41).
   Additionally, another study has shown that H101 exhibits favorable
   efficacy in the treatment of malignant ascites ([105]42). However,
   there are relatively few reports on the use of H101 oncolytic virus in
   the treatment of pancreatic cancer. Given IRE’s ability to preserve
   tumor vasculature and enhance the permeability of cell membranes, we
   hypothesized that it could facilitate the penetration of H101 virus
   into tumor tissues, improving infection efficiency and cytotoxicity.
   Experiments confirmed that RE alone did not inhibit tumor growth but
   enhanced migration, while the H101 virus significantly suppressed tumor
   proliferation and migration. Combined treatment amplified these
   effects, reducing tumor growth more effectively than either approach
   alone. Immunohistochemical analysis revealed lower Ki-67 expression in
   the combination group, indicating reduced tumor cell proliferation.

   To further elucidate the mechanism by which RE combined with H101
   oncolytic virus kills tumor cells compared to RE alone, we collected
   tumor tissues from mice and performed bulk RNA-seq and scRNA-seq. The
   GO and KEGG enrichment analysis results from bulk RNA-seq presented the
   top 10 pathways in descending order of significance. The results
   indicated that both the MAPK and apoptosis pathways are significantly
   activated. This conclusion was further corroborated by Gene Set
   Enrichment Analysis (GSEA) and KEGG pathway analysis results from
   scRNA-seq. Subsequent protein analysis revealed that combined treatment
   specifically upregulated JNK phosphorylation, with no changes in ERK or
   p38. JNK, a key regulator of the MAPK pathway, plays a central role in
   apoptosis ([106]43, [107]44). Flow cytometry confirmed increased
   apoptotic cell proportions in the combination group, while Western blot
   analysis showed elevated pro-apoptotic BAX and reduced anti-apoptotic
   Bcl-2 expression. It is noteworthy that with the addition of the JNK
   inhibitor (SP600125), the apoptotic effect of the combined treatment
   group was attenuated, and the expression changes of apoptosis-related
   proteins were significantly reversed. This clearly indicates that JNK
   plays a crucial and direct role in activating the intrinsic apoptotic
   pathway and initiating the intracellular death program in the combined
   treatment group.

   This study has certain limitations. Due to the slow growth of mouse
   tumors and the extended experimental period, the number of tumor
   specimens meeting sequencing requirements was limited. In addition, the
   high cost of single-cell sequencing further limits the ability to
   perform large-scale sample analysis. As a result, there may be
   potential selection bias in the sample selection and comparison
   process. In addition, this study primarily focused on the cytotoxic
   effects of H101 on tumor cells following infection, without further
   investigating its replication ability within cells. This mechanism
   warrants further investigation in future studies to optimize the
   application of H101 oncolytic virus and enhance its clinical efficacy.

5. Conclusion

   In conclusion, for tumor cells surviving IRE treatment due to uneven
   electric field distribution, the combined use of the H101 oncolytic
   virus offers a promising adjuvant strategy. This combination enhances
   therapeutic efficacy by activating the JNK-MAPK pathway, leading to
   increased tumor cell apoptosis. Further clinical studies are warranted
   to validate this approach and explore its potential in improving
   outcomes for pancreatic cancer patients.

Acknowledgments