After delivering high-voltage electric pulses to tumor cells, it kills them via a variety of mechanisms including cell membrane perforation, mitochondrial damage, reactive oxygen species (ROS), and DNA damage (4, 12, 13). Firstly, IRE, nsPEF, and H-FIRE all cause damage to cell membranes, resulting in osmotic imbalance and cell swelling (14, 15). And electrical pulses can also lead to DNA damage, but whether the direct effect or the indirect effect induced by apoptosis is not clear (16–18). ROS is also one of the mechanisms of damage. High levels of ROS were found after PEF treated melanoma cells (19). What needs to be emphasized is that mitochondrial damage is more studied in nsPEF, because nsPEF has shorter pulse width, increasing the possibility of causing damage to organelles, and nsPEF causes mitochondrial damage by the loss of mitochondrial membrane potential (14, 20). Thus, damage to cells through different mechanisms may lead to changes in cellular signaling pathways.

Some articles have focused on the effects of electrical pulses on cellular signaling pathways. According to one study, applying nsPEF to the human pancreatic carcinoma cell line (PANC-1) can change the protein expression of the Wnt/β-catenin signaling pathway, matrix metalloproteinases (MMP) family, and vascular endothelial growth factor (VEGF). The downstream signals of the Wnt/β-catenin signaling pathway, including hDPR1, β-catenin, and c-Myc, are dose-dependently decreased by nanosecond pulses (21). Wnt/β-Catenin has two pathways, the canonical pathway and the non-canonical, and the canonical pathway can lead to the transcription of target genes such as myc and cyclin D1, nanosecond pulses inhibit the transcription of target genes through this pathway, thereby inhibiting the proliferation of tumor cells (22). In addition to Wnt/β-catenin pathway, the expression of NF-κB pathway proteins including IKK-α, IKK-β, IκB-α, NF-κB p-65, and p-p65 is also significantly reduced (21). Not only that, the expression of proapoptotic lymphocytes/leukemia-2 (Bcl-2) family proteins (Bax, Bim, and BID) is promoted, and the expression of antiapoptotic Bcl-2 family proteins phosphorylated Bcl-2 protein (p-Bcl-2), Bcl-xL and myeloid leukemia-1 (Mcl-1) are inhibited (22, 23). The MMPs family and VEGF are also lower than those of the control group. Downgrading of MMPs and VEGF can inhibit tumor invasion and metastasis. It is explained in detail in “4. Vascularity, stroma and chemical environment “.

Sun S et al. performed IRE on human pancreatic cancer cell line AsPC-1 and BxPC-3 in vitro and found that IRE can trigger ROS-dependent apoptosis in pancreatic cancer through the PI3K/Akt pathway (11). Another study found that the gene expression of KRAS and EGFR pathway signaling molecules changed significantly after IRE treatment on pancreatic tumors. EGFR signaling was inhibited: (i) causing a decrease in AKT, NF-kB, and VEGF expression, which inhibited tumor growth and invasion, metastasis, etc. (ii) leading to the inhibition of JAK and STAT3, thus providing inhibition of G0 to G1 phase transformation and reducing tumor cell replication. While K-RAS was inhibited, MEK1/2, JNK, and ERK1/2 expression were down-regulated, thus inhibiting cell replication and proliferation. IRE significantly altered the cancer hallmarks and immunosuppressive biological pathways in the PDX pancreatic tumor model. And necrosis, regeneration/repair, and inflammatory signaling were significantly increased after IRE (23).

Wnt/β-Catenin, KRAS, EGFR, as well as downstream cellular pathways like MMP and VEGF were found to be downregulated after electrical pulses were applied to pancreatic cancer, and then cancer biology, including proliferation, cell death, invasion, and metastasis, all changed ( Figure 1 ). Both IRE and nsPEF can exert anti-tumor effects by inhibiting cell replication, increasing the expression of pro-apoptotic proteins and suppressing the expression of antiapoptosis proteins, but there is not enough evidence to prove a significant difference between IRE and nsPEF in causing changes in cellular pathways.

Pulsed electric field ablation is known for its ability to cause apoptosis-a kind of programmed cell death. Because pro-apoptotic and anti-apoptotic factors regulate cell apoptosis, the increase in Bax, Bim, and BID and decrease in p-Bcl-2, Bcl-XL, and McL-1 after an electric pulse suggests that electroporation can promote cell apoptosis (18, 22, 24–26). Significantly increased cleavaged and active caspase 3, 7, and 9 were also detected after IRE (4, 21, 26–29), which are the markers of apoptosis. Cells exhibit the pathological characteristics of apoptosis after electrical impulses: nuclear pyknosis, nucleolysis, nuclear fragmentation, and apoptotic bodies were observed (21, 30–33).

However, during the delivery of electrical pulses, some heat will inevitably be generated. Tissues and cells exhibit distinct death features depending on their distance from the electrode needle. Generally speaking, the closer to the needle track, the easier it is to necrosis, the middle part shows irreversible electroporation, and the cells far away from the needle track are easy to form reversible perforation, which may be related to temperature, the closer the needle track is to the more heated the tissue, the more serious the thermal damage caused, which is characterized by zones of white coagulation (30, 34). The necrosis zone shows endoplasmic reticulum and nuclear membrane expansion and random DNA degradation (4, 16).

Pyroptosis and necroptosis belong to immunogenic cell death (ICD) that rely on the release of damage associated molecular patterns (DAMPs) to drive local immune responses. Pyroptosis forms intracellular inflammatory vesicles and activates caspase-1, gasdermin D (GSDMD) channels are formed on the cell surface and interleukin (IL)-1β, IL-18, and DAMP molecules are released from the cell via GSDMD pores, where they stimulate an immune response. Water and ion can also influx the cell from GSDMD, causing edema of the cell (4). Activation of caspase-1 and GSDMD was observed in rat liver tissue at 6 and 24 hours after electroporation, illustrating that IRE can cause pyroptosis (16). Necroptosis is initiated by the necrosome and activates the receptor interacting serine/threonine kinase 3 (RIPK3), which activates mixed lineage kinase domain-like pseudokinase (MLKL). Activated MLKL molecules aggregate and form pores in the cell membrane, allowing the release of DAMPs and the influx of water and ions, causing cellular edema and cell membrane disintegration, similar to the morphological manifestation of necrosis (4). Elevated RIP3 and MLKL were harvested after IRE, and cell morphology was observed with loss of the plasma membrane and release of organelles and chromatin, which is consistent with the morphology of necroptosis (21). Multiple modes of cell death may exist in the target area after electrical pulses, but they can change over time, and genetic analysis revealed that apoptosis was the predominant mode of cell death after H-FIRE (2000V, 100μs, bipolar pulses, a 2μs positive pulse, 5μs inter-pulse delay, 2;μs negative pulse, and a 5μs inter-pulse delay) was applied to the mouse 4T1 mammary tumor at 2 hours, while necrosis and pyroptosis were predominant by 24 hours (27). In addition, the mode of cell death can change with parameters, more energy may have greater thermal damage, more necrosis. Brock et al. conducted IRE on utilizing patient-derived xenograft (PDX) models, and found that apoptosis was evident at 500 V/cm but necrosis was more prominent at 2500 V/cm (23).

Common DAMPs include the non-histone chromatin protein high mobility group box 1 (HMGB1), cell surface calcium reticulum protein (CRT), and other endoplasmic reticulum (ER) proteins, and adenosine triphosphate (ATP), which are associated with cell death. CD91, toll-like receptor 4 (TLR4), and The P2X7 receptor (P2RX7) are expressed by dendritic cells (DCs) and promote phagocytosis of dead cells, presentation of tumor antigens, and production of IL-1β, respectively (35). The release of DAMPs (ATP, calreticulin, nucleic acids and uric acid) increases with increasing pulse amplitude after IRE on cells in vitro (12, 29, 36–39) and causes massive immune cell aggregation in post-electroporation pancreatic cancer tissue in vivo (36) ( Table 1 ). The release of DAMPs is related to the parameters of the pulses, at IRE (500-1500 V, 100 μs, 8-24 pulses) with increasing voltage, the release of DAMP increases (29), similarly, the number of DAMP releases is related to the number of pulses, after IRE (1000 V, 100 μs, 8/40/80 pulses), CRT, ATP, and HMGB1 were released most at 40 pulses and less at 8 and 80 pulses, suggesting that there may be a suitable number of pulses, neither too less nor too more, that would allow the most DAMP release, Go EJ et al. speculated that low pulses (<40) would not induce ICD and high pulses (>40) would lead to rapid cell death, thus limiting DAMP expression (38). Most of the studies about DAMP are in vitro, and the appropriate parameters, as well as the intensity-release dependence, may require further studies.

(i) CRT is the most abundant in the endoplasmic reticulum. After activation of ICD-related signaling pathways, it transfers from the endoplasmic reticulum to the cell membrane surface and can interact with transmembrane receptors including CD49, CD69, CD91 (also known as the low density lipoprotein (LDL) receptor-related protein-1 (LRP1)), and integrins. The most important is the CD91 molecule. CRT releases effective phagocytic signals to CD91-positive cells (mainly macrophages and DCs) and causes the production of pro-inflammatory cytokines (including IL-6 and TNF-α) (35, 40). (ii)In addition to participating in purinergic neurotransmission, ATP released from damaged cells can bind to the P2Y2 receptor of macrophages, promoting the infiltration of macrophages in tumor sites, and can also bind to the P2RX7 of DC cells, leading to DC maturation and release of IL-1β. (iii) HMGB1 can bind to protein toll-like receptor 4 (TLR-4) and receptor for advanced glycation end products (RAGE) to activate monocytes/macrophages. HMGB1 can also upregulate costimulatory molecules and major histocompatibility complex (MHC) class II to transfer immature DC to mature DC (35, 41, 42). HMGB1 stimulates neutrophils and monocytes, enabling these cells to adhere to activated vascular endothelium and migrate to inflamed tissues (43).

Electrical pulse stimulation triggers the release of DAMPs, which acts as a “find me” signal, enhances tumor immunogenicity and subsequently induces antigen-presenting cells (APC) activation. These signals enhance the ability of APC to phagocytose, process, and present tumor-derived antigens to T cells, thereby facilitating the induction of tumor-specific adaptive immunity. So, the level of these DAMPs and cells increases after pulsed electric field (29, 36–39).

A large number of immunosuppressive cells are present in tumors, including T regulatory cells (Tregs), tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), and myeloid-derived suppressor cells (MDSCs), and the upregulation of these cell types in tumors depends on the reciprocal signaling between these cells and tumor cells.

The production of Treg (usually CD4+CD25⁺Foxp3⁺ T cells) depends mainly on transforming growth factor-β (TGF-β) and IL-2, which negatively regulate immunity and can produce TGF-β and IL-10 to suppress immune responses (55, 57, 58). And Tregs’ infiltration is negatively correlated with median survival OS in many patients with solid tumors (59). Tregs can effectively suppress effector T lymphocytes and can inhibit the function of B, NK, dendritic cells, and macrophages through different mechanisms (58, 60).

TAM has an M2 macrophage-like phenotype and promotes tumor progression through several mechanisms: secretion of VEGF, which promotes tumor angiogenesis; promotion of tumor invasion mainly through the release of metalloproteinases, matrix remodeling enzymes, and chemotactic growth factors from the environment; and suppression of innate immune responses (61).

There are mainly two types of MDSC: polymorphonuclear MDSC (P-MDSC) which resemble neutrophils morphologically and phenotypically, and monocyte MDSC (M-MDSC) which resemble monocytes. MDSC has potent immunosuppressive activity through multiple pathways: promoting Tregs’ production and promoting fibroblast differentiation into cancer-associated fibroblasts (CAF) depleting L-arginine eliminates key trophic factors required for T cell proliferation, nitrates chemokines and blocks CD8+ T cells from entering the tumor, and produces immunosuppressive cytokines such as IL-10 and TGF-β (61, 62).

Unlike normal myofibroblasts, CAF does not undergo apoptosis and can release various cytokines and MMPs to hydrolyze extracellular matrix, stimulate angiogenesis and promote tumor growth and invasion (63). (As described in 4. Vasculature, extracellular matrix, and chemical environment).

Reduction of systemic Tregs in locally advanced pancreatic cancer (LAPC) patients 2 weeks after IRE was found in clinical trials (64). However, a transient increase in Tregs on day three followed by a decrease on day seven was found in the clinical trial by He C (46). Similar results were also found in Harshul et al.’s study, where LAPC patients could have a procedure-mediated Treg attenuation between the third and fifth day after IRE (65). A reduction in Li^- CD³³⁺ HLA^-DR^- early myeloid-derived suppressor cells (eMDSC) was observed 2 weeks after IRE treatment (64). IRE combined with OX40 agonist induced a significant reduction in MDSC in primary and distant tumors (66). H-FIRE resulted in a reduction of MDSCs and TAMs in the tumor microenvironment of mammary carcinoma in mice 2 days after procedure (27). NsPEF treated with C57 malignant melanoma reduced Treg cells from 4.3% to 2.4% and MDSC by 39.0% to 19.7%, which was observed 4 days later (67). NsPEF can act on mice with pancreatic cancer after 3 days postoperative, 7 days postoperative decreased the percentage of nMDSCs and mMDSCs in the spleen, although Tregs slightly increased at 3 days postoperatively, but significantly decreased at 7 days postoperative (53), indicates that the immunosuppressed state can be reversed in this period of time, which would facilitate the combination with immunotherapy.

Therefore, electrical pulses can inhibit the proliferation of tumor-associated immune cells in the tumor microenvironment and promote anti-tumor responses to create an immune environment conducive to tumor suppression. However, the reversion of immunosuppression after IRE or nsPEF is time-dependent and this may start after day 3, but a longer and more subtle follow-up is needed to determine the time window for combination with immunotherapy.

Adaptive immunity is achieved through regulated interactions between APC and T and B cells. Circulating antigens or APC-treated antigens are presented to T and B cells, eliciting cellular and humoral immunity, respectively. The largest T cell population in the body is the CD4+αβ T cell receptor (TCR) population. Most of these cells have a helper function and are called helper T (Th) cells, which produce many cytokines. CTL is a type of CD8+ T cells that kill target host cells through a contact-dependent mechanism: increased expression of FasL on CTL binds to Fas receptors in target tissues, participates in apoptosis, and acts on target cells by releasing substances such as perforin and granzyme. Adaptive humoral immunity is mediated by antibodies produced by plasma cells (55).

Several studies have found that electrical pulses acting on cells induce increased circulation and ablation foci of CD8+ T cells (24, 37, 46, 64, 68–70), and some experiments have found elevated CD4+ levels (38, 46, 55, 56), however, some studies has also shown no significant increase in CD4+ levels (10, 23) ( Table 1 ). Zhao et al. found an increased CD8+ T cells and CD4+ T after nanosecond pulses acting on pancreatic cancer in mice, and a significantly higher CD8/CD3 ratio in tumors compared to controls (53). He et al. found an increase of effector CD8+ T cells, effector CD4+ T cells, and memory T cells at 7 days after IRE, despite decrease at day 3, so it can effectively induce the activation of T cells over a period of time, and the experiment also found that IRE can inhibit the growth of potential tumors through the distant effect (50). However, Dai et al. implied that IRE treatment significantly inhibited HCC growth by more CD8+ T and dendritic cells, but not CD4+ T or B cells infiltrating into the peri-ablative region. CD8+ depleted T cells induced local tumor regeneration and distant metastasis after IRE (10). Most of the IRE or nsPEF studies have activated the proliferation of CD8+T, but the proliferation of CD4+T is not obvious in some studies, revealing that CD8+ T-mediated cellular immunity plays a great role in electric pulses induced immunity. Effective T cell initiation requires several events, including: release of endogenous antigens from cancer cells, release of “danger signals” from damaged cells, processing of cancer antigens, antigens presented to naive T cells by APC, activation and proliferation of cancer-specific cytotoxic T cells (55, 69, 71). The current results suggest that pulsed electric field can promote cellular immunity through these sessions: 1) induce immunogenic death, resulting in the massive release of DAMP (29, 36, 38, 39); 2) Proliferation and activation of antigen presenting cells (29, 36, 38); 3) Activation, proliferation and function of cancer-specific cytotoxic T cells (36, 64, 66, 67, 70). In addition, Shao et al. compared IRE, thermal therapy (Heat), cryosurgery (Cryo) in vitro, and found that IRE can cause more protein release than other ablation. Although the released protein has 40% denatureation, T cell proliferation is still 2-3 times higher than Cryo (69). IRE induces OX40 expression in CD8+ T cells in vivo, and OX40 acts as a co-stimulatory molecule to increase T cell expansion and cytokine secretion (66). The combination of IRE and TLR 3/9 agonists and PD-1 blockade can effectively reverse the depletion of intratumoral CD8+T and enhance local immunity against tumors (72). Brandon et al. made a deeper exploration by combining anti-T-lymphocyte-associated protein-4 (anti-CTLA-4) therapy prior with IRE on prostate cancer to promote neoantigen-specific T-cell responses, resulting in increased numbers of splenic systemic SPAS-1+ T cells concentrated in tumors and distant sites. Circulating memory CD8+ T cells, in addition to central memory (T_CM) and effector memory (T_EM), have tissue-resident memory (T_RM). Endogenous SPAS-1 neoantigen-specific CD8+ T cells were increased in number and enriched in tumors following TRAMP-C2 tumor cell were attack and generated CD8+ T_RM cells in different tissues (68). In addition, Shi et al. treated hepatocellular carcinoma (HCC) with IRE in combination with an anti-PD-L1 monoclonal antibody and found enhanced off-target necrosis and inflammatory infiltration, with IRE significantly increasing the inflammatory infiltration index and increasing CD8+ T infiltration not only in target tissues but also in non-target tissues (untreated tumors) (70). Immunotherapy Combined IRE induced more CD8+ T proliferation and enrichment in tumors as well as other sites than immunotherapy alone, probably because: 1) IRE increased its immunogenicity: IRE caused immunogenic death of tumor tissues, massive release of DAMPs, causing activation of APCs and presentation to T cells, leading to tumor specific T-cell population expansion and enhanced systemic antitumor effects; 2) Reversal of the immune tolerant tumor microenvironment, with M1 macrophages polarizing CD4+ Th1 cell differentiation to enhance CD8+ T cell survival and tumor infiltration; 3) IRE-induced regulation of the tumor stroma, extracellular matrix, and/or vascular system may be another reason (21, 36, 46, 53, 68, 73).

Several studies have demonstrated the protective effect of ablation foci on large vessels (9, 16, 74). For example, researchers followed 158 vessels with a mean distance of 2.3 ± 2.5 mm from the treatment area and found only 7 (4.4%) with abnormal vascular changes, including stenosis and thrombosis (9). However, the effect of IRE on microvessels is uncertain, and in some studies, microvessels remain histopathologically preserved in the area after ablation and the structure is still present (75), but can show microvascular distortion, occlusion, and thrombosis when observed under electron microscopy (32), and after disruption of vascular continuity there can be hemorrhagic necrosis with infiltration of surrounding neutrophils (76), and endothelial cells are damaged significantly. Thereafter, the disrupted vessel can be recognized by new endothelial cells derived from neighboring cells and/or circulating endothelial progenitor cells (32). Non-thermal irreversible electroporation can cause a decellularizing effect of the vessel at 3 days, the vessel skeleton survives while cells are shed, however, at 7 days this skeleton has endothelial ingrowth (74).

The changes of the microvasculature after IRE are: immediate congestion (75); necrosis of endothelial cells, hemorrhage, and peripheral inflammatory response (32, 76); and there can be regeneration of new vessels (32). It is worth mentioning that in Lv et al.’s theoretical study of the effect of perforation on tumor vasculature and normal vasculature, by establishing a multilayer dielectric model, explored that rich vascular smooth muscle cells (VSMCs) might have a protective effect on normal vasculature, thus demonstrated that electroporation may have a stronger destructive effect on tumor vasculature (77).

At the level of regulation of angiogenesis, tumor growth requires nutritional support from blood vessels, and angiogenesis is influenced by the expression of pro-angiogenic factors and anti-angiogenic factors; the VEGF family, composed of six growth factors (VEGFA-F), is essential for angiogenesis (78, 79), and angiopoietin 1-2 (Ang1-2) is independent of VEGF, while Ang-2 is mainly present in vascular expressed in remodeled tissues and in the hypoxic tumor microenvironment (80). VEGF can also exert inhibitory effects on DC cells and effector T cells in driving neoangiogenesis, as well as increase TAM infiltration and the expansion of Tregs and MDSCs (78, 81–84). However, due to the overexpression of pro-angiogenic factors and less in tumors, tumor vessels exhibit functional abnormalities with abnormal leakage, rapid growth, high tortuosity, and little perivascular pericytes and smooth muscle cells coverage (78, 79). A decrease in VEGF and CD34 proteins can be detected 1 hour after nanosecond pulse treatment of pancreatic cancer (21). He et al. also found increased expression of CD31 in tumor after IRE (53). In addition, nsPEFs and everolimus (The mammalian target of rapamycin (mTOR) inhibitor) synergistically inhibited angiogenesis by decreasing the expression of vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR), and CD34 (85). In addition to inhibiting the expression of pathological proangiogenic factors, a study by Zhao et al. found a transient increase in CD31 calculated tumor microvascular density microvessel density (MVD) followed by a decrease four days after IRE treatment of pancreatic cancer and an increase in microvascular permeability determined by fluorescein isothiocynate (FITC)-bound dextran (73). Therefore, pulsed electric field can inhibit the growth of tumor pathological blood vessels and blood supply around the tumor, and also preserve the permeability of functional blood vessels to a certain extent, which is conducive to the infiltration of immune cells and factors.

In the tumor microenvironment, not only tumor cells proliferate rapidly, but also stromal deposition and remodeling as well as cancer cells and stromal cells increase, and CAFs form the main support structure of tumor tissues (1, 2). CAFs also promote cancer development by secreting growth-promoting factors such as TGF-β, stromal degrading enzymes and angiogenic factors such as MMP or VEGF, α smooth muscle actin (α-SMA) is a reliable biomarker for CAFs, and fibroblast activating protein α (FAP-α, seprase) is a surface glycoprotein that is selectively expressed on solid tumor fibroblasts. MMP hydrolyzes the extracellular matrix and its expression correlates with the aggressive phenotype of tumor cells and tumor progression (86).

Extracellular matrix and collagen structures can exist intact after IRE action because IRE acts on phospholipid bilayers (3, 74).

MMPs family proteins (MMP1, MMP2, MMP9, MMP11, MMP12, MMP14, and MMP21) are expressed at different levels of nsPEF intensity (21). In a study by Zhao et al. collagen matrix or αSMA+ CAFs were not affected by IRE, and FAP-α, hyaluronic acid (indicated by HABP1 expression levels) and lysyl oxidase (LOX, a marker of extracellular matrix stiffness) were decreased to varying degrees (36). Vasculature and collagen were still present in IRE-treated lung tissue 2 days after treatment and 28 days after a significant increase, indicating remodeling and regeneration of the mesenchyme, but decorin and heparan sulfate decreased after ablation (87).

Therefore, when electric pulses cause irreversible electroporation of cells, the presence of stromal and collagen structures can be observed histopathologically, but they can also microscopically modulate the cellular matrix and reduce the levels of CAFs and MMPs ( Table 1 ). With the preservation of functional vessels and increased vascular permeability, softened extracellular matrix is beneficial to infiltration of inflammation and distant effects (16, 36, 53, 67).

Tumor vessels show characteristics of tortuous, twisted, and easily occluded, and the tumor presents a relatively hypoxic state due to the rapid proliferation of tumor cells and the increase of extracellular matrix leading to the increase of tumor tissue pressure. Hypoxia leads to the accumulation of hypoxia-inducible factor 1-α (HIF-1α), which promotes further tumor angiogenesis and suppresses T-cell function (2, 88). Moreover, hypoxia increases anaerobic enzymes and lactate accumulation further reduces T and NK cell activation (89). Reversal of intratumoral hypoxia effectively increases the infiltration of immune cells. The downregulation of HIF-1α and carbonic anhydrase 9 (CA-IX) and increased vascular permeability after IRE suggest that IRE may also increase the number and action of local T cells, NK cells by alleviating tumor hypoxia (36).