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Induction of long-term memory T cells and antitumor immunity after NPS treatment. (a) CD41 or CD81 memory T cells in the spleens of mice. Tcm: CD44 1 CD62L1 T cells; Tem: CD44 1 CD62L-T cells. Note: percentage of memory cells 5 gated CD41 or CD81 T cells (%) 3 gated Tcm or Tem (%) (seen gating strategy in Supporting Information, Fig. S4). (b,c) A representative histogram of intracellular cytokine staining for each group after 6 hr incubation with plate bound anti-CD3. (d,e) IFN-g producing cells from splenocytes after 6 hr incubation with plate bound anti-CD3. (f) IFN-g product of splenocytes after 24 hr incubation with tumor lysate. Groups, Ctr: na€ ıve control mice at age of 15-16 weeks (n 5 3); Tumor: tumor mice at the end point for euthanasia (age 15-16 weeks, n 5 4); NPS: mice with tumor free at least for 4 months after NPS treatment (n 5 5). *p < 0.05, **p < 0.01 and ***p < 0.001 (one-way ANOVA).
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Nano-Pulse Stimulation (NPS) as a developing technology has been studied for minimally invasive, non-thermal local cancer elimination for more than a decade. Here we show that a single NPS treatment results in complete regression of the poorly immunogenic, metastatic 4T1-Luc mouse mammary carcinoma. Impressively, spontaneous distant organ metastase...
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... or surpassed levels of control mice, especially central memory CD41 and CD81 T cells. Memory T cells in spleens were greatly increased in the tumor-free mice compared to na€ ıve control mice with CD41 (3.61% vs 0.62%) and CD81 T-cells (1.38% vs 0.2%) and compared to tumor-bearing mice with CD41 (3.61% vs 0.22%) and CD81 (1.38% vs 0.06%) T-cells (Fig. 4a and Supporting Information, Fig. S4). after tumor inoculation. Ctr: orthotopic breast cancer without NPS (n 5 4); 300 p (n 5 6) and 1,000 p (n 5 4): 4T1-Luc orthotopic breast can- cer treated with 300 pulses or 1,000 pulses with 100 ns and 50 kV/cm at 1 Hz. Tx: treatment Day 11. *p < 0.05 for Ctr versus 300 p or 1000 p by one-way ...
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... both of CD41 and CD81 T memory cells in the blood or spleen of treated, tumor-free mice were recovered or surpassed levels of control mice, especially central memory CD41 and CD81 T cells. Memory T cells in spleens were greatly increased in the tumor-free mice compared to na€ ıve control mice with CD41 (3.61% vs 0.62%) and CD81 T-cells (1.38% vs 0.2%) and compared to tumor-bearing mice with CD41 (3.61% vs 0.22%) and CD81 (1.38% vs 0.06%) T-cells (Fig. 4a and Supporting Information, Fig. S4). after tumor inoculation. ...
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... antibody for 6 hr, IFN- g producing CD41 (0.242%) and CD81 T cells (0.364%) from spleens of tumor-free mice were significantly increased whereas IFN-g producing T cells from tumor-bearing mice (0.029% for CD41 and 0.031% for CD81 T cells) were largely decreased in contrast to na€ ıve control mice (0.135% for CD41 and 0.116% for CD81 T cells) (Figs. 4d-e). The significant decrease of cytotoxic T cells in tumor-bearing mice can be explained with immune suppressive status evi- denced in the following studies. Furthermore, tumor specific IFN-g production after splenocytes co-culture with tumor lysate was increased as well (Fig. 4f). The significantly high amount of IFN-g was released from ...
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... na€ ıve control mice (0.135% for CD41 and 0.116% for CD81 T cells) (Figs. 4d-e). The significant decrease of cytotoxic T cells in tumor-bearing mice can be explained with immune suppressive status evi- denced in the following studies. Furthermore, tumor specific IFN-g production after splenocytes co-culture with tumor lysate was increased as well (Fig. 4f). The significantly high amount of IFN-g was released from splenocytes of tumor- free mice (75.9 pg/ml vs 28.5 pg/ml in the media control) in response to tumor antigens for 24 hr but not in the na€ ıve mice (31.5 pg/ml) or tumor mice (21.1 pg/ml). ...
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... is consistent with the vaccine-like effect in previous studies with the orthotopic N1-S1 HCC model where following tumor elimination for 7 weeks, all tumor-free rats were protected (21/21) from ortho- topic challenge injections of the same tumor line. 8 Yet more impressive, for tumors that exhibited partial responses, NPS treatment resulted in a significant reduction in spontaneous Supporting Information, Fig. S4). (b,c) A representative histogram of intracellular cytokine staining for each group after 6 hr incubation with plate bound anti-CD3. ...
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It has been reported that chemo-radiotherapy can induce immunogenic tumor cell death (ICD), which triggers T-cell immunity mainly mediated by high-mobility group box 1 protein (HMGB1) and calreticulin. However, there is still limited information to support this theory relating to chemotherapy alone. In the present study, the expression of HMGB1 and...
The ability of anticancer treatments to promote the activation of tumor-reactive adaptive immune responses is emerging as a critical requirement underlying their clinical effectiveness. We investigated the ability of Hemidesmus indicus, a promising anticancer botanical drug, to stimulate immunogenic cell death in a human colorectal cancer cell line...
Citations
... The BCG approach presented no cancer antigens but activated the immune system. In addition to BCG, toll-like receptor (TLR) agonists, oncolytic viruses, cytokines, radiation, and hypothermia [10], emerging novel drugs/technologies, including checkpoint inhibitors, nanoparticles, electrochemotherapy (ECT) [11,12], gene electro-transfer (GET) [13,14], and nano-pulse treatment (NPT) [15][16][17], can serve as ISV approaches as well. The most documented but rare phenomenon is what radiation oncologists term the abscopal effect. ...
... Nano-pulse treatment (NPT), also known as nano-pulse stimulation (NPS) or nanosecond pulsed electric fields or electric pulses (nsPEFs/nsEPs), is a pulsed-power technology that compresses electric energy and releases it in high-powered (generally 100 megawatts) nanosecond (1~999 ns)duration electric pulses [20,21]. NPT has been demonstrated to effectively ablate tumors in multiple animal models [15,[22][23][24][25]. In our previous studies, NPT ablated localized tumors in poorly immunogenic cancer models, including orthotopic 4T1-luc mouse triple-negative breast cancer [15], N1S1 rat liver models, and ectopic [24] Pan02 mouse pancreatic cancer [17]. ...
... NPT has been demonstrated to effectively ablate tumors in multiple animal models [15,[22][23][24][25]. In our previous studies, NPT ablated localized tumors in poorly immunogenic cancer models, including orthotopic 4T1-luc mouse triple-negative breast cancer [15], N1S1 rat liver models, and ectopic [24] Pan02 mouse pancreatic cancer [17]. Importantly, after the complete regression of primary tumors, NPT resulted in a 75-100% rejection of secondary live tumor challenges in these three cancer models, demonstrating its strong ISV effects. ...
We previously reported that nano-pulse treatment (NPT), a pulsed power technology, resulted in 4T1-luc mammary tumor elimination and a strong in situ vaccination, thereby completely protecting tumor-free animals against a second live tumor challenge. The mechanism whereby NPT mounts effective antitumor immune responses in the 4T1 breast cancer predominantly immunosuppressive tumor microenvironment (TME) remains unanswered. In this study, orthotopic 4T1 mouse breast tumors were treated with NPT (100 ns, 50 kV/cm, 1000 pulses, 3 Hz). Blood, spleen, draining lymph nodes, and tumors were harvested at 4-h, 8-h, 1-day, 3-day, 7-day, and 3-month post-treatment intervals for the analysis of frequencies, death, and functional markers of various immune cells in addition to the suppressor function of regulatory T cells (Tregs). NPT was verified to elicit strong in situ vaccination (ISV) against breast cancer and promote both acute and long-term T cell memory. NPT abolished immunosuppressive dominance systemically and in the TME by substantially reducing Tregs, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs). NPT induced apoptosis in Tregs and TAMs. It also functionally diminished the Treg suppression capacity, explained by the downregulation of activation markers, particularly 4-1BB and TGFβ, and a phenotypic shift from predominantly activated (CD44⁺CD62L⁻) to naïve (CD44⁻CD62L⁺) Tregs. Importantly, NPT selectively induced apoptosis in activated Tregs and spared effector CD4⁺ and CD8⁺ T cells. These changes were followed by a concomitant rise in CD8⁺CD103⁺ tissue-resident memory T cells and TAM M1 polarization. These findings indicate that NPT effectively switches the TME and secondary lymphatic systems from an immunosuppressive to an immunostimulatory state, allowing cytotoxic T cell function and immune memory formation to eliminate cancer cells and account for the NPT in situ vaccination.
... Nanopulse Treatment (NPT), also known as nano-pulse stimulation (NPS) or nanosecond pulsed electric fields or electric pulses/ (nsPEFs/nsEPs), is a pulsed-power technology that compresses electric energy and releases it in high powered (generally 100 megawatts) nanosecond (1~999 ns) duration electric pulses [10,11]. NPT has been demonstrated to effectively ablate tumors in multiple animal models [12][13][14][15][16]. In our previous studies, NPT ablated localized tumors in poorly immunogenic cancer models including orthotopic 4T1-luc mouse triple negative breast [12], N1S1 rat liver models and ectopic [15] Pan02 mouse pancreatic cancer [17]. ...
... NPT has been demonstrated to effectively ablate tumors in multiple animal models [12][13][14][15][16]. In our previous studies, NPT ablated localized tumors in poorly immunogenic cancer models including orthotopic 4T1-luc mouse triple negative breast [12], N1S1 rat liver models and ectopic [15] Pan02 mouse pancreatic cancer [17]. Importantly, after the complete regression of primary tumors, NPT resulted in a 75-100% rejection of secondary live tumor challenges in these three cancer models, demonstrating its strong ISV effects. ...
... As shown in Figure 1B, a single NPT elicited a strong ISV effect with 81.5% (22/27) of the animals protected against a second live tumor challenge. This result validated our previous report that a single NPT achieved 100% (11/11) ISV protection in the same 4T1 model [12]. In contrast, none of age-matched naïve mice (0/14) were able to reject the secondary tumor challenges. ...
We previously reported nano-pulse treatment (NPT), a pulsed power technology, resulted in 4T1-luc mammary tumor elimination and a strong in situ vaccination, thereby completely protecting tumor-free animals against a second live tumor challenge. The mechanism whereby NPT mounts effective antitumor immune responses in the 4T1 breast cancer predominantly immunosuppressive tumor microenvironment (TME) remains unanswered. In this study, orthotopic 4T1 mouse breast tumors were treated with NPT (100 ns, 50 kV/cm, 1000 pulses, 3 Hz). Blood, spleen, draining lymph nodes and tumors were harvested at 4-hour, 8-hour, 1-, 3-, 7-day. Additional studies were conducted over 3-month posttreatment intervals for the analysis of frequencies, death and functional markers of various immune cells in addition to suppressor function of regulatory T cells (Tregs). NPT was verified to elicit strong in situ vaccination (ISV) against breast cancer and promoted both acute and long-term T cell memory. NPT abolished immunosuppressive dominance systemically and in the TME by substantially reducing Tregs, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs). NPT induced apoptosis in Tregs and TAMs. It also functionally diminished the Treg suppression capacity, likely explained by the downregulation of activation markers, particularly 4-1BB and TGFβ, and a phenotypic shift from predominantly activated (CD44+CD62L-) to naïve (CD44-CD62L+) Tregs. Importantly, NPT selectively induced apoptosis in activated Tregs and spared effector CD4+ and CD8+ T cells. These changes were followed by a concomitant rise in CD8+CD103+ tissue-resident memory T cells and TAM M1 polarization. These findings indicate that NPT effectively switches the TME and secondary lymphatic systems from an immunosuppressive to an immunostimulatory state allowing cytotoxic T-cell function and immune memory formation to eliminate cancer cells and account for the NPT in situ vaccination.
... PEF protocols currently in use in the clinic were developed to maximize tumor tissue ablation (IRE and nsPEFs) or to achieve the highest drug uptake or the most persistent gene expression (ECT and GET). Recent research in both healthy and tumor tissue indicates that PEFs have a potent immune stimulatory effect (5,7,3336), a novel dimension that merits consideration during treatment planning. Activation of a robust immune response against cancerous cells or a pathogen Ag can be a significant advantage for any anticancer therapeutic modality or vaccine. ...
... Although longer pulses of 100 ms are routinely used in the clinic for cancer applications, including IRE and ECT, treatment with nsPEFs is a newer technology that has been investigated mostly in preclinical settings using pulses ranging from 100 to 300 ns (114). Multiple studies, including ours, have shown that nsPEFs induce an immunogenic form of cell death that assists tumor eradication and prevents the formation of new tumors (5,7,34,35,82,115120). Although the inflammasome has been investigated most often in the context of inflammation and innate immunity, there is growing evidence that inflammasome products, particularly IL-1 family cytokines, have an essential role in stimulating adaptive immune responses, including those involved in anticancer immunosurveillance (121). ...
Although electric field–induced cell membrane permeabilization (electroporation) is used in a wide range of clinical applications from cancer therapy to cardiac ablation, the cellular- and molecular-level details of the processes that determine the success or failure of these treatments are poorly understood. Nanosecond pulsed electric field (nsPEF)–based tumor therapies are known to have an immune component, but whether and how immune cells sense the electroporative damage and respond to it have not been demonstrated. Damage- and pathogen-associated stresses drive inflammation via activation of cytosolic multiprotein platforms known as inflammasomes. The assembly of inflammasome complexes triggers caspase-1–dependent secretion of IL-1β and in many settings a form of cell death called pyroptosis. In this study we tested the hypothesis that the nsPEF damage is sensed intracellularly by the NLRP3 inflammasome. We found that 200-ns PEFs induced aggregation of the inflammasome adaptor protein ASC, activation of caspase-1, and triggered IL-1β release in multiple innate immune cell types (J774A.1 macrophages, bone marrow–derived macrophages, and dendritic cells) and in vivo in mouse skin. Efflux of potassium from the permeabilized cell plasma membrane was partially responsible for nsPEF-induced inflammasome activation. Based on results from experiments using both the NRLP3-specific inhibitor MCC950 and NLRP3 knockout cells, we propose that the damage created by nsPEFs generates a set of stimuli for the inflammasome and that more than one sensor can drive IL-1β release in response to electrical pulse stimulation. This study shows, to our knowledge, for the first time, that PEFs activate the inflammasome, suggesting that this pathway alarms the immune system after treatment.
... High-intensity (tens of MV/m) pulses of short duration (hundreds of ns) showed excellent capabilities to induce cellular death of cancerous cells within in vitro [1][2][3] and in vivo [4,5] models, as well as in humans, as reported in human trials [6,7]. In addition, the unique capability of nsPEF to bypass the cell plasma membrane may elicit specific intracellular responses, including the activation of regulated cell death and the initiation of an adaptive immune response [8,9] showing high efficacy in different in vivo tumor models, including murine hepatocellular carcinoma [10], melanoma [11][12][13][14], or breast cancer [15,16]. Compared to other cancer ablation modalities, nsPEF exposure allows direct and localized tumor ablation, minimizing the damage to healthy tissues surrounding the tumor, avoiding the use of chemotherapeutic drugs, and avoiding associated adverse reactions. ...
Cellular response upon nsPEF exposure depends on different parameters, such as pulse number and duration, the intensity of the electric field, pulse repetition rate (PRR), pulsing buffer composition, absorbed energy, and local temperature increase. Therefore, a deep insight into the impact of such parameters on cellular response is paramount to adaptively optimize nsPEF treatment. Herein, we examined the effects of nsPEF ≤ 10 ns on long-term cellular viability and growth as a function of pulse duration (2–10 ns), PRR (20 and 200 Hz), cumulative time duration (1–5 µs), and absorbed electrical energy density (up to 81 mJ/mm3 in sucrose-containing low-conductivity buffer and up to 700 mJ/mm3 in high-conductivity HBSS buffer). Our results show that the effectiveness of nsPEFs in ablating 3D-grown cancer cells depends on the medium to which the cells are exposed and the PRR. When a medium with low-conductivity is used, the pulses do not result in cell ablation. Conversely, when the same pulse parameters are applied in a high-conductivity HBSS buffer and high PRRs are applied, the local temperature rises and yields either cell sensitization to nsPEFs or thermal damage.
... Intense nanosecond pulses have been shown to induce diverse biological responses, such as membrane permeabilization [4], DNA damage, and activation of signaling pathways [5,6]. Nanosecond pulsed electric fields (nsPEFs) have emerged as a promising tool for various biomedical applications such as tissue treatment [7,8], atrium ablation for heart defibrillation [9,10], and immune response expression for cancer treatment [11][12][13][14]. ...
... While effects depend on different factors, in all studies of nsPEFs no cell line or tumor type has shown resistances to nsPEF elimination. Two models have shown vaccine effects as vaccinations [13,38,39], meaning that tumor-free animals are resistant to regrowing the treated cancer again. In the viability studies, like that seen for the PM responses, the PFL had a lower IC 50 value for viability than the BL pulser, as shown by requiring fewer pulses and requiring a lower electric field. ...
High-intensity nanosecond pulse electric fields (nsPEF) can preferentially induce various effects, most notably regulated cell death and tumor elimination. These effects have almost exclusively been shown to be associated with nsPEF waveforms defined by pulse duration, rise time, amplitude (electric field), and pulse number. Other factors, such as low-intensity post-pulse waveform, have been completely overlooked. In this study, we show that post-pulse waveforms can alter the cell responses produced by the primary pulse waveform and can even elicit unique cellular responses, despite the primary pulse waveform being nearly identical. We employed two commonly used pulse generator designs, namely the Blumlein line (BL) and the pulse forming line (PFL), both featuring nearly identical 100 ns pulse durations, to investigate various cellular effects. Although the primary pulse waveforms were nearly identical in electric field and frequency distribution, the post-pulses differed between the two designs. The BL’s post-pulse was relatively long-lasting (~50 µs) and had an opposite polarity to the main pulse, whereas the PFL’s post-pulse was much shorter (~2 µs) and had the same polarity as the main pulse. Both post-pulse amplitudes were less than 5% of the main pulse, but the different post-pulses caused distinctly different cellular responses. The thresholds for dissipation of the mitochondrial membrane potential, loss of viability, and increase in plasma membrane PI permeability all occurred at lower pulsing numbers for the PFL than the BL, while mitochondrial reactive oxygen species generation occurred at similar pulsing numbers for both pulser designs. The PFL decreased spare respiratory capacity (SRC), whereas the BL increased SRC. Only the PFL caused a biphasic effect on trans-plasma membrane electron transport (tPMET). These studies demonstrate, for the first time, that conditions resulting from low post-pulse intensity charging have a significant impact on cell responses and should be considered when comparing the results from similar pulse waveforms.
... These findings agree with the pancreatic cancer irreversible electroporation treatment study [53]. Moreover, earlier studies have demonstrated robust immune protection achieved through nsEP tumor ablation in mouse breast [54] and rat hepatocellular cancers [55]. ...
... Our findings suggest that the use of nanosecond pulses could have some effect on long-term immune memory formation. Similar induction of splenic memory T cell response following nsEP treatment was reported by Guo et al. [54]. Moreover, comparable tendencies of effector and memory T cells dynamics after the treatment were reported in our study with irreversible calcium electroporation [26] and in He et al.'s research on irreversible electroporation with pancreatic cancer [56]. ...
... A significant decrease in splenic MDSC was noticed after µsCaEP treatment compared to CTRL, while after nsCaEP treatment decrease was not observed. Our results agree with the literature-reported data that IRE, ECT, and CaEP treatment decrease the MDSC [19,44,54]. Moreover, an increased percentage of splenic NK cells after µsCaEP treatment might be related to diminished tumor growth and better survival rates, which is in agreement with Lisec et al. study [35]. ...
Calcium electroporation (CaEP) is an innovative approach to treating cancer, involving the internalization of supraphysiological amounts of calcium through electroporation, which leads to cell death. CaEP enables the replacement of chemotherapeutics (e.g., bleomycin). Here, we present a standard microsecond (μsCaEP) and novel high-frequency nanosecond protocols for calcium electroporation (nsCaEP) for the elimination of carcinoma tumors in C57BL/6J mice. We show the efficacy of CaEP in eliminating tumors and increasing their survival rates in vivo. The antitumor immune response after the treatment was observed by investigating immune cell populations in tumors, spleens, lymph nodes, and blood, as well as assessing antitumor antibodies. CaEP treatment resulted in an increased percentage of CD4+ and CD8+ central memory T cells and decreased splenic myeloid-derived suppressor cells (MDSC). Moreover, increased levels of antitumor IgG antibodies after CaEP treatment were detected. The experimental results demonstrated that the administration of CaEP led to tumor growth delay, increased survival rates, and stimulated immune response, indicating a potential synergistic relationship between CaEP and immunotherapy.
... This release of cytochrome c activated the apoptotic protein caspase, leading to apoptosis [27,54]. Additionally, the permeabilization of the mitochondrial membrane resulted in ATP depletion, which acts as a critical damageassociated molecular pattern (DAMP) and triggers immunogenic cell death, including necrosis [55,56]. The endoplasmic reticulum (ER) is a large membrane system responsible for protein and lipid synthesis required by the cell, as well as intracellular material transfer. ...
... The osmotic pressure inside mitochondria and the ER is higher than that of the cytoplasmic environment. Therefore, the pulse electric field increases the permeability of the mitochondrial and ER membranes, leading to calcium ion release and the swelling of these organelles [56,59]. The physical connection between mitochondria and the ER is responsible for mediating various physiological functions, including autophagy, intracellular calcium dynamic balance, phospholipid synthesis and transport, and metabolic signal transduction. ...
... The physical connection between mitochondria and the ER is responsible for mediating various physiological functions, including autophagy, intracellular calcium dynamic balance, phospholipid synthesis and transport, and metabolic signal transduction. Pulse electric fields disrupt this physical connection, thereby inhibiting the aforementioned cellular functions [56]. ...
In order to explore the bioelectric effect of 5 ns pulsed electric fields on tumor cells, a spherical single-cell multiphysics model was first established based on the finite element simulation platform. In consideration of the dielectric relaxation of the biological plasma membrane under the high-frequency electric fields, the electroporation and Maxwell stress tensors on the cell membrane and nuclear envelope were analyzed; secondly, taking MDA-MB-231 cells as the research object, combined with fluorescent probe technology, the state change and fluorescence dissipation of its subcellular structure exposed to pulse fields were studied. The results showed that 5 ns pulsed electric fields directly acted inside the cell, causing an electroporation effect and tensile stress on the nuclear envelope, destroying the integrity and order of the cytoskeleton, and damaging the functions of subcellular structures including endoplasmic reticulum, mitochondria, etc. This study provides theoretical and experimental evidence for the research and application of a high-voltage short pulse in the field of biomedical engineering.
... nsPEF, a technology that emerged in 1995 [1], has seen a significant surge in research interest since 2005 [2]. nsPEF's ability to elicit specific cellular effects [3] has resulted in an impressive range of applications, including the activation of neurons [4][5][6][7][8][9] and myocytes [10][11][12][13], wound healing [14][15][16][17], the manipulation of phenotype [18], the modulation of gene expression [19][20][21][22][23][24], antiparasitic effects [25][26][27], enhancement of the immune response [28][29][30][31][32][33], cell proliferation [18,[34][35][36], improved fermentation [37,38], sterilization for the food industry [39][40][41], seed germination [42][43][44], and most notably, the development of novel cancer therapies [2]. Recently, nsPEF has been proposed for virus inactivation [45]. ...
This study takes a step in understanding the physiological implications of the nanosecond pulsed electric field (nsPEF) by integrating molecular dynamics simulations and machine learning techniques. nsPEF, a state-of-the-art technology, uses high-voltage electric field pulses with a nanosecond duration to modulate cellular activity. This investigation reveals a relatively new and underexplored phenomenon: protein-mediated electroporation. Our research focused on the voltage-sensing domain (VSD) of the NaV1.5 sodium cardiac channel in response to nsPEF stimulation. We scrutinized the VSD structures that form pores and thereby contribute to the physical chemistry that governs the defibrillation effect of nsPEF. To do so, we conducted a comprehensive analysis involving the clustering of 142 replicas simulated for 50 ns under nsPEF stimuli. We subsequently pinpointed the representative structures of each cluster and computed the free energy between them. We find that the selected VSD of NaV1.5 forms pores under nsPEF stimulation, but in a way that significant differs from the traditional VSD opening. This study not only extends our understanding of nsPEF and its interaction with protein channels but also adds a new effect to further study.
... So far, activation of immune response through the release of DAMP molecules (such as ATPadenosine triphosphate, HMGB1-High mobility group box 1, calreticulin, nucleic acids, uric acid) has been confirmed in electroporation studies of ECT [24], GET [30] and IRE [31,32]. Furthermore, DAMP molecules were detected also when using HFIRE pulses [33] and nsPEF [34][35][36]. The release of DAMP molecules increases with increasing pulse amplitude, number and duration. ...
... For ICD prediction, DAMP molecules such as ATP, HMBG1 and calreticulin are usually detected, as they are known as the gold standard for predicting ICD in cancer cells [29]. Presence of DAMPs after electroporation has been identified in numerous studies [24,[30][31][32][33][34][35]46,47]. In our previous study, we showed that release of DAMP molecules correlates strongly with IRE, although a weaker correlation was also observed with reversible electroporation [31]. ...
... The presence of HMGB1 as a DAMP molecule has been confirmed in in vitro and in vivo electroporation studies [24,[30][31][32][33][34][35]. Nevertheless, whether the release of HMGB1 depends on pulse duration or pulse type remains unknown. ...
Traditionally, electroporation-based therapies such as electrochemotherapy (ECT), gene electrotransfer (GET) and irreversible electroporation (IRE) are performed with different but typical pulse durations-100 microseconds and 1-50 milliseconds. However, recent in vitro studies have shown that ECT, GET and IRE can be achieved with virtually any pulse duration (millisecond, microsecond, nanosecond) and pulse type (monopolar, bipolar-HFIRE), although with different efficiency. In electroporation-based therapies, immune response activation can affect treatment outcome, and the possibility of controlling and predicting immune response could improve the treatment. In this study, we investigated if different pulse durations and pulse types cause different or similar activations of the immune system by assessing DAMP release (ATP, HMGB1, calreticulin). Results show that DAMP release can be different when different pulse durations and pulse types are used. Nanosecond pulses seems to be the most immunogenic, as they can induce the release of all three main DAMP molecules-ATP, HMGB1 and calreticulin. The least immunogenic seem to be millisecond pulses, as only ATP release was detected and even that assumingly occurs due to increased permeability of the cell membrane. Overall, it seems that DAMP release and immune response in electroporation-based therapies can be controlled though pulse duration.
... Data in this figure were analyzed by one-way ANOVA followed by Tukey's test to determine differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001, ****, p < 0.0001) role in the induction of immune response. Accumulating evidence suggests that local treatment of established tumors with ICD inducer can result in remission of distant tumors, signifying the establishment of systemic immunity [51]. Here we also showed that local administration of miR-CVB3 + CpGMel can effectively delay the progression of distant tumors, suggesting a strong capacity of the developed treatment to induce systemic immunity against tumor cells. ...
Background
Immunotherapy has emerged as an efficient therapeutic approach for cancer management. However, stimulation of host immune system against cancer cells often fails to achieve promising clinical outcomes mainly owing to the immunosuppressive characteristics of the tumor microenvironment (TME). Combination therapeutics that can trigger sustained immunogenic cell death (ICD) have provided new opportunities for cancer treatment.
Methods
In this study, we designed and applied an ICD inducer regimen, including a genetically engineered oncolytic virus (miRNA-modified coxsackieviruses B3, miR-CVB3), a pore-forming lytic peptide (melittin, found in bee venom), and a synthetic toll-like receptor 9 ligand (CpG oligodeoxynucleotides), for breast cancer and melanoma treatment. We compared the anti-tumor efficacy of miR-CVB3 and CpG-melittin (CpGMel) alone and in combination (miR-CVB3 + CpGMel) and investigated possible mechanisms involved.
Results
We demonstrated that miR-CVB3 + CpGMel had no major impact on viral growth, while enhancing the cellular uptake of CpGMel in vitro. We further showed that combination therapy led to significant increases in tumor cell death and release of damage-associated molecular patterns compared with individual treatment. In vivo studies in 4T1 tumor-bearing Balb/c mice revealed that both primary and distant tumors were significantly suppressed, and the survival rate was significantly prolonged after administration of miR-CVB3 + CpGMel compared with single treatment. This anti-tumor effect was accompanied by increased ICD and immune cell infiltration into the TME. Safety analysis showed no significant pathological abnormalities in Balb/c mice. Furthermore, the developed therapeutic regimen also demonstrated a great anti-tumor activity in B16F10 melanoma tumor-bearing C57BL/6 J mice.
Conclusions
Overall, our findings indicate that although single treatment using miR-CVB3 or CpGMel can efficiently delay tumor growth, combining oncolytic virus-based therapy can generate even stronger anti-tumor immunity, leading to a greater reduction in tumor size.
