Figure 7 - uploaded by Franck Michel Andre
Content may be subject to copyright.
ecT is much more efficacious in immunocompetent mice than in immunodeficient animals. (A and B) comparison of electrochemotherapy (ecT) efficiency in wild-type and in nude BalB/c mice bearing established cT26 tumors. Once the mean volume of tumors reached approximately 60 mm 3 , electrochemotherapy was applied by i.v. injection of 15 µg of bleomycin in 100 µl PBs into each anesthetized mouse and 4 min later, 8 electric pulses of 100 µs at 1300 V/cm were delivered to the tumors by cliniporator. (A) ecT treatment in wild-type mice (n = 7 untreated mice and n = 8 treated mice). (B) ecT treatment in immunocompromised nude mice (n = 8 mice per group). Dotted bold lines represent the mean volume of the untreated tumors. The volume of each ecT-treated tumor is depicted with a thin full line.
Source publication
Electrochemotherapy (ECT) is a local cancer treatment that has been used over the course of more than 2 decades for the removal of cutaneous and subcutaneous tumors. Several lines of evidence support the premise that the immune system is an important factor underlying anticancer treatment efficacy, potentially including patient responses to ECT. Th...
Contexts in source publication
Context 1
... order to mimic the treatment of human disease, and to determine the necessity for an intact immune system in eliciting ECT-mediated anticancer effects, ECT was per- formed on established CT26 tumors in both immunocom- petent wild-type and immunodeficient nude BALB/c mice. As shown in Figure 7, we observed that 7 out of 8 immunocompe- tent mice were disease-free 24 d after the ECT treatment whereas all the treated nude mice presented progressive disease. ...
Context 2
... we demonstrated that ECT treatment performed in immunocompetent mice was much more efficient than those that were immunodeficient (refer to Fig. 7). Indeed, a large number (7 out of 8) of complete responses was observed in wild-type BALB/c mice whereas all the tumors progressed in nude mice, despite a temporary decrease in volume and an ini- tial arrest in tumor development caused by the debulking effect Figure 6. Vaccination with electrochemotherapy-treated cancer cells elicits a ...
Similar publications
Electroporation is used in cancer treatment because of its ability to increase local cytotoxicity of e.g. bleomycin (electrochemotherapy) and calcium (calcium electroporation). Calcium electroporation is a novel anticancer treatment that selectively kills cancer cells by necrosis, a cell death pathway that stimulates the immune system due to high r...
Citations
... 20,48,80,81 Electrochemotherapy can induce immunogenic cell death through the release of damage-associated molecular patterns (DAMP) which serve as a signal to stimulate the immune system. [82][83][84] Massive liberation of tumor antigens together with DAMPs can activate the antigenpresenting dendritic cells. 85,86 Multiple studies in canine [87][88][89] , in mice 47 and human patients 90 have thus been testing ECT in combination with gene electrotransfer (GET) of plasmid DNA encoding for interleukin-12 (IL-12), which stimulates the immune system. ...
Background
Electrochemotherapy (ECT) is a treatment involving the administration of chemotherapeutics drugs followed by the application of 8 square monopolar pulses of 100 μs duration at a repetition frequency of 1 Hz or 5000 Hz. However, there is increasing interest in using alternative types of pulses for ECT. The use of high-frequency short bipolar pulses has been shown to mitigate pain and muscle contractions. Conversely, the use of millisecond pulses is interesting when combining ECT with gene electrotransfer for the uptake of DNA-encoding proteins that stimulate the immune response with the aim of converting ECT from a local to systemic treatment. Therefore, the aim of this study was to investigate how alternative types of pulses affect the efficiency of the ECT.
Materials and methods
We performed in vitro experiments, exposing Chinese hamster ovary (CHO) cells to conventional ECT pulses, high-frequency bipolar pulses, and millisecond pulses in the presence of different concentrations of cisplatin. We determined cisplatin uptake by inductively coupled plasma mass spectrometry and cisplatin cytotoxicity by the clonogenic assay.
Results
We observed that the three tested types of pulses potentiate the uptake and cytotoxicity of cisplatin in an equivalent manner, provided that the electric field is properly adjusted for each pulse type. Furthermore, we quantified that the number of cisplatin molecules, resulting in the eradication of most cells, was 2−7 × 10 ⁷ per cell.
Conclusions
High-frequency bipolar pulses and millisecond pulses can potentially be used in ECT to reduce pain and muscle contraction and increase the effect of the immune response in combination with gene electrotransfer, respectively.
... (45) further enhancing transport of the therapeutic agents into cells. This results in the induction of a number of cell death pathways and release of a number of damage-associated molecular patterns (46,47) which may enhance recognition of the tumor by the immune system (48). In veterinary oncology, ECT is an emerging approach for the treatment of dermal malignancies including equine skin tumors (1) along with feline and canine sarcomas (49, 50). ...
Introduction
Integrated time nanosecond pulse irreversible electroporation (INSPIRE) is a novel tumor ablation modality that employs high voltage, alternating polarity waveforms to induce cell death in a well-defined volume while sparing the underlying tissue. This study aimed to demonstrate the in vivo efficacy of INSPIRE against spontaneous melanoma in standing, awake horses.
Methods
A custom applicator and a pulse generation system were utilized in a pilot study to treat horses presenting with spontaneous melanoma. INSPIRE treatments were administered to 32 tumors across 6 horses and an additional 13 tumors were followed to act as untreated controls. Tumors were tracked over a 43–85 day period following a single INSPIRE treatment. Pulse widths of 500ns and 2000ns with voltages between 1000 V and 2000 V were investigated to determine the effect of these variables on treatment outcomes.
Results
Treatments administered at the lowest voltage (1000 V) reduced tumor volumes by 11 to 15%. Higher voltage (2000 V) treatments reduced tumor volumes by 84 to 88% and eliminated 33% and 80% of tumors when 500 ns and 2000 ns pulses were administered, respectively.
Discussion
Promising results were achieved without the use of chemotherapeutics, the use of general anesthesia, or the need for surgical resection in regions which are challenging to keep sterile. This novel therapeutic approach has the potential to expand the role of pulsed electric fields in veterinary patients, especially when general anesthesia is contraindicated, and warrants future studies to demonstrate the efficacy of INSPIRE as a solid tumor treatment.
... When electroporation is used for the local treatment of tumours by enhancing the toxicity of chemotherapeutic agents, it is called electrochemotherapy. This is an ablative technique that causes the reversible permeability of the cell membrane so that chemotherapeutic agents such as cisplatin and bleomycin can penetrate the tumour cells and cause immunogenic cell death and the activation of the immune cell response [1,6,9,10,12,13]. Recent studies indicate that ECT also has a systemic remote effect on untreated tumours [10,[13][14][15][16]. ...
... This is an ablative technique that causes the reversible permeability of the cell membrane so that chemotherapeutic agents such as cisplatin and bleomycin can penetrate the tumour cells and cause immunogenic cell death and the activation of the immune cell response [1,6,9,10,12,13]. Recent studies indicate that ECT also has a systemic remote effect on untreated tumours [10,[13][14][15][16]. It induces immunogenic cell death, leading to the release of tumour antigens and educating immune cells, resulting in a systemic effect. ...
Simple Summary
This study focused on identifying and evaluating the use of selected potential biomarkers in predicting the treatment response of mast cell tumours (MCT) in dogs using a combination of electrochemotherapy (ECT) and interleukin 12 gene electrotransfer (IL-12 GET). In the study of 48 patients with 86 MCTs, blood samples were taken before, one month and six months after treatment to identify reliable biomarkers that predict response to treatment. An increased plasma nucleosome concentration and increased serum lactate dehydrogenase (LDH) activity one month after treatment correlated with a more effective local response, such as necrosis and swelling. These biomarkers proved to be potential early indicators of treatment success. However, the serum ferritin concentration did not prove to be meaningful. The results of this study provide crucial insights into the use of the plasma nucleosome concentration and serum LDH activity as potential indicators of treatment efficacy in veterinary oncology. Ultimately, this study emphasises the need to identify and validate predictive biomarkers to improve treatment outcomes in dogs with MCT and other malignancies treated similarly.
Abstract
Electrochemotherapy (ECT) in combination with the gene electrotransfer of interleukin 12 (IL-12 GET) has been successfully used in veterinary medicine for the treatment of mast cell tumours (MCT), but the biomarkers that could predict response to this treatment have not yet been investigated. The aim of this study was to determine the plasma nucleosome and serum ferritin concentrations, as well as the lactate dehydrogenase (LDH) activity, in the serum of treated patients before and one and six months after treatment to evaluate their utility as potential biomarkers that could predict response to the combined treatment. The study was conducted in 48 patients with a total of 86 MCTs that we treated with the combined treatment. The blood samples used for analysing the potential predictive biomarkers were taken before treatment and one and six months after treatment, when the response to treatment was also assessed. The Nu. Q® Vet Cancer Test, the Canine Ferritin ELISA Kit, and the RX Daytona+ automated biochemical analyser were used to analyse the blood samples. The results showed that the plasma nucleosome concentration (before treatment (BT): 32.84 ng/mL (median); one month after treatment (1 M AT): 58.89 ng/mL (median); p = 0.010) and serum LDH activity (BT: 59.75 U/L (median); 1 M AT: 107.5 U/L (median); p = 0.012) increased significantly one month after treatment and that the increase correlated significantly with the presence of a more pronounced local reaction (necrosis, swelling, etc.) at that time point for both markers (nucleosome: BT (necrosis): 21.61 ng/mL (median); 1 M AT (necrosis): 69.92 ng/mL (median), p = 0.030; LDH: BT (necrosis): 54.75 U/L (median); 1 M AT (necrosis): 100.3 U/L (median), p = 0.048). Therefore, both the plasma nucleosome concentration and serum LDH activity could serve as early indicators of the effect of the treatment. In this context, the serum ferritin concentration showed no significant predictive potential for treatment response (p > 0.999 for all comparisons). In conclusion, this study provides some new and important observations on the use of predictive biomarkers in veterinary oncology. Furthermore, it emphasises the need for the continued identification and validation of potential predictive biomarkers in dogs with MCT and other malignancies undergoing ECT treatment in combination with IL-12 GET to ultimately improve treatment outcomes.
... Initiation of cell signaling pathways has also been observed in melanoma cells, leading to increased expression of heat shock proteins due to structural changes induced by electrical eld pulse application in the cell membrane [15,16]. The translocation of calreticulin to the cell membrane in murine colon cancer cells, both with and without the use of bleomycin, has been demonstrated [17]. Electrical pulse application triggers the release of ATP from cancer cells [17][18][19], which can generate reactive oxygen species (ROS), potentially activating apoptotic pathways in cancer cells [20,21]. ...
... The translocation of calreticulin to the cell membrane in murine colon cancer cells, both with and without the use of bleomycin, has been demonstrated [17]. Electrical pulse application triggers the release of ATP from cancer cells [17][18][19], which can generate reactive oxygen species (ROS), potentially activating apoptotic pathways in cancer cells [20,21]. These ndings suggest that the effects of electrical eld pulses can activate multiple cell death pathways in cancer cells. ...
Purpose
Novel, alternative and combinatorial approaches to combat cancers with minimal side effects is the need of the hour, owing to significant therapy-associated adverse effects. Natural molecule curcumin has shown to have significant anticancer action in lung cancers, a major cause of mortality world over. However, usage of curcumin is limited by its poor bioavailability. High voltage microsecond pulse electric field (HV-µsPEF) therapy is an emerging alternative option in cancer therapy which can be used as an effective adjuvant anti-cancer therapy. In the current investigation, we assessed the combinatorial effect of HV-µsPEF with low-dose curcumin on A549 lung cancer cell line with the main objective of minimization of the curcumin dose required for its anti-cancer action.
Method
HV-µsPEF was generated using a previously reported pulse-generator which was combined with low doses of curcumin to assess effects on A549 cell line. Phase-contrast microscopy based cellular morphology analysis, flow-cytometry based analyses to assess the mode of cell death, uptake of curcumin, assessment of reactive oxygen species and that of mitochondrial membrane potential (MMP) loss were carried out.
Results
Our work demonstrates a highly synergistic cell death induction in A549 cells as assessed by phase contrast microscopy and flow-cytometry based analysis due to the increased uptake of curcumin by cancer cells in the presence of HV-µsPEF. This led to increased ROS production and loss of MMP in cancer cells with the combinatorial treatment.
Conclusion
This study shows promising anti-cancer effects in the usage of low doses of curcumin, a natural molecule, with HV-µsPEF. Combined treatment of HV-µsPEF with curcumin in A549 cells increased the uptake of curcumin in the cells leading to effective induction of cell death via increased ROS production and MMP loss effectively minimizing the requirement of high doses of curcumin which are practically not achievable.
... It was shown that not only ECT combined with the chemotherapeutic drug bleomycin can induce ICD, but that also ECT with other drugs (cisplatin and oxaliplatin) triggers ICD to a similar degree [42]. Electric pulses and chemotherapeutics alone do not always induce the release of DAMP (HMGB1 was released only in the presence of chemotherapeutics alone, whereas calreticulin was externalized after electric pulses alone) [43]. ...
These authors contributed equally to this work. Abstract: High-Intensity Pulsed Electromagnetic Fields (HI-PEMF) treatment is an emerging noninvasive and contactless alternative to conventional electroporation, since the electric field inside the tissue is induced remotely by an externally applied pulsed magnetic field. Recently, HI-PEMF has been successfully used in the transfer of plasmid DNA and siRNA in vivo, with no or minimal infiltration of immune cells. In addition to gene electrotransfer, treatment with HI-PEMF has also shown potential for electrochemotherapy, where activation of the immune response contributes to the treatment outcome. The immune response can be triggered by immunogenic cell death that is characterized by the release of damage-associated molecular patterns (DAMPs) from damaged or/and dying cells. In this study, the release of the best-known DAMP molecules, i.e., adenosine triphosphate (ATP), calreticulin and high mobility group box 1 protein (HMBG1), after HI-PEMF treatment was investigated in vitro on three different cell lines of different tissue origin and compared with conventional electroporation treatment parameters. We have shown that HI-PEMF by itself does not cause the release of HMGB1 or calreticulin, whereas the release of ATP was detected immediately after HI-PEMF treatment. Our results indicate that HI-PEMF treatment causes no to minimal release of DAMP molecules, which results in minimal/limited activation of the immune response.
... ECT can induce immunogenic cell death (ICD), stimulating the immune system, mainly dendritic cells (DCs) and macrophages, through damageassociated molecular patterns (DAMPs) such as calreticulin (CRT), heat shock proteins, high-mobility group box 1 (HMGB1), type I IFN, and ATP [97,98]. Furthermore, it has been demonstrated that T cells play a key role in the immune response for the local and systemic effects of ECT [99][100][101]. Roux et al., demonstrated that ECT followed by TLR-9 ligands, CpG oligodeoxynucleotides (CpG ODN), in three murine tumor models synergized and enhanced the local effect and systemic T-cell-dependent antitumor activity [100]. Furthermore, it has also been demonstrated that ECT can modify the DC and T cell populations in the lesions of melanoma patients who have undergone bleomycin ECT. ...
Melanoma is one of the deadliest skin tumors, accounting for almost 90% of skin cancer mortality. Although immune therapy and targeted therapy have dramatically changed the prognosis of metastatic melanoma, many patients experience disease progression despite the currently available new treatments. Skin metastases from melanoma represent a relatively common event as first sign of advanced disease or a sign of recurrence. Skin metastases are usually asymptomatic, although in advanced stages, they can present with ulceration, bleeding, and superinfection; furthermore, they can cause symptoms related to compression on nearby tissues. Treatments vary from simple surgery resections to topical or intralesional local injections, or a combination of these techniques with the most recent systemic immune or target therapies. New research and studies should focus on the pathogenesis and molecular mechanisms of the cutaneous metastases of melanoma in order to shed light on the mechanisms underlying the different behavior and prognoses of different patients.
... From preclinical studies, both ECT and CaEP have been shown to give rise to an anticancer immune response and to repress distant tumor growth outside of the treated area (Calvet et al. 2014;Di Gennaro et al. 2016;Falk 2008Falk , 2017Tremble et al. 2019). ECT is known to induce immunogenic cell death by releasing High Mobility Group Box 1 Protein (HMGB1), calreticulin, and ATP (Calvet et al. 2014). ...
... From preclinical studies, both ECT and CaEP have been shown to give rise to an anticancer immune response and to repress distant tumor growth outside of the treated area (Calvet et al. 2014;Di Gennaro et al. 2016;Falk 2008Falk , 2017Tremble et al. 2019). ECT is known to induce immunogenic cell death by releasing High Mobility Group Box 1 Protein (HMGB1), calreticulin, and ATP (Calvet et al. 2014). In murine models, ECT has been shown to induce a systemic effect and suppress distant metastases (Roux et al. 2008;Tremble et al. 2019). ...
Purpose:
In this study, we aim to investigate gene expression changes in tumor samples obtained from patients with esophageal cancer treated with calcium electroporation. Previously, local treatment with calcium electroporation has been shown to induce gene expression alterations, potentially contributing to a more tumor-hostile microenvironment.
Methods:
In this sub-study of a phase I clinical trial, we included five patients with esophageal cancer treated with calcium electroporation. We compared cancer-associated gene expression patterns in tumor samples before and after treatment. Furthermore, we used linear support vector regression to predict the cellular composition of tumor samples.
Results:
Using differential expression analysis, we identified the downregulation of CXCL14 and upregulation of CCL21, ANGPTL4, and CRABP2 genes. We also found a decreased predicted proportion of dendritic cells while the proportion of neutrophils was increased.
Conclusion:
This study provides evidence that calcium electroporation for esophageal cancer induces local transcriptional changes and possibly alters the cellular composition of the tumor microenvironment. The results are explorative, larger studies are needed to confirm and further correlate our findings with clinical outcomes.
... Finally, the last advancement in deep-seated tumor treatment with ECT is a combination strategy using other techniques in addition to ECT. ECT alone induces a local immune response and has been considered by several reviews to be a type of in situ vaccination [51,52]. However, in addition to the ECT procedure, combining immunotherapeutic drugs helps to enhance this local and systemic immune response after ECT treatment. ...
The primary aim of this study was to analyze studies that use electrochemotherapy (ECT) in “deep-seated” tumors in solid organs (liver, kidney, bone metastasis, pancreas, and abdomen) and understand the similarities between patient selection, oncologic selection, and use of new procedures and technology across the organ systems to assess response rates. A literature search was conducted using the term “Electrochemotherapy” in the title field using publications from 2017 to 2023. After factoring in inclusion and exclusion criteria, 29 studies were analyzed and graded based on quality in full. The authors determined key patient and oncologic selection characteristics and ECT technology employed across organ systems that yielded overall responses, complete responses, and partial responses of the treated tumor. It was determined that key selection factors included: the ability to be administered bleomycin, life expectancy greater than three months, unrespectability of the lesion being treated, and a later stage, more advanced cancer. Regarding oncologic selection, all patient cohorts had received chemotherapy or surgery previously but had disease recurrence, making ECT the only option for further treatment. Lastly, in terms of the use of technology, the authors found that studies with better response rates used the ClinporatorTM and updated procedural guidelines by SOP. Thus, by considering patient, oncologic, and technology selection, ECT can be further improved in treating lesions in solid organs.
... Moreover, a clinical study with CaEP indicated a systemic immune response after the treatment. Patients with malignant melanoma had complete remission in both treated and untreated metastases, even without any additional treatment [43,44]. ...
... The tumor luminescent visualization confirms the obtained tumor volumetric measurement results. Our results on ESOPE pulses are in agreement with CaEP effects reported in other studies [5,12,26,35,43,44]. More importantly, we have presented novel data on MHz nanosecond pulses, which can be used as a better alternative for ECT. ...
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 effect lasts longer in neoplastic areas, enhancing tumor exposure to drugs and in turn making chemotherapeuticinduced mitotic cell death the most potent mechanism in ECT (123). Apoptosis and pyroptosis have also been commonly observed in BLM-mediated ECT, and necroptosis has been observed in both BLM and CDDP ECT (124,125). ...
Over the past decade, the increased adoption of electroporation-based technologies has led to an expansion of clinical research initiatives. Electroporation has been utilized in molecular biology for mammalian and bacterial transfection; for food sanitation; and in therapeutic settings to increase drug uptake, for gene therapy, and to eliminate cancerous tissues. We begin this article by discussing the biophysics required for understanding the concepts behind the cell permeation phenomenon that is electroporation. We then review nano- and microscale single-cell electroporation technologies before scaling up to emerging in vivo applications.
Expected final online publication date for the Annual Review of Biomedical Engineering, Volume 25 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
