Irreversible electroporation promotes a pro-inflammatory tumor microenvironment and anti-tumor immunity in a mouse pancreatic cancer model.

Pancreatic cancer is a significant cause of cancer-related mortality and often presents with limited treatment options. Pancreatic tumors are also notorious for their immunosuppressive microenvironment. Irreversible electroporation (IRE) is a non-thermal tumor ablation modality that employs high-voltage microsecond pulses to transiently permeabilize cell membranes, ultimately inducing cell death. However, the understanding of IRE's impact beyond the initiation of focal cell death in tumor tissue remains limited. In this study, we demonstrate that IRE triggers a unique mix of cell death pathways and orchestrates a shift in the local tumor microenvironment driven, in part, by reducing the myeloid-derived suppressor cell (MDSC) and regulatory T cell populations and increasing cytotoxic T lymphocytes and neutrophils. We further show that IRE drives induce cell cycle arrest at the G0/G1 phase in vitro and promote inflammatory cell death pathways consistent with pyroptosis and programmed necrosis in vivo. IRE-treated mice exhibited a substantial extension in progression-free survival. However, within a span of 14 days, the tumor immune cell populations reverted to their pre-treatment composition, which resulted in an attenuation of the systemic immune response targeting contralateral tumors and ultimately resulting in tumor regrowth. Mechanistically, we show that IRE augments IFN- Î³ signaling, resulting in the up-regulation of the PD-L1 checkpoint in pancreatic cancer cells. Together, these findings shed light on potential mechanisms of tumor regrowth following IRE treatment and offer insights into co-therapeutic targets to improve treatment strategies.

Properties of tissue within prostate tumors and treatment planning implications for ablation therapies.

Irreversible electroporation (IRE) is a promising alternative therapy for the local treatment of prostate tumors. The procedure involves the direct insertion of needle electrodes into the target zone, and subsequent delivery of short but high-voltage pulses. Successful outcomes rely on adequate exposure of the tumor to a threshold electrical field. To aid in predicting this exposure, computational models have been developed, yet often do not incorporate the appropriate tissue-specific properties. This work aims to quantify electrical conductivity behavior during IRE for three types of tissue present in the target area of a prostate cancer ablation: the tumor tissue itself, the surrounding healthy tissue, and potential areas of necrosis within the tumor. Animal tissues were used as a stand-in for primary samples. The patient-derived prostate tumor tissue showed very similar responses to healthy porcine prostate tissue. An examination of necrotic tissue inside the tumors revealed a large difference, however, and a computational model showed that a necrotic core with differing electrical properties can cause unexpected inhomogeneities within the treatment region.

Histotripsy Ablation Alters the Tumor Microenvironment and Promotes Immune System Activation in a Subcutaneous Model of Pancreatic Cancer.

Pancreatic cancer is a significant cause of cancer-related deaths in the United States with an abysmal five-year overall survival rate that is under 9%. Reasons for this mortality include the lack of late-stage treatment options and the immunosuppressive tumor microenvironment. Histotripsy is an ultrasound-guided, noninvasive, nonthermal tumor ablation therapy that mechanically lyses targeted cells. To study the effects of histotripsy on pancreatic cancer, we utilized an in vitro model of pancreatic adenocarcinoma and compared the release of potential antigens following histotripsy treatment to other ablation modalities. Histotripsy was found to release immune-stimulating molecules at magnitudes similar to other nonthermal ablation modalities and superior to thermal ablation modalities, which corresponded to increased innate immune system activation in vivo. In subsequent in vivo studies, murine Pan02 tumors were grown in mice and treated with histotripsy. Flow cytometry and rtPCR were used to determine changes in the tumor microenvironment over time compared to untreated animals. In mice with pancreatic tumors, we observed significantly increased tumor-progression-free and general survival, with increased activation of the innate immune system 24 h posttreatment and decreased tumor-associated immune cell populations within 14 days of treatment. This study demonstrates the feasibility of using histotripsy for pancreatic cancer ablation and provides mechanistic insight into the initial innate immune system activation following treatment. Further work is needed to establish the mechanisms behind the immunomodulation of the tumor microenvironment and immune effects.

Establishing an immunocompromised porcine model of human cancer for novel therapy development with pancreatic adenocarcinoma and irreversible electroporation.

New therapies to treat pancreatic cancer are direly needed. However, efficacious interventions lack a strong preclinical model that can recapitulate patients' anatomy and physiology. Likewise, the availability of human primary malignant tissue for ex vivo studies is limited. These are significant limitations in the biomedical device field. We have developed RAG2/IL2RG deficient pigs using CRISPR/Cas9 as a large animal model with the novel application of cancer xenograft studies of human pancreatic adenocarcinoma. In this proof-of-concept study, these pigs were successfully generated using on-demand genetic modifications in embryos, circumventing the need for breeding and husbandry. Human Panc01 cells injected subcutaneously into the ears of RAG2/IL2RG deficient pigs demonstrated 100% engraftment with growth rates similar to those typically observed in mouse models. Histopathology revealed no immune cell infiltration and tumor morphology was highly consistent with the mouse models. The electrical properties and response to irreversible electroporation of the tumor tissue were found to be similar to excised human pancreatic cancer tumors. The ample tumor tissue produced enabled improved accuracy and modeling of the electrical properties of tumor tissue. Together, this suggests that this model will be useful and capable of bridging the gap of translating therapies from the bench to clinical application.

Multi-Tissue Analysis on the Impact of Electroporation on Electrical and Thermal Properties.

OBJECTIVE: Tissue electroporation is achieved by applying a series of electric pulses to destabilize cell membranes within the target tissue. The treatment volume is dictated by the electric field distribution, which depends on the pulse parameters and tissue type and can be readily predicted using numerical methods. These models require the relevant tissue properties to be known beforehand. This study aims to quantify electrical and thermal properties for three different tissue types relevant to current clinical electroporation. METHODS: Pancreatic, brain, and liver tissue were harvested from pigs, then treated with IRE pulses in a parallel-plate configuration. Resulting current and temperature readings were used to calculate the conductivity and its temperature dependence for each tissue type. Finally, a computational model was constructed to examine the impact of differences between tissue types. RESULTS: Baseline conductivity values (mean 0.11, 0.14, and 0.12 S/m) and temperature coefficients of conductivity (mean 2.0, 2.3, and 1.2 % per degree Celsius) were calculated for pancreas, brain, and liver, respectively. The accompanying computational models suggest field distribution and thermal damage volumes are dependent on tissue type. CONCLUSION: The three tissue types show similar electrical and thermal responses to IRE, though brain tissue exhibits the greatest differences. The results also show that tissue type plays a role in the expected ablation and thermal damage volumes. SIGNIFICANCE: The conductivity and its changes due to heating are expected to have a marked impact on the ablation volume. Incorporating these tissue properties aids in the prediction and optimization of electroporation-based therapies.

Starting a Fire Without Flame: The Induction of Cell Death and Inflammation in Electroporation-Based Tumor Ablation Strategies.

New therapeutic strategies and paradigms are direly needed for the treatment of cancer. While the surgical removal of tumors is favored in most cancer treatment plans, resection options are often limited based on tumor localization. Over the last two decades, multiple tumor ablation strategies have emerged as promising stand-alone or combination therapeutic options for patients. These strategies are often employed to treat tumors in areas where surgical resection is not possible or where chemotherapeutics have proven ineffective. The type of cell death induced by the ablation modality is a critical aspect of therapeutic success that can impact the efficacy of the treatment and systemic anti-tumor immune system responses. Electroporation-based ablation technologies include electrochemotherapy, irreversible electroporation, and other modalities that rely on pulsed electric fields to create pores in cell membranes. These pores can either be reversible or irreversible depending on the electric field parameters and can induce cell death either alone or in combination with a therapeutic agent. However, there have been many controversial findings among these technologies as to the cell death type initiated, from apoptosis to pyroptosis. As cell death mechanisms can impact treatment side effects and efficacy, we review the main types of cell death induced by electroporation-based treatments and summarize the impact of these mechanisms on treatment response. We also discuss potential reasons behind the variability of findings such as the similarities between cell death pathways, differences between cell-types, and the variation in electric field strength across the treatment area.

EView: An electric field visualization web platform for electroporation-based therapies.

BACKGROUND AND OBJECTIVES: Electroporation is the phenomenon by which cell membrane permeability to ions and macromolecules is increased when the cell is briefly exposed to high electric fields. In electroporation-based treatments, such exposure is typically performed by delivering high voltage pulses across needle electrodes in tissue. For a given tissue and pulsing protocol, an electric field magnitude threshold exists that must be overreached for treatment efficacy. However, it is hard to preoperatively infer the treatment volume because the electric field distribution intricately depends on the electrodes' positioning and length, the applied voltage, and the electric conductivity of the treated tissues. For illustrating such dependencies, we have created EView (https://eview.upf.edu), a web platform that estimates the electric field distribution for arbitrary needle electrode locations and orientations and overlays it on 3D medical images. METHODS: A client-server approach has been implemented to let the user set the electrode configuration easily on the web browser, whereas the simulation is computed on a dedicated server. By means of the finite element method, the electric field is solved in a 3D volume. For the sake of simplicity, only a homogeneous tissue is modeled, assuming the same properties for healthy and pathologic tissues. The non-linear dependence of tissue conductivity on the electric field due to the electroporation effect is modeled. The implemented model has been validated against a state of the art finite element solver, and the server has undergone a heavy load test to ensure reliability and to report execution times. RESULTS: The electric field is rapidly computed for any electrode and tissue configuration, and alternative setups can be easily compared. The platform provides the same results as the state of the art finite element solver (Dice = 98.3 ± 0.4%). During the high load test, the server remained responsive. Simulations are computed in less than 2 min for simple cases consisting of two electrodes and take up to 40 min for complex scenarios consisting of 6 electrodes. CONCLUSIONS: With this free platform we provide expert and non-expert electroporation users a way to rapidly model the electric field distribution for arbitrary electrode configurations.

Development of a Multi-Pulse Conductivity Model for Liver Tissue Treated With Pulsed Electric Fields.

Pulsed electric field treatment modalities typically utilize multiple pulses to permeabilize biological tissue. This electroporation process induces conductivity changes in the tissue, which are indicative of the extent of electroporation. In this study, we characterized the electroporation-induced conductivity changes using all treatment pulses instead of solely the first pulse as in conventional conductivity models. Rabbit liver tissue was employed to study the tissue conductivity changes caused by multiple, 100 µs pulses delivered through flat plate electrodes. Voltage and current data were recorded during treatment and used to calculate the tissue conductivity during the entire pulsing process. Temperature data were also recorded to quantify the contribution of Joule heating to the conductivity according to the tissue temperature coefficient. By fitting all these data to a modified Heaviside function, where the two turning points (E 0, E 1) and the increase factor (A) are the main parameters, we calculated the conductivity as a function of the electric field (E), where the parameters of the Heaviside function (A and E 0) were functions of pulse number (N). With the resulting multi-factor conductivity model, a numerical electroporation simulation can predict the electrical current for multiple pulses more accurately than existing conductivity models. Moreover, the saturating behavior caused by electroporation can be explained by the saturation trends of the increase factor A in this model. The conductivity change induced by electroporation has a significant increase at about the first 30 pulses, then tends to saturate at 0.465 S/m. The proposed conductivity model can simulate the electroporation process more accurately than the conventional conductivity model. The electric field distribution computed using this model is essential for treatment planning in biomedical applications utilizing multiple pulsed electric fields, and the method proposed here, relating the pulse number to the conductivity through the variables in the Heaviside function, may be adapted to investigate the effect of other parameters, like pulse frequency and pulse width, on electroporation.

Patient Derived Xenografts Expand Human Primary Pancreatic Tumor Tissue Availability for ex vivo Irreversible Electroporation Testing.

New methods of tumor ablation have shown exciting efficacy in pre-clinical models but often demonstrate limited success in the clinic. Due to a lack of quality or quantity in primary malignant tissue specimens, therapeutic development and optimization studies are typically conducted on healthy tissue or cell-line derived rodent tumors that don't allow for high resolution modeling of mechanical, chemical, and biological properties. These surrogates do not accurately recapitulate many critical components of the tumor microenvironment that can impact in situ treatment success. Here, we propose utilizing patient-derived xenograft (PDX) models to propagate clinically relevant tumor specimens for the optimization and development of novel tumor ablation modalities. Specimens from three individual pancreatic ductal adenocarcinoma (PDAC) patients were utilized to generate PDX models. This process generated 15-18 tumors that were allowed to expand to 1.5 cm in diameter over the course of 50-70 days. The PDX tumors were morphologically and pathologically identical to primary tumor tissue. Likewise, the PDX tumors were also found to be physiologically superior to other in vitro and ex vivo models based on immortalized cell lines. We utilized the PDX tumors to refine and optimize irreversible electroporation (IRE) treatment parameters. IRE, a novel, non-thermal tumor ablation modality, is being evaluated in a diverse range of cancer clinical trials including pancreatic cancer. The PDX tumors were compared against either Pan02 mouse derived tumors or resected tissue from human PDAC patients. The PDX tumors demonstrated similar changes in electrical conductivity and Joule heating following IRE treatment. Computational modeling revealed a high similarity in the predicted ablation size of the PDX tumors that closely correlate with the data generated with the primary human pancreatic tumor tissue. Gene expression analysis revealed that IRE treatment resulted in an increase in biological pathway signaling associated with interferon gamma signaling, necrosis and mitochondria dysfunction, suggesting potential co-therapy targets. Together, these findings highlight the utility of the PDX system in tumor ablation modeling for IRE and increasing clinical application efficacy. It is also feasible that the use of PDX models will significantly benefit other ablation modality testing beyond IRE.

Dynamics of Cell Death After Conventional IRE and H-FIRE Treatments.

High-frequency irreversible electroporation (H-FIRE) has emerged as an alternative to conventional irreversible electroporation (IRE) to overcome the issues associated with neuromuscular electrical stimulation that appear in IRE treatments. In H-FIRE, the monopolar pulses typically used in IRE are replaced with bursts of short bipolar pulses. Currently, very little is known regarding how the use of a different waveform affects the cell death dynamics and mechanisms. In this study, human pancreatic adenocarcinoma cells were treated with a typical IRE protocol and various H-FIRE schemes with the same energized time. Cell viability, membrane integrity and Caspase 3/7 activity were assessed at different times after the treatment. In both treatments, we identified two different death dynamics (immediate and delayed) and we quantified the electric field ranges that lead to each of them. While in the typical IRE protocol, the electric field range leading to a delayed cell death is very narrow, this range is wider in H-FIRE and can be increased by reducing the pulse length. Membrane integrity in cells suffering a delayed cell death shows a similar time evolution in all treatments, however, Caspase 3/7 expression was only observed in cells treated with H-FIRE.

High-frequency irreversible electroporation is an effective tumor ablation strategy that induces immunologic cell death and promotes systemic anti-tumor immunity.

BACKGROUND: Despite promising treatments for breast cancer, mortality rates remain high and treatments for metastatic disease are limited. High-frequency irreversible electroporation (H-FIRE) is a novel tumor ablation technique that utilizes high-frequency bipolar electric pulses to destabilize cancer cell membranes and induce cell death. However, there is currently a paucity of data pertaining to immune system activation following H-FIRE and other electroporation based tumor ablation techniques. METHODS: Here, we utilized the mouse 4T1 mammary tumor model to evaluate H-FIRE treatment parameters on cancer progression and immune system activation in vitro and in vivo. FINDINGS: H-FIRE effectively ablates the primary tumor and induces a pro-inflammatory shift in the tumor microenvironment. We further show that local treatment with H-FIRE significantly reduces 4T1 metastases. H-FIRE kills 4T1 cells through non-thermal mechanisms associated with necrosis and pyroptosis resulting in damage associated molecular pattern signaling in vitro and in vivo. Our data indicate that the level of tumor ablation correlates with increased activation of cellular immunity. Likewise, we show that the decrease in metastatic lesions is dependent on the intact immune system and H-FIRE generates 4T1 neoantigens that engage the adaptive immune system to significantly attenuate tumor progression. INTERPRETATION: Cell death and tumor ablation following H-FIRE treatment activates the local innate immune system, which shifts the tumor microenvironment from an anti-inflammatory state to a pro-inflammatory state. The non-thermal damage to the cancer cells and increased innate immune system stimulation improves antigen presentation, resulting in the engagement of the adaptive immune system and improved systemic anti-tumor immunity.

Post-treatment analysis of irreversible electroporation waveforms delivered to human pancreatic cancer patients.

Irreversible electroporation (IRE) is a focal ablation therapy that uses high voltage, short electrical pulses to destroy tumor tissue. The success of treatment directly depends on exposure of the entire tumor to a lethal electric field magnitude. However, this exposure is difficult to predict ahead of time and it is challenging for clinicians to determine optimal treatment parameters. One method clinicians rely upon for the cessation of pulse delivery is to monitor the resistance value of the tissue, as the cells within the tissue will undergo changes during electroporation. This work presents a computational model which incorporates human pancreatic tumor conductivity, and compares predicted and measured output currents from IRE treatments of human patients. The measured currents vary widely from patient to patient, suggesting there may areas of high local conductivity in the treatment area.

Electrical Characterization of Human Biological Tissue for Irreversible Electroporation Treatments.

Irreversible electroporation (IRE) is a cancer therapy that uses short, high-voltage electrical pulses to treat tumors. Due to its predominantly non-thermal mechanism and ability to ablate unresectable tumors, IRE has gained popularity in clinical treatments of both liver and pancreatic cancers. Existing computational models use electrical properties of animal tissue that are quantified a priori to predict the area of treatment in three dimensions. However, the changes in the electrical properties of human tissue during IRE treatment are so far unexplored. This work aims to improve models by characterizing the dynamic electrical behavior of human liver and pancreatic tissue. Fresh patient samples of each tissue type, both normal and tumor, were collected and IRE pulses were applied between two parallel metal plates at various voltages. The electrical conductivity was determined from the resistance using simple relations applicable to cylindrical samples. The results indicate that the percent change in conductivity during IRE treatments varies significantly with increasing electric field magnitudes. This percent change versus applied electric field behavior can be fit to a sigmoidal curve, as proposed in prior studies. The generic conductivity data from human patients from this work can be input to computational software using patient-specific geometry, giving clinicians a more accurate and personalized prediction of a given IRE treatment.