CpG and Interleukin-15 Synergize to Enhance IFN-gamma Production by Activated CD8(+) T Cells

Interleukin-15 (IL-15) regulates the development and maintenance of memory CD8(+) T cells. Paradoxically, we previously reported that IL-15 could enhance CD8(+) T-cell responses to IL-12, a proinflammatory cytokine required for optimal priming of effector CD8(+) T cells. To expand the physiological relevance of these findings, we tested IL-15 for its ability to enhance T-cell responses to bacterial CpG. Expectedly, CpG enhanced the production of IFN-gamma by CD8(+) T cells polyclonally activated with anti-CD3. However, addition of IL-15 to CpG-stimulated cultures led to a striking increase in IFN-gamma production. The effect of CpG and IL-15 was also evident with CD8(+) T cells recovered from mice infected with the parasite Trypanosoma cruzi (T. cruzi) and restimulated with antigen. The observed synergy between CpG and IL-15 occurred in an IL-12-dependent manner, and this effect could even be demonstrated in cocultures of activated CD8(+) T cells and CD4(+)CD25(+) regulatory T cells. Although IFN-gamma was not essential for CpG-induced IL-12, the ability of CpG and IL-15 to act on CD8(+) T cells required expression of the IFN-gamma-inducible transcription factor T-bet. These data have important implications for development of vaccines and design of therapies to boost CD8(+) T-cell responses to infectious agents and tumors

Moderate Heat Application Enhances the Efficacy of Nanosecond Pulse Stimulation for the Treatment of Squamous Cell Carcinoma

Nanosecond pulse stimulation as a tumor ablation therapy has been studied for the treatment of various carcinomas in animal models and has shown a significant survival benefit. In the current study, we found that moderate heating at 43°C for 2 minutes significantly enhanced in vitro nanosecond pulse stimulation-induced cell death of KLN205 murine squamous cell carcinoma cells by 2.43-fold at 600 V and by 2.32-fold at 900 V, as evidenced by propidium iodide uptake. Furthermore, the ablation zone in KLN205 cells placed in a 3-dimensional cell-culture model and pulsed at a voltage of 900 V at 43°C was 3 times larger than in cells exposed to nanosecond pulse stimulation at room temperature. Application of moderate heating alone did not cause cell death. A nanosecond pulse stimulation electrode with integrated controllable laser heating was developed to treat murine ectopic squamous cell carcinoma. With this innovative system, we were able to quickly heat and maintain the temperature of the target tumor at 43 degrees C during nanosecond pulse stimulation. Nanosecond pulse stimulation with moderate heating was shown to significantly extend overall survival, delay tumor growth, and achieve a high rate of complete tumor regression. Moderate heating extended survival nearly 3-fold where median overall survival was 22 days for 9.8 kV without moderate heating and over 63 days for tumors pulsed with 600, 100 ns pulses at 5 Hz, at voltage of 9.8 kV with moderate heating. Median overall survival in the control groups was 24 and 31 days for mice with untreated tumors and tumors receiving moderate heat alone, respectively. Nearly 69% (11 of 16) of tumor-bearing mice treated with nanosecond pulse stimulation with moderate heating were tumor free at the completion of the study, whereas complete tumor regression was not observed in the control groups and in 9.8 kV without moderate heating. These results suggest moderate heating can reduce the necessary applied voltage for tumor ablation with nanosecond pulse stimulation

Gene Electrotransfer Enhanced by Nanosecond Pulsed Electric Fields

The impact of nanosecond pulsed electric fields (nsPEFs) on gene electrotransfer has not been clearly demonstrated in previous studies. This study was conducted to evaluate the influence of nsPEFs on the delivery of plasmids encoding luciferase or green fluorescent protein and subsequent expression in HACAT keratinocyte cells. Delivery was performed using millisecond electric pulses (msEPs) with or without nsPEFs. In contrast to reports in the literature, we discovered that gene expression was significantly increased up to 40-fold by applying nsPEFs to cells first followed by one msEP but not in the opposite order. We demonstrated that the effect of nsPEFs on gene transfection was time restricted. The enhancement of gene expression occurred by applying one msEP immediately after nsPEFs and reached the maximum at posttreatment 5 minutes, slightly decreased at 15 minutes and had a residual effect at 1 hour. It appears that nsPEFs play a role as an amplifier without changing the trend of gene expression kinetics due to msEPs. The effect of nsPEFs on cell viability is also dependent on the specific pulse parameters. We also determined that both calcium independent and dependent mechanisms are involved in nsPEF effects on gene electrotransfer

Nano-Pulse Stimulation for the Treatment of Pancreatic Cancer and the Changes in Immune Profile

A Pancreatic cancer is a notorious malignant neoplasm with an extremely poor prognosis. Current standard of care is rarely effective against late-stage pancreatic cancer. In this study, we assessed nanopulse stimulation (NPS) as a local treatment for pancreatic cancer in a syngeneic mouse Pan02 pancreatic cancer model and characterized corresponding changes in the immune profile. A single NPS treatment either achieved complete tumor regression or prolonged overall survival in animals with partial tumor regression. While this is very encouraging, we also explored if this local ablation effect could also result in immune stimulation, as was observed when NPS led to the induction of immune-mediated protection from a second tumor challenge in orthotopic mouse breast and rat liver cancer models. In the Pan02 model, there were insufficient abscopal effects (1/10) and vaccine-like protective effects (1/15) suggesting that NPS-induced immune mechanisms in this model were limited. To evaluate this further, the immune landscape was analyzed. The numbers of both T regulatory cells (Tregs) and myeloid derived suppressor cells (MDSCs) in blood were significantly reduced, but memory (CD44+) T-cells were absent. Furthermore, the numbers of Tregs and MDSCs did not reduce in spleens compared to tumor-bearing mice. Very few T-cells, but large numbers of MDSCs were present in the NPS treated tumor microenvironment (TME). The number of dendritic cells in the TME was increased and multiple activation markers were upregulated following NPS treatment. Overall, NPS treatments used here are effective for pancreatic tumor ablation, but require further optimization for induction of immunity or the need to include effective combinational NPS therapeutic strategy for pancreatic cancer

Controllable Moderate Heating Enhances the Therapeutic Efficacy of Irreversible Electroporation for Pancreatic Cancer

Irreversible electroporation (IRE) as a non-thermal tumor ablation technology has been studied for the treatment of pancreatic carcinoma and has shown a significant survival benefit. We discovered that moderate heating (MH) at 43°C for 1-2 minutes significantly enhanced ex vivo IRE tumor ablation of Pan02 cells by 5.67-fold at 750 V/cm and by 1.67-fold at 1500 V/cm. This amount of heating alone did not cause cell death. An integrated IRE system with controllable laser heating and tumor impedance monitoring was developed to treat mouse ectopic pancreatic cancer. With this novel IRE system, we were able to heat and maintain the temperature of a targeted tumor area at 42°C during IRE treatment. Pre-heating the tumor greatly reduced the impedance of tumor and its fluctuation. Most importantly, MHIRE has been demonstrated to significantly extend median survival and achieve a high rate of complete tumor regression. Median survival was 43, 46 and 84 days, for control, IRE with 100 μs, 1 Hz, 90 pulses and electric fields 2000-2500 V/cm and MHIRE treatment respectively. 55.6% of tumor-bearing mice treated with MHIRE were tumor-free, whereas complete tumor regression was not observed in the control and IRE treatment groups

Challenges in Bioelectrics for Cancer Immunotherapy

Major advances have been made in cancer immunotherapy. One example is checkpoint blockade immunotherapy which has been approved by FDA to treat numerous cancer types resulting in a substantial increase in the number of lives saved. However, two major hurdles; low response rates, especially to poorly immunogenic cancers and immune-related adverse events (irAEs), limit its broader applications. Differently, a number of tumor ablation modalities have been studied to improve antitumor immunity, nevertheless, low potency is a common issue even in combination with systemic immunotherapy. Emerging bioelectric technologies including electrochemotherapy, irreversible electroporation, gene electro-transfer (GET), nanosecond electrical pulse (nsEP), non-thermal plasma, etc. have been studied to treat cancer in animal models and clinical trials. GET immunotherapy has been demonstrated a safe and effective therapy for metastatic melanoma in clinical trials. Immunological effects have been observed in other bioelectric modalities as well. Hence, bioelectric technologies as cancer immunotherapies have been proposed and studied. However, there are a number of challenges that need to be addressed before bioelectric cancer immunotherapies could benefit patients. These challenges include technological limitation, tumor heterogeneity, animal model, research capacity, etc. Our group has been focusing on nsEPs induced immunological effects. The challenges in nsEPs as cancer immunotherapy are discussed. Various solutions are proposed to overcome these challenges

Synergistic Effect of Nanopulse and Cold Plasma in Pancreatic Cancer

Pancreatic adenocarcinoma is a highly aggressive malignancy with disease-related mortality almost equaling its incidence. The notorious resistance of pancreatic cancer not only to conventional cytotoxic therapies but also to almost all targeted agents developed to date continues to puzzle the oncological community and represents one of the biggest hurdles to reduce the death toll from this ominous disease. Currently, we have been focusing on developing nanosecond electric pulse or nano-pulse (NP) technology for cancer therapy. However, due to the demand for high electric fields, all currently available designs of NP delivery systems are only able to treat tumors with small sizes (4-7 mm). To solve this problem, we propose a combination of NP with non-thermal plasma or cold plasma (CP) to treat large tumors. Our hypothesis is that the synergistic effect between NP and CP-produced reactive plasma species can be utilized to treat large tumors with sizes relevant to human cancer. First, the dose-dependent cytotoxicity of NP or CP to Pan02 pancreatic cancer cells was examined by cells treated with different doses of NP or CP. Second, we selected one specific dose of NP for further combination evaluation. The combination treatment was carried out either by treating cells with NP first then CP, or CP first then NP. The synergistic effect was assessed with cell viability and impedance measurement. Our results showed that NP at a very low dose can sensitize cancer cells that will then be killed by CP. Our future goals are to develop a prototype engineering system for both NP and CP delivery to tumor tissue, to evaluate the synergistic effect of NP and CP in vitro and in vivo, and to explore the potential molecular mechanisms of the synergistic effect of NP and CP

Modulation of ROS in Nanosecond-Pulsed Plasma-Activated Media for Dosage-Dependent Cancer Cell Inactivation \u3ci\u3ein vitro\u3c/i\u3e

Dosage control of reactive oxygen and nitrogen species (RONS) is critical to low-temperature plasma applications in cancer therapy. Production of RONS by atmospheric pressure, nonequilibrium plasmas in contact with liquid may be modulated via plasma conditions including plasma treatment time and pulse voltage and repetition frequency. In this study, a terephthalic acid-based probe was used to measure hydroxyl radicals [OHaq] in water exposed to plasma and to demonstrate that the OHag concentration increases linearly with treatment time. Fluorometric measurements of hydrogen peroxide concentration in plasma-activated water show a linear relationship between the H2O2 production rate and the pulse repetition frequency of the plasma. In vitro plasma treatment of cancer cells shows that pancreatic (Pan02) and breast (4T1-Luc) cancer cells have different sensitivities to plasma exposure. The dependence of Pan02 cell viability on plasma treatment time or pulse voltage is nonlinear. The system described here for generation and delivery of reactive oxygen species from a nanosecond pulsed plasma jet represents a promising alternative approach to cancer therapy

Evaluation of Electric Property Changes in Cancer Cells in Vitro Induced by Cold Plasma and Electric Field Treatment

Nonthermal atmospheric pressure plasma jet (APPJ) is a promising method for medical applications, especially in the treatment of tumors in vitro. Its main mechanism of action is it induces temporary or permanent damage to tissue known as reversible electroporation (RE) and irreversible electroporation (IRE), respectively. It has been previously shown that APPJ treatment reduces the proliferation of cancer cells, reduces the size of the tumor, and induces oxidative stress in cancer cells that can lead to the death of the cells via apoptosis [1]. Furthermore, nanosecond pulsed electric field (nsPEF) has equally been reported to cause increase in permeability of cell membrane and/or oxidative stress on cells, which is accompanied with a decrease in the impedance of the cells. It is found that the impedance of compromised or dying cells are different, typically lower than healthy cells [2]. It is therefore possible to analyze the electrical properties of the cells before, during and after treatment to better monitor the treatment outcome on the cells in real time. Here, we report the conductivity change of pancreatic cancer cells (Pan02) in response to the treatment of 200-ns, 9 kV pulsed plasma jet, 60-ns, 50 kV/cm nsPEF and the combination in vitro. The dosage dependence is evaluated by varying the treatment time of the plasma, and pulse numbers of the nsPEF, followed by the comparison of the impedance change of the monolayer Pan02 cells. [1] C. Jiang et al., Synergistic Effects of an Atmospheric-Pressure Plasma Jet and Pulsed Electric Field on Cells and Skin, in IEEE Transactions on Plasma Science. 49(11) (2021). [2] E. Oshin, S. Guo, and C. Jiang “Determining Tissue Conductivity in Tissue Ablation by Nanosecond Pulsed Electric Fields,” Bioelectrochemistry. 143 (2022)