Nanoelectroablation of Murine Tumors Triggers a CD8-Dependent Inhibition of Secondary Tumor Growth.

We have used both a rat orthotopic hepatocellular carcinoma model and a mouse allograft tumor model to study liver tumor ablation with nanosecond pulsed electric fields (nsPEF). We confirm that nsPEF treatment triggers apoptosis in rat liver tumor cells as indicated by the appearance of cleaved caspase 3 and 9 within two hours after treatment. Furthermore we provide evidence that nsPEF treatment leads to the translocation of calreticulin (CRT) to the cell surface which is considered a damage-associated molecular pattern indicative of immunogenic cell death. We provide direct evidence that nanoelectroablation triggers a CD8-dependent inhibition of secondary tumor growth by comparing the growth rate of secondary orthotopic liver tumors in nsPEF-treated rats with that in nsPEF-treated rats depleted of CD8+ cytotoxic T-cells. The growth of these secondary tumors was severely inhibited as compared to tumor growth in CD8-depleated rats, with their average size only 3% of the primary tumor size after the same one-week growth period. In contrast, when we depleted CD8+ T-cells the second tumor grew more robustly, reaching 54% of the size of the first tumor. In addition, we demonstrate with immunohistochemistry that CD8+ T-cells are highly enriched in the secondary tumors exhibiting slow growth. We also showed that vaccinating mice with nsPEF-treated isogenic tumor cells stimulates an immune response that inhibits the growth of secondary tumors in a CD8+-dependent manner. We conclude that nanoelectroablation triggers the production of CD8+ cytotoxic T-cells resulting in the inhibition of secondary tumor growth.

First-in-human trial of nanoelectroablation therapy for basal cell carcinoma: proof of method.

This nanoelectroablation therapy effectively treats subdermal murine allograft tumors, autochthonous basal cell carcinoma (BCC) tumors in Ptch1+/-K14-Cre-ER p53 fl/fl mice, and UV-induced melanomas in C57/BL6 HGF/SF mice. Here, we described the first human trial of this modality. We treated 10 BCCs on three subjects with 100-1000 electric pulses 100 ns in duration, 30 kV/cm in amplitude, applied at 2 pulses per second. Seven of the 10 treated lesions were completely free of basaloid cells when biopsied and two partially regressed. Two of the 7 exhibited seborrheic keratosis in the absence of basaloid cells. One of the 10 treated lesions recurred by week 10 and histologically had the appearance of a squamous cell carcinoma. No scars were visible at the healed sites of any of the successfully ablated lesions. One hundred pulses were sufficient for complete ablation of BCCs with a single, 1-min nanoelectroablation treatment.

Nanosecond pulsed electric field stimulation of reactive oxygen species in human pancreatic cancer cells is Ca(2+)-dependent.

The cellular response to 100 ns pulsed electric fields (nsPEF) exposure includes the formation of transient nanopores in the plasma membrane and organelle membranes, an immediate increase in intracellular Ca(2+), an increase in reactive oxygen species (ROS), DNA fragmentation and caspase activation. 100 ns, 30 kV/cm nsPEF stimulates an increase in ROS proportional to the pulse number. This increase is inhibited by the anti-oxidant, Trolox, as well as the presence of Ca(2+) chelators in the intracellular and extracellular media. This suggests that the nsPEF-triggered Ca(2+) increase is required for ROS generation.

Nanoelectroablation of human pancreatic carcinoma in a murine xenograft model without recurrence.

We have identified an effective nanoelectroablation therapy for treating pancreatic carcinoma in a murine xenograft model. This therapy initiates apoptosis in a nonthermal manner by applying low energy electric pulses 100 ns long and 30 kV/cm in amplitude to the tumor. We first identified the minimum pulse number required for complete ablation by treating 30 tumors. We found that the minimum number of pulses required to ablate the tumor with a single treatment is between 250 and 500 pulses. We settled on a single application of either 500 or 1,000 pulses to treat pancreatic carcinomas in 19 NIH-III mice. Seventeen of the 19 treated tumors exhibited complete regression without recurrence. Three mice died of unknown causes within 3 months after treatment but 16 lived for 270-302 days at which time we sacrificed them for histological analysis. In the 17 untreated controls, the tumor grew so large that we had to sacrifice all of them within 4 months.

Nanoelectroablation therapy for murine basal cell carcinoma.

When skin tumors are exposed to non-thermal, low energy, nanosecond pulsed electric fields (nsPEF), apoptosis is initiated both in vitro and in vivo. This nanoelectroablation therapy has already been proven effective in treating subdermal murine allograft tumors. We wanted to determine if this therapy would be equally effective in the treatment of autochthonous BCC tumors in Ptch1(+/-)K14-Cre-ER p53 fl/fl mice. These tumors are similar to human BCCs in histology [2,20] and in response to drug therapy [19]. We have treated 27 BCCs across 8 mice with either 300 pulses of 300 ns duration or 2700 pulses of 100 ns duration, all at 30 kV/cm and 5-7 pulses per second. Every nsPEF-treated BCC began to shrink within a day after treatment and their initial mean volume of 36 ± 5 (SEM) mm(3) shrunk by 76 ± 3% over the ensuing two weeks. After four weeks, they were 99.8% ablated if the size of the treatment electrode matched the tumor size. If the tumor was larger than the 4mm wide electrode, multiple treatments were needed for complete ablation. Treated tumors were harvested for histological analysis at various times after treatment and exhibited apoptosis markers. Specifically, pyknosis of nuclei was evident as soon as 2 days after nsPEF treatment, and DNA fragmentation as detected via TUNEL staining was also evident post treatment. Nanoelectroablation is effective in triggering apoptosis and remission of radiation-induced BCCs with a single 6 min-long treatment of 2700 pulses.

Non-thermal nanoelectroablation of UV-induced murine melanomas stimulates an immune response.

Non-thermal nanoelectroablation therapy completely ablates UV-induced murine melanomas. C57/BL6-HGF/SF transgenic mice were exposed to UV radiation as pups and began to develop visible melanomas 5-6 months later. We have treated 27 of these melanomas in 14 mice with nanosecond pulsed electric field (nsPEF) therapy delivering 2000 electric pulses each 100 ns long and 30 kV/cm at a rate of 5-7 pulses per second. All nanoelectroablated melanoma tumors began to shrink within a day after treatment and gradually disappeared over a period of 12-29 days. Pyknosis of nuclei was evident within 1 h of nsPEF treatment, and DNA fragmentation as detected by TUNEL staining was evident by 6 h after nsPEF treatment. In a melanoma allograft system, nsPEF treatment was superior to tumor excision at accelerating secondary tumor rejection in immune-competent mice, suggesting enhanced stimulation of a protective immune response by nsPEF-treated melanomas. This is supported by the presence of CD4(+) -T cells within treated tumors as well as within untreated tumors located in mice with other melanomas that had been treated with nanoelectroablation at least 19 days earlier.

Optimized nanosecond pulsed electric field therapy can cause murine malignant melanomas to self-destruct with a single treatment.

We have identified a new, nanosecond pulsed electric field (nsPEF) therapy capable of eliminating murine melanomas located in the skin with a single treatment. When these optimized parameters are used, nsPEFs initiate apoptosis without hyperthermia. We have developed new suction electrodes that are compatible with human skin and have applied them to a xenograft nude mouse melanoma model system to identify the optimal field strength, pulse frequency and pulse number for the treatment of murine melanomas. A single treatment using the optimal pulse parameters (2,000 pulses, 100 ns in duration, 30 kV/cm in amplitude at a pulse frequency of 5-7 pulses/sec) eliminated all 17 melanomas treated with those parameters in 4 mice. This was the highest pulse frequency that we could use without raising the treated skin tumor temperature above 40 degrees C. We also demonstrate that the effects of nsPEF therapy are highly localized to only cells located between electrodes and results in very little scarring of the nsPEF-treated skin.