Cutaneous papilloma and squamous cell carcinoma therapy utilizing nanosecond pulsed electric fields (nsPEF)

Abstract

Nanosecond pulsed electric fields (nsPEF) induce apoptotic pathways in human cancer cells. The potential therapeutic effective of nsPEF has been reported in cell lines and in xenograft animal tumor model. The present study investigated the ability of nsPEF to cause cancer cell death in vivo using carcinogen-induced animal tumor model, and the pulse duration of nsPEF was only 7 and 14 nano second (ns). An nsPEF generator as a prototype medical device was used in our studies, which is capable of delivering 7-30 nanosecond pulses at various programmable amplitudes and frequencies. Seven cutaneous squamous cell carcinoma cell lines and five other types of cancer cell lines were used to detect the effect of nsPEF in vitro. Rate of cell death in these 12 different cancer cell lines was dependent on nsPEF voltage and pulse number. To examine the effect of nsPEF in vivo, carcinogen-induced cutaneous papillomas and squamous cell carcinomas in mice were exposed to nsPEF with three pulse numbers (50, 200, and 400 pulses), two nominal electric fields (40 KV/cm and 31 KV/cm), and two pulse durations (7 ns and 14 ns). Carcinogen-induced cutaneous papillomas and squamous carcinomas were eliminated efficiently using one treatment of nsPEF with 14 ns duration pulses (33/39 = 85%), and all remaining lesions were eliminated after a 2nd treatment (6/39 = 15%). 13.5% of carcinogen-induced tumors (5 of 37) were eliminated using 7 ns duration pulses after one treatment of nsPEF. Associated with tumor lysis, expression of the anti-apoptotic proteins Bcl-xl and Bcl-2 were markedly reduced and apoptosis increased (TUNEL assay) after nsPEF treatment. nsPEF efficiently causes cell death in vitro and removes papillomas and squamous cell carcinoma in vivo from skin of mice. nsPEF has the therapeutic potential to remove human squamous carcinoma.

Figure 1. Treatment of cell lines in vitro with nsPEF.

nsPEF exposure of Jurkat cells (Human T cell leukemia, 2×10⁶/mL) to 50, 100 or 200 pulses of 30 ns duration, at 50 Hz and a varying peak voltage (Panel A, left), and at a fixed peak voltage resulting in a field of 30 kV/cm and a varying number of pulses (Panel A, right). nsPEF exposure of 11 solid tumor cell lines (2×10⁶/mL in 1 mm cuvette): glioblastoma multiforme (GBM) cells (U118, T98G, U373); colon cancer cells ( HCT116); skin cancer cells (SRB-1, SRB-12, SCC-13, Colo-16, HaCaT); as well as early transformed cells [AK (actinic keratosis), KA (keratoacanthoma)]. Cells were exposed to 100 or 200 pulses of 30 ns duration, at 50 Hz and a varying peak voltage, and at a fixed peak voltage resulting in a field of 30 kV/cm and a varying number of pulses (Panels B. C. D. E). The effect of nsPEF exposure on cell viability is represented by the percentage of viable cells remaining after exposure calculated as a fraction of viable control cells, not exposed but handled similarly. Trypan blue was used to measure viable cells after nsPEF exposure at one hour. The dashed lines indicate either the pulse number or peak voltage associated with a 50% (ED50) reduction in cell viability. Results are values obtained from three experiments under identical conditions (mean + SD).

Figure 2. Treatment of induced papillomas and squamous cell carcinomas in vivo with nsPEF (14ns).

Tumors were exposed to either 50, 200 or 400 pulses of 14 ns duration, 50 Hz and a 40 kV/cm nominal electric field (Panel A, n = 6, median size = 10.4 mm³ before treatment; B, n = 11, median size = 15.9 mm³ before treatment; C, n = 15, median size = 11.1 mm³ before treatment;) and to 200 pulses of 14 ns duration 50 Hz and a 31 kV/cm nominal electric field (Panel D, n = 7, median size = 11.9 mm³ before treatment). Unexposed control skin (Panel E, n = 6, median size = 7.3 mm³ before untreatment, 20.4 mm³ after untreatment). Tumor size was measured prior to exposure and 1-week post exposure. The solid lines depict mean tumor size in each group.

Figure 3. Treatment of induced papillomas and squamous cell carcinomas in vivo with nsPEF (7ns).

nsPEF exposure (7 ns) of carcinogen induced squamous cell carcinomas in vivo. Induced tumors were exposed to 50, 100, 200 or 400 pulses of 7 ns duration, at 50 Hz and 40 kV/cm peak nominal electric field (Panels A, n = 7, median size = 6.4 mm³ before ureatment, 19.2 mm³ after treatment; B, n = 5, median size = 9.6 mm³ before ureatment, 25.6 mm³ after treatment; C, n = 10, median size = 23.9 mm³ before ureatment, 11.2 mm³ after treatment; D, n = 15, median size = 19.2 mm³ before ureatment, 2.1 mm³ after treatment). Tumor sizes were measured prior to and at 1-week post exposure. The solid lines depict mean tumor size in each group. Images (Panel E) are representative of the experiments and show pre- and one week post-exposure to either 200 or 400 pulses.

Figure 4. Visual changes over time following nsPEF exposure of induced papillomas and squamous cell carcinomas.

Solid circle surround squamous cell carcinomas and dash lines surround normal skin. Appearance at 24 hours (d1) post-exposure is shown following nsPEF at 200 and 400 pulses of 14 ns duration, 50 Hz and 40 kV/cm peak nominal electric field and at 200 pulses of 14ns duration, 50 Hz and 31 kV/cm peak nominal electric field. Additional images are shown on alternate days up to one week (d3, d5 and d7) for nsPEF exposure of 200 pulses of 14ns duration, 50 Hz and 40 kV/cm nominal electric field.

Figure 5. Histology and immunohistochemistry examination.

Histopathology (Hematoxylin and Eosin stain) of unaffected normal mouse skin and induced papillomas and squamous cell carcinomas (prior to nsPEF exposure and 5 weeks following effective exposure to 200 pulses of 14 ns duration, 50 Hz, 40 kV/cm) (Panel A). Carcinogen induced tumors were treated with nsPEF (40 kV/cm, 50 Hz, 1.75 mm tip 200p). After 3, 6 and 24 hours, immunohistochemistry was performed to detect the anti-apoptotic proteins Bcl-2 (brown in Panel B) and Bcl-xl (brown in Panel C), as well as, apoptosis as shown by TUNEL assay (Terminal deoxynucleotidyl transferase dUTP nick end labeling) (red in Panel D). Scale bar: 50 μm.

Figure 6. Expression of Bcl-xl in vitro and in vivo.

Western blot analyzed the Bcl-xl expression post nsPEF exposure. Glioblastoma multiforme cell line (U118) was exposed to varying numbers of pulses in vitro [20 ns duration, 50 Hz, and 30 kV/cm) in 1 mm cuvette]. 1 hour post nsPEF exposure, Bcl-xl expression was measured (Panel A). Squamous carcinoma cell line (SRB-12) was injected subcutaneously into immunocompromised mice. After one week, established tumors were exposed to nsPEF 200 pulses of 14 ns duration, 50 Hz and 40 kV/cm nominal electric field. One hour later, cells were harvested and western blot was performed to measure Bcl-xl expression (Panel B). Three induced tumors were either untreated or treated with nsPEF (40 kV/cm nominal electric field, 50 Hz, 1.75 mm tip 200p). Protein was extracted from these tumors after 1 and 3 hours, followed by Western blotted and probed with antibody to Bcl-xl (Panel C). GAPDH was used as loading control.

Figure 7. Nanosecond pulsed electric field generator.

Image showing the experimental nanosecond pulsed electric field generator and hand piece for delivery of nsPEF exposure to skin tumors in vivo (Panel A). Enlarged image showing nsPEF delivery tip; the tip is placed into the tumor during exposure. Significant components of the tip include five short 30-gauge needle electrodes, the center electrode being the positive with four surrounding return electrodes, each spaced 1.75 mm from the center electrode (Panel B). Image showing typical pulse waveform, with 7KV amplitude and 14 ns pulse width (FWHM) delivered into a fixed 100 ohm load (Panel C).