Schoenbach, Beebe and Buescher first observed the effects of NPSs on mammalian cells at Old Dominion University (ODU) and Eastern Virginia Medical School (EVMS).They applied NPS to human white blood cells containing granules stained by eosin (eosinophils) in vitro. When NPS was applied to human eosinophils, intracellular vesicles were modified without permanent disruption of the plasma membrane. The main conclusion from this model was that shortening the pulse duration and rise time of intense electric field pulses allows manipulation of membranes of internal cell structures, which could be applicable to all cell types. This opens the potential for new applications in influencing cellular secretion, apoptosis induction, gene delivery to the nucleus, or other altered cell functions, depending on the electrical pulse conditions.
Scientists at ODU demonstrated NPSs can be used as a new, drug-free therapy for treating solid skin melanomas. This research indicated some important findings set forth below.
a) Certain pulse parameters were capable of penetrating the interior of tumor cells and cause tumor cell nuclei to rapidly shrink and tumor blood flow to stop.
b) Within two months of the initial treatment, the melanomas were undetectable by transillumination, ultrasound, or serial section histological investigation.
c) The results of this research showed that melanomas shrank by 90% within two weeks following treatment with NPS. This new technique provides a highly localized targeting of tumor cells with only minor effects on overlying skin, compared to other technologies that use electric fields (i.e. radiofrequency or microwave devices that kill cells via hyperthermia).
Garon et al. evaluated cell viability of a wide range of malignant cell types in vitro and in vivo. Five hematologic and 16 solid tumor cell lines were pulsed in vitro. Additionally, a single human subject with basal cell carcinoma was treated with NPS and had a complete pathologic response. This study demonstrated that NPS was able to ablate a wide variety of human cancer cells in vitro, induce tumor regression in vivo and show efficacy in a single human patient. The research indicated that different pulsing regimens led to different responses; cell lines displayed significant variability in response to NPS therapy.
Pulse Biosciences’ predecessor investigators demonstrated that the NPS-ablated melanomas did not recur. NPS was used on murine melanomas in vivo which triggered both necrosis and apoptosis, resulting in complete tumor remission within an average of 47 days in the 17 animals treated. The study was terminated 4 months after all tumors had been eliminated with no recurrence during that period.
Pulse Biosciences’ predecessor eliminated all melanomas in transgenic mice developing the melanomas within their own skin.All 27 NPS-treated melanomas in 14 mice began to shrink within a day after treatment and gradually disappeared over a period of 12–29 days. These mice were euthanized at different times after melanoma treatment in order to gather histological data and some were followed for over 100 days.
NPS generated a vaccine-like effect after being used to treat liver tumors in an orthotopic animal model. Rats with successfully ablated tumors failed to re-grow tumors when implanted in the same or different liver lobe that harbored the original tumor. Given this protective effect, infiltration of immune cells and the presence of granzyme B expressing cells within days of treatment suggest the possibility of an anti-tumor adaptive immune response. The authors concluded that NPS not only eliminates HCC tumors, but also induced an immuno-protective effect against recurrences of the same cancer.
Figure 1 Survival curves for rats in which the primary liver tumor was either untreated (green) or NPS treated (red). At the arrow, a second tumor was injected into the liver of either the NPS-treated rat (red) or naive rats (blue). No tumors grew in rats whose primary tumor had been ablated by NPS.
Pulse Biosciences replicated this immuno-protection result in another rat strain and also demonstrated that this protection requires the presence of cytotoxic T cells (CD8+). This is the best indication thus far that NPS triggers an adaptive immune response.13
Pulse Biosciences published data demonstrating the vaccine-effect of NPS-treated tumor cell lines. Pulse Biosciences demonstrated for the first time that NPS-treated fibrosarcoma cells could be used as a vaccine to protect mice against fibrosarcoma subdermal allografts. When the NPS-treated cells are injected under the skin of naïve, isogeneic mice, and three weeks are allowed for the immune system to generate cytotoxic T cells specific to these fibrosarcoma cells, subsequent healthy tumor cell injections fail to grow a tumor. Moreover, if CD8+ T cells are greatly reduced by the addition of CD8 antibodies when the healthy tumor cells are injected, the tumor growth was normal. This provides strong evidence that the NPS-treated tumor cells produced a CD8+-dependent immune response that prevented tumor growth.
Figure 2 Vaccination of mice with NPS-treated tumor cells triggers an immune response in three weeks that blocks tumor growth
 Sun Y, Vernier PT, Behrend M, Wang J, Thu MM, Gundersen M, and Marcu L. Fluorescence microscopy imaging of electroperturbation in mammalian cells. J Biomed Opt 2006; 11(2):024010.
 Walker K III, Pakhomova ON, Kolb JF, Schoenbach KH, Stuck BE, Murphy MR, and Pakhomov AG. Oxygen enhances lethal effect of high-intensity, ultra-short electrical pulses. Bioelectromagn J 2006;27: 221–225.
 Vernier PT, Ziegler MJ, Sun Y, Chang WV, Gundersen MA, Tieleman DP. Nanopore formation and phosphatidylserine externalization in a phospholipid bilayer at high transmembrane potential. J Am Chem Soc 2006; 128 (19): 6288-9.
 Nuccitelli R, Chen X, Pakhomov AG, Baldwin WH, Sheikh S, Pomicter JL, Ren W et al. A new pulsed electric field therapy for melanoma disrupts the tumor’s blood supply and causes complete remission without recurrence. Int’l J Cancer 2009; 125: 438–445.
 Ren W, Sain NM, Beebe SJ. Nanosecond pulsed electric fields (nsPEFs) activate intrinsic caspase-dependent and caspase-independent cell death in Jurkat cell. Biochem Biophys Res Comm 2012; 421: 808-812.
 Chen R, Sain NM, Harlow KT, Chen YJ, Shires PK, Heller R, Beebe SJ. A protective effect after clearance of orthotopic rat hepatocellular carcinoma by nanosecond pulsed electric fields. European Journal of Cancer 2014; 5 (15):2705-2713.
 Schoenbach KH, Beebe SJ, and Buescher ES. Intracellular effect of ultrashort pulses. Bioelectromagnetics 2001; 22: 440–448.
 Nuccitelli R, Pliquett U, Chen X, Ford W, James SR, Beebe SJ, Kolb JF, and Schoenbach KH. Nanosecond pulsed electric fields cause melanomas to self-destruct. Biochem Biophys Res Comm 2006; 343(2): 351-60.
 Garon EB, Sawcer D, Vernier PT, Tang T, Sun Y, Marcu L, Gundersen MA, Koeffler HP. In vitro and in vivo evaluation and a case report of intense nanosecond pulsed electric field as a local therapy for human malignancies. Int’l J Cancer 2007; 121(3): 675-82.
 Nuccitelli R, Chen X, Pakhomov AG, Baldwin WH, Sheikh S, Pomicter JL, Ren W, Osgood C, Swanson RJ, Kolb JF, Beebe SJ, Schoenbach KH. A new pulsed electric field therapy for melanoma disrupts the tumor’s blood supply and causes complete remission without recurrence. Int’l J Cancer 2009; 125(2): 438–445.
 Nuccitelli R, Tran K, Lui K, Huynh J, Athos B, Kreis M, Nuccitelli P, De Fabo EC. Non-thermal nanoelectroablation of UV-induced murine melanomas stimulates an immune response. Pigment Cell Melanoma Res 2012; 25(5): 618-29.
 Chen R, Sain NM, Harlow KT, Chen YJ, Shires PK, Heller R, Beebe SJ. A protective effect after clearance of orthotopic rat hepatocellular carcinoma by nanosecond pulsed electric fields. Eur J of Cancer 2014; 5(15): 2705-2713.
 Nuccitelli R, Berridge JC, Mallon Z, Kreis M, Athos B, Nuccitelli P. Nanoelectroablation of murine tumors triggers a CD8-dependent inhibition of secondary tumor growth. PLoS ONE 2015; 10(7): e013436