3. Nanosecond electric pulses for the treatment of pancreatic cancer

#### 3.1. Background

An electrical engineering technology, nanosecond electric pulses (nsEPs), has been developed and studied by Dr. Schoenbach's [42] and other groups [43]. NsEPs are assumed nonthermal if the appropriate parameters especially the low frequencies are selected. Similar to IRE, nsEPs have been utilized to treat cancer in animal models for local tumor ablation [44–46]. Beyond the local tumor ablation, a vaccine-like protective effect has been observed by two groups [44, 47]. The vaccine like-protection effect has been demonstrated by our group [48] in a poorly immunogenic breast cancer model as well. We have demonstrated that local nsEP tumor ablation elicits an anti-tumor immunity to prevent distant metastases, reject established distant tumors and protect animals from secondary tumor challenge. Thus, nsEP therapy shows additional advantages, in addition to local tumor eradication.

NsEPs have been reported for the treatment of pancreatic cancer in two studies [36, 49]. However, whether immune protection is induced by the nsEP treatment is unknown because xenograft tumors in immune deficient animals have been used in both studies. To assess if nsEP ablation could induce antitumor immunity and achieve additional benefits beyond local ablation for pancreatic cancer, a syngeneic mouse Pan02 pancreatic cancer model was utilized in this study.

#### 3.2. Experimental design

However, as is known, most tumors in patients are larger than 1 cm, especially for later stage cancers, which are the targets of the IRE treatment. This limitation of treatable size can be addressed with the adjustment of the electrode configuration, to cover a larger area. Meanwhile, the depth of laser heating and its thermal distribution needs to be profiled, and the refined MHIRE system will be calibrated/reprogrammed and optimized in an in vivo pancreatic cancer

Figure 4. Kaplan-Meier survival curves of mice treated with IRE or MHIRE. Pan02 pancreatic tumors with the size of 8– 10 mm were treated with IRE or MHIRE at day 31 indicated by arrow. IRE parameters: pulse duration 100 μs, frequency 1 Hz, pulse number 90 and applied electric fields 2000-2500 V/cm. Ctr: No treatment (n = 8 mice per treatment group); IRE: Treated with IRE (n = 8); MHIRE: Tumor preheated with laser at 42C with IRE (n = 9). \*\*\*: p < 0.001 (LogRank test).

Figure 3. Pancreatic tumor growth after IRE or MHIRE treatment. Pan02 pancreatic tumors with the size of 8–10 mm were treated with IRE or MHIRE at day 31 indicated by black arrow. IRE parameters: pulse duration 100 μs, frequency 1 Hz, pulse number 90 and applied electric fields 1500 V/cm. Ctr: no treatment (n = 4); MH: tumor heated with laser at 42C for 2 min; 1500 V: IRE at 1500 V/cm (n = 4 mice); 1500 V + MH: Tumor preheated with laser at 42C with IRE at

1500 V/cm (n = 8). \*: p < 0.05, or p < 0.01 or p < 0.001 for MHIRE vs. IRE or Ctr (one way ANOVA).

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A syngeneic mouse Pan02 pancreatic cancer model was established as above mentioned. Tumors were treated when it reached 5–7 mm or 8–10 mm in diameter with an average tumor volume of 40–120 mm<sup>3</sup> (small) or 250–300 mm<sup>3</sup> (large). The nsEP parameters were pulse duration 100 or 200 ns, frequency 1–3 Hz, pulse number 600–1200, and electric fields 30–50 kV/cm. Pancreatic tumors were treated with either a four-needle electrode with gaps of 5 7 mm or a pitch electrode, which was selected from three configurations including 2 mm gap with 6 mm in diameter, 3 mm gap with 8 mm in diameter and 4 mm gap with 10 mm in diameter. In comparison, pancreatic tumors were also treated with IRE. The IRE parameters and the choice of electrode were described in the previous section.

To assess if a vaccine-like protection occurred after pancreatic cancer was treated with nsEPs, tumor free mice were challenged with 0.5 million live Pan02 tumor cells on the right flank. Tumor growth was monitored as above mentioned.

#### 3.3. Results and discussion

#### 3.3.1. NsEP treatment resulted in complete tumor regression or extension of survival for animals with incomplete tumor regression

As shown in Figure 5, a single nsEP treatment achieved 50–100% complete tumor regression dependent on the doses of nsEPs. In contrast to the IRE treatment, muscle contraction was greatly reduced with the nsEP treatment. Both pitch electrode and two-plate suction electrodes were safe and no mortality was found. A minor issue was that scab was formed after the nsEP treatment. It usually shed within 2–3 weeks and left a small scar or no visual changes on the skin.

Figure 6. Survival extension in animals with incomplete tumor regression after the nsEP treatment. Pan02 pancreatic cancer was treated with IRE or nsEP. Only animals with incomplete tumor regression were included here. A, tumor size with 5–7 mm was treated (n = 13, 12 or 4 for Ctr, IRE or nsEP). Tx: Treatment at day 7 (IRE) or 11 (nsEP). B, tumor size with 8–10 mm was treated. Ctr: No treatment; IRE: Treatment with IRE; nsEP: Treatment with 200 ns, 2 Hz, 30 kV/cm and 600–

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Figure 7. Pancreatic cancer growth after treatment with IRE or nsEP\*. Control (n = 13): no treatment. IRE (n = 12): treated with IRE. Tx: treatment day 7. nsEP (n = 7): treated with nsEP (200 ns, 30 kV/cm, 2 Hz with 800–1000 pulses), Tx: treatment day 31. \*: Only animals with partial tumor regression were included to assess the effect of treatment on tumor

1200 pulses (n = 13, 6 or 7 for Ctr, IRE or nsEP). Tx: treatment at day 31.

regrowth.

Extension of survival was achieved even with partial tumor ablation regardless of whether pancreatic cancer with small size (5–7 mm) or big size (8–10 mm) was treated, and median survival was extended to 63 days (Figure 6A) if small tumors were treated, or to 68 days (Figure 6B) if large tumors were treated, in contrast to 45 days for the control animals. However, the survival benefit was only present in large tumors treated with IRE but not in small tumors if the tumors were partially ablated. Median survival was extended to 50 days if large tumor was treated, in contrast to 45 days for the control animals (Figure 6B). Actually, the median survival was shortened to 40 days if the small tumors were not completely ablated with IRE. Obviously, tumor growth was accelerated and lost heterogeneous pattern after partial IRE ablation (Figure 7). The same phenomenon was reported in literature and explained as cancer stem cell activation [50]. Nevertheless, this was not seen in the animals treated with nsEPs. It suggests different cell death mechanisms or possible inhibition of immune responses may occur.

#### 3.3.2. A vaccine-like protective effect was resulted from the nsEP treatment

As shown in Figure 8, tumor free mice after the nsEP treatment were able to impede or prevent the growth of challenging tumors. Noticeably, there was a significant difference between the two nsEP protocols. Majority of tumor free mice (66.7%) after the 100 nsEP treatment were successfully protected from the second live tumor challenge whereas no protection but only growth inhibition of tumor was observed in animals treated with the 200 nsEPs. Nevertheless, neither protection nor growth inhibition was seen in the animals treated with IRE.

Figure 5. Pancreatic tumor growth after the nsEP treatment. Pan02 pancreatic tumors with the size of 5–7 mm were treated with nsEPs at day 11 indicated by black arrow. nsEP parameters: 200 ns, 2 Hz, 30 kV/cm, and pulse numbers 600, 800, 1000 or 1200, indicated by 600p, 800p, 1000p or 1200p, separately. Number of tumor free mice vs. total number of treated mice was indicated.

3.3. Results and discussion

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incomplete tumor regression

immune responses may occur.

treated mice was indicated.

3.3.2. A vaccine-like protective effect was resulted from the nsEP treatment

3.3.1. NsEP treatment resulted in complete tumor regression or extension of survival for animals with

As shown in Figure 5, a single nsEP treatment achieved 50–100% complete tumor regression dependent on the doses of nsEPs. In contrast to the IRE treatment, muscle contraction was greatly reduced with the nsEP treatment. Both pitch electrode and two-plate suction electrodes were safe and no mortality was found. A minor issue was that scab was formed after the nsEP treatment. It usually shed within 2–3 weeks and left a small scar or no visual changes on the skin. Extension of survival was achieved even with partial tumor ablation regardless of whether pancreatic cancer with small size (5–7 mm) or big size (8–10 mm) was treated, and median survival was extended to 63 days (Figure 6A) if small tumors were treated, or to 68 days (Figure 6B) if large tumors were treated, in contrast to 45 days for the control animals. However, the survival benefit was only present in large tumors treated with IRE but not in small tumors if the tumors were partially ablated. Median survival was extended to 50 days if large tumor was treated, in contrast to 45 days for the control animals (Figure 6B). Actually, the median survival was shortened to 40 days if the small tumors were not completely ablated with IRE. Obviously, tumor growth was accelerated and lost heterogeneous pattern after partial IRE ablation (Figure 7). The same phenomenon was reported in literature and explained as cancer stem cell activation [50]. Nevertheless, this was not seen in the animals treated with nsEPs. It suggests different cell death mechanisms or possible inhibition of

As shown in Figure 8, tumor free mice after the nsEP treatment were able to impede or prevent the growth of challenging tumors. Noticeably, there was a significant difference between the two nsEP protocols. Majority of tumor free mice (66.7%) after the 100 nsEP treatment were successfully protected from the second live tumor challenge whereas no protection but only growth inhibition of tumor was observed in animals treated with the 200 nsEPs. Nevertheless,

Figure 5. Pancreatic tumor growth after the nsEP treatment. Pan02 pancreatic tumors with the size of 5–7 mm were treated with nsEPs at day 11 indicated by black arrow. nsEP parameters: 200 ns, 2 Hz, 30 kV/cm, and pulse numbers 600, 800, 1000 or 1200, indicated by 600p, 800p, 1000p or 1200p, separately. Number of tumor free mice vs. total number of

neither protection nor growth inhibition was seen in the animals treated with IRE.

Figure 6. Survival extension in animals with incomplete tumor regression after the nsEP treatment. Pan02 pancreatic cancer was treated with IRE or nsEP. Only animals with incomplete tumor regression were included here. A, tumor size with 5–7 mm was treated (n = 13, 12 or 4 for Ctr, IRE or nsEP). Tx: Treatment at day 7 (IRE) or 11 (nsEP). B, tumor size with 8–10 mm was treated. Ctr: No treatment; IRE: Treatment with IRE; nsEP: Treatment with 200 ns, 2 Hz, 30 kV/cm and 600– 1200 pulses (n = 13, 6 or 7 for Ctr, IRE or nsEP). Tx: treatment at day 31.

Figure 7. Pancreatic cancer growth after treatment with IRE or nsEP\*. Control (n = 13): no treatment. IRE (n = 12): treated with IRE. Tx: treatment day 7. nsEP (n = 7): treated with nsEP (200 ns, 30 kV/cm, 2 Hz with 800–1000 pulses), Tx: treatment day 31. \*: Only animals with partial tumor regression were included to assess the effect of treatment on tumor regrowth.

challenge or to diminish its growth. An induction of antitumor immunity following the nsEP treatment is highly suggested to account for this vaccine-like protective effect. For both MHIRE and nsEPs for the treatment of pancreatic cancer, our data are preliminary and more studies are needed to further optimize these technologies, elucidate the underlying mechanisms and

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This work was supported by a grant award from Pulse Biosciences, Inc. (S. Guo). The authors

The data present in the second section have been published in or modified from the journal of

S.G. conceived, designed, and supervised the studies. S.G., K.S., R.H, and C.J. designed and developed the MHIRE system. N.B., C.E., J.H. S.B., and S.G. conducted the experiments. S.G. analyzed and interpreted the data. All authors contributed to writing, editing, and review of

, Chelsea M. Edelblute<sup>1</sup>

1 Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, Virginia,

2 Department of Electrical and Computer Engineering, Batten College of Engineering and

, Richard Heller<sup>1</sup> and Stephen J. Beebe<sup>1</sup>

, James Hornef<sup>2</sup>

, Chunqi Jiang1,2,

would like to thank the SoBran personnel who manage the ODU animal facility.

R. Heller and S.J. Beebe own stock in Pulse Biosciences, Inc. All other authors declared no potential conflicts of interest.

evaluate their translational feasibility.

Acknowledgements

Conflict of interest

Scientific Report (see [27]).

Author contributions

the manuscript.

Author details

Karl Schoenbach<sup>1</sup>

\*, Niculina I. Burcus<sup>1</sup>

\*Address all correspondence to: s2guo@odu.edu

Technology, Old Dominion University, Norfolk, Virginia, USA

Siqi Guo<sup>1</sup>

USA

Notes

Figure 8. A vaccine-like protection effect after the nsEP treatment. Growth curves of second challenge pancreatic tumors in tumor-free animals after IRE or nsEPs. Primary pancreatic tumors were treated with IRE (IRE), nsEPs with 200 ns, 2 Hz, 30 kV/cm and 600 -1200 pulses (nsEP-200 ns), or nsEPs with 100 ns, 2 Hz, 50 kV/cm and 800 -1200 pulses (nsEP-100 ns). Number of protective mice vs. total number of challenged mice was indicated. p < 0.05 for nsEP-200 ns vs. IRE and p = 0.001 for nsEP-200 ns vs. nsEP-100 ns (Chi Square test).

Surprisingly, the protective rates between two sets of nsEP parameters are very different. A high rate of protection from the second live tumor challenge, 100%, has been observed in both mouse breast cancer [48] and rat hepatocellular cancer models [44] after the same 100 nsEP treatment. Does this mean 100 nsEPs are more favorable to induce immune protection than 200 nsEPs? The answer is not clear because 100 nsEPs has eradicated local mouse lung squamous cell cancer (KLN205) but has failed to result in any vaccine-like protection (0/19 protection in our unpublished data). It's very likely that cancer cell types and distinctive tumor microenvironments play a critical role on the induction of immunity following the nsEP tumor ablation.

The growth inhibition of local recurring tumors and the second challenging tumors suggests that underlying common immune responses are induced after the nsEP treatment. It's critical to understand the mechanisms causing the differential responses and outcomes between IRE and nsEPs or among various nsEP parameters, so it is possible forresearchers to design more effective therapeutic strategies, such as further optimization of the system or a combination therapy with other immunomodulators. Currently, we are investigating cell death mechanisms, local and systemic immune responses, and the changes of tumor microenvironments following the nsEP tumor ablation.

#### 4. Conclusion

Two electric pulse-based technologies have been studied to treat pancreatic cancer in a syngeneic mouse pancreatic cancer model. A novel MHIRE system has been developed. This MHIRE system has three functions including controllable tumor heating, impedance monitoring and electric pulse delivery. MH has been demonstrated to decrease the impedance of tumor, to enlarge the tumor ablation zone of IRE ex vivo and to enhance the complete tumor ablation of the IRE treatment in vivo. The MHIRE treatment significantly improves the therapeutic efficacy of the IRE treatment. In contrast to the IRE treatment, nsEP tumor ablation showed distinctive outcomes and potential advantages. If partial ablation occurred after either the IRE or the nsEP treatment, animals treated with nsEPs received survival benefit. If complete local ablation was achieved, animals treated with nsEPs but not with IRE were able to reject secondary tumor challenge or to diminish its growth. An induction of antitumor immunity following the nsEP treatment is highly suggested to account for this vaccine-like protective effect. For both MHIRE and nsEPs for the treatment of pancreatic cancer, our data are preliminary and more studies are needed to further optimize these technologies, elucidate the underlying mechanisms and evaluate their translational feasibility.
