Abstract 4780: Comparison of safety and efficacy outcomes with nonthermal plasma and tirapazamine in porcine skin and mouse melanoma models
Authors: Yehl Matthew , Kucharski Dominik , Eubank Michelle , Ramadan Xavier , Dianat Ahmad , Pham Bianca , Hutcherson Timothy C. , Sexton Sandra , Zucker Shoshanna N. 
Published: April 2023

Abstract
A novel preclinical experimental therapy for various cancer types including melanoma is the combination of nonthermal plasma (NTP) and tirapazamine (TPZ) delivered in situ. This patented combination therapy delivers reactive oxygen species (ROS) and reactive nitrogen species (RNS) from the atmosphere that are activated and ionized with helium gas to create the state of matter known as plasma. The combination with the prodrug, TPZ, that only activates to become a ROS in hypoxic environments, shows synergistic targeting of cancer cells. In this study, we assayed the effects of the dual therapy on porcine skin, which is the closest model to represent human skin and is commonly used for testing drug therapy. The results demonstrated that porcine skin showed no significant cell killing with the therapy. We also compared the combination therapy in normal skin keratinocytes and 1205Lu cells derived from a melanoma metastasis to the lung. The cells were treated with NTP and TPZ and maintained in a hypoxic chamber. Four days following therapy the keratinocytes continued to grow in culture, whereas the melanoma cells expressing endogenous gap junctions, or overexpressed Cx43 gap junctions showed decreased viability. However, the melanoma cells with a dominant negative Cx43 mutant did not show this loss in viability. This confirms our previous report that gap junctions mediate the passage of derivatives of ROS to increase the extent of cell death. To determine the effects of NTP on regulating the lethal dose of TPZ, we performed IC50 curves for TPZ in the presence or absence of NTP in B16-F10 murine metastatic melanoma cells. We demonstrated a decrease in the IC50 from 28 μM without NTP to 17.4 μM with NTP. In further analysis, the effects of treating with NTP in the presence or absence of tissue culture media were compared. The results showed a highly significant increased efficacy of NTP in the absence of media. The combination therapy was more effective than each independent treatment. To further confirm the experimental analysis, we induced tumor formation with the B16-F10 cells in a syngeneic C57BL/6 mouse model. The tumors from mice that were untreated or treated with NTP or TPZ alone, showed a significant increase in tumor volume as compared to the tumors from mice receiving the combination treatment in situ. In comparison of the tumor growth rates, the control, NTP-, or TPZ-treated tumors had increased by approximately 350% by day 10, whereas the tumors in the mice that received the dual therapy only increased by an average of 70%. The results from these studies collectively implicate the combination of NTP and TPZ as a potential synergistic treatment for in situ melanomas that offer selectivity as compared to normal tissue.
Citation Format: Matthew Yehl, Dominik Kucharski, Michelle Eubank, Xavier Ramadan, Ahmad Dianat, Bianca Pham, Timothy C. Hutcherson, Sandra Sexton, Shoshanna N. Zucker. Comparison of safety and efficacy outcomes with nonthermal plasma and tirapazamine in porcine skin and mouse melanoma models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4780.