Ionized gas at room temperature or non-thermal plasma (NTP) may be a novel method for ablation of solid tumors. Our laboratory has demonstrated the efficacy of NTP in a pre-clinical model of neuroblastoma. We investigated the production of reactive oxygen species (ROS) with NTP treatment as a potential mechanism for the tumor ablation observed.
Neuro2a murine neuroblastoma cells in culture were exposed to increasing amounts of NTP (0, 15, 30, and 60J) and ROS were measured by dihydroethidium staining. Annexin-V assay was used to quantify apoptosis in treated cells. N-acetylcysteine, a ROS scavenger, was utilized to determine if elimination of ROS hindered NTP efficacy. In vivo, a 10mm neuroblastoma was treated with 60J of transdermal NTP and compared to an untreated control. The tumors were sectioned into thirds from the outer surface down. Sections were stained with dihydroethidium and cells were also suspended and analyzed by flow cytometry to determine the production of ROS in the treated tumor.
An increase in ROS formation in Neuro2a cells corresponded to the intensity of NTP treatment (Figure). Apoptotic cell counts also increased concurrently with intensity of treatment. Neuro2a cells pretreated with N-acetylcysteine prior to NTP demonstrated improved viability when compared to control cells. When pretreated with N-acetylcysteine followed by NTP, cells showed resistance to NTP-induced cytotoxicity. (p=.0024). Dihydroethidium median fluorescence intensity as an indicator of ROS production was much higher (34.1) in the outer third of the treated tumor than the mid (2.75) and lower (6.63) thirds. The untreated tumor demonstrated low fluorescence in all thirds (10.7, 7.16, and 6.31).
A correlation between increased ROS and the onset of apoptosis was observed following plasma treatment in-vitro and in-vivo. Non thermal plasma induction of ROS may be a unique method of local tumor ablation.