Abstract: Targeting the redox balance of malignant cells via the delivery of high oxidative stress unlocks a potential therapeutic strategy against glioblastoma (GBM). We investigated a novel reactive oxygen species (ROS)-inducing combination treatment strategy, by increasing exogenous ROS via cold atmospheric plasma and inhibiting the endogenous protective antioxidant system via auranofin (AF), a thioredoxin reductase 1 (TrxR) inhibitor. The sequential combination treatment of AF and cold atmospheric plasma-treated PBS (pPBS), or AF and direct plasma application, resulted in a synergistic response in 2D and 3D GBM cell cultures, respectively. Differences in the baseline protein levels related to the antioxidant systems explained the cell-line-dependent sensitivity towards the combination treatment. The highest decrease of TrxR activity and GSH levels was observed after combination treatment of AF and pPBS when compared to AF and pPBS monotherapies. This combination also led to the highest accumulation of intracellular ROS. We confirmed a ROS-mediated response to the combination of AF and pPBS, which was able to induce distinct cell death mechanisms. On the one hand, an increase in caspase-3/7 activity, with an increase in the proportion of annexin V positive cells, indicates the induction of apoptosis in the GBM cells. On the other hand, lipid peroxidation and inhibition of cell death through an iron chelator suggest the involvement of ferroptosis in the GBM cell lines. Both cell death mechanisms induced by the combination of AF and pPBS resulted in a significant increase in danger signals (ecto-calreticulin, ATP and HMGB1) and dendritic cell maturation, indicating a potential increase in immunogenicity, although the phagocytotic capacity of dendritic cells was inhibited by AF. In vivo, sequential combination treatment of AF and cold atmospheric plasma both reduced tumor growth kinetics and prolonged survival in GBM-bearing mice. Thus, our study provides a novel therapeutic strategy for GBM to enhance the efficacy of oxidative stress-inducing therapy through a combination of AF and cold atmospheric plasma.

Abstract: Pancreatic ductal adenocarcinoma (PDAC) and glioblastoma multiforme (GBM) are two of the most malignant solid tumor types with poor survival rates, which underscore the urgency of novel and efficacious treatment strategies. Within the last decade, immunotherapy has been established as a breakthrough in cancer therapy. This mainly has been driven by the clinical data and approval associated with several immune checkpoint inhibitors (e.g. anti-CTLA-4 and anti-PD-1/L1). Despite the clinical benefit in specific tumor types, these inhibitors have not yet fulfilled their promise in low immunogenic tumors such as PDAC and GBM. Oxidative stress in cancer cells due to elevated reactive oxygen species (ROS) and an inability to balance intracellular redox state has recently been highlighted as promising target for anticancer treatment strategies with possible immunogenic effects. In this PhD dissertation, I investigated novel oxidative stress-mediated treatment approaches to target PDAC and GBM and to enhance immunogenicity by inducing immunogenic cell death (ICD). In the first part of this thesis (chapter 2), I reviewed the mechanistic responses of cancer cells towards different oxidative stress-inducing treatment strategies and their immunomodulating effects. The resulting literature demonstrated that different exogenous and endogenous ROS-inducing therapies show direct and indirect immunomodulating effects, which can be either immunostimulatory or immunosuppressive. One of the indirect immunostimulatory effects of the ROS-mediating therapies is the capacity of inducing immunogenic cell death (ICD) in tumor cells, which can increase the immunogenicity and consequently can trigger an antitumoral immune response. In chapter 3, I investigated a novel exogenous ROS-inducing treatment method, namely cold atmospheric plasma, to determine the therapeutic and ICD-inducing effects in PDAC, in vitro. I revealed that plasma-treated PBS (pPBS) has the potential to induce ICD in pancreatic cancer cells (PCCs) and to reduce the immunosuppressive tumor microenvironment (TME) by attacking the tumor supportive pancreatic stellate cells (PSCs). Although the cell death induced in PSCs was non-immunogenic as seen by the lack of danger-associated molecular patterns (DAMPs) emission and DC activation, I showed that pPBS could disrupt the physical barrier and lower the immunosuppressive secretion profile (lower TGF-β) of PSCs. In contrast, DAMPs were released by PCCs after treatment with pPBS which resulted in activation and maturation of DCs and a more immunostimulatory secretion profile (higher TNF-α, IFN-γ). Hence, indirect plasma treatment via pPBS has the potential to enhance immunogenicity in PDAC by triggering ICD and by attacking the immunosuppressive PSCs. Tumor cells can evolve adaptation mechanisms to protect themselves against intrinsic oxidative stress by upregulation of pro-survival molecules and their antioxidant defense system to maintain the redox balance. As such, tumor cells can become resistant towards exogenous ROS-inducing therapies, like plasma. Dual targeting of the redox balance of tumor cells by increasing exogenous levels of ROS and inhibiting the antioxidant defense system can maximally exploit ROS-mediated cell death mechanisms as therapeutic anticancer strategy. In this regard, cold atmospheric plasma was combined with auranofin, a thioredoxin reductase inhibitor, in GBM (chapter 4). A synergistic effect was shown after this combination treatment in 2D and 3D, however, in 3D only high concentrations of auranofin synergized with plasma treatment. I confirmed a ROS-mediated response after combination treatment, which was able to induce distinct cell death mechanisms, specifically apoptosis and ferroptosis. Additionally, the auranofin and plasma combined treatment strategy induced cell death, which resulted in an increased release of DAMPs. Together with the observed DC maturation, these results indicates the potential increase in immunogenicity, though, the phagocytotic capacity of DCs was inhibited by auranofin. In chapter 5, I evaluated this promising oxidative stress combination therapy in GBM, in vivo. A decrease in tumor kinetics and an increased survival in GBM-bearing mice was observed when auranofin was sequentially combined with direct plasma treatment. No T cell infiltration was observed after auranofin monotherapy. However, further characterization of the TME after the combination therapy is necessary to provide more insight in the immunogenic effects in vivo. In conclusion, this PhD dissertation comprises novel and important therapeutic and immunogenic insights in cold atmospheric plasma and auranofin as promising oxidative stress-mediated treatment strategies for low immunogenic tumors, like PDAC and GBM. These preclinical results provide a solid basis for future research towards combinations with immunotherapeutic approaches.

Abstract: A packed bed dielectric barrier discharge plasma reactor is computationally studied with a fluid model. Two different complementary axisymmetric 2D geometries are used to mimic the intrinsic 3D problem. It is found that a packing enhances the electric field strength and electron temperature at the contact points of the dielectric material due to polarization of the beads by the applied potential. As a result, these contact points prove to be of direct importance to initiate the plasma. At low applied potential, the discharge stays at the contact points, and shows the properties of a Townsend discharge. When a high enough potential is applied, the plasma will be able to travel through the gaps in between the beads from wall to wall, forming a kind of glow discharge. Therefore, the inclusion of a so-called ‘channel of voids’ is indispensable in any type of packed bed modelling.

Abstract: A packed bed dielectric barrier discharge (DBD) was studied by means of fluid modelling, to investigate the influence of the dielectric constant of the packing on the plasma characteristics, for two different gap sizes. The electric field strength and electron temperature are much more enhanced in a microgap reactor than
in a mm-gap reactor, leading to more current peaks per half-cycle, but also to non-quasineutral plasma. Increasing the dielectric constant enhances the electric field further, but only up to a certain value of dielectric constant, being 9 for a microgap and 100 for a mm-gap reactor. The enhanced electric field results in a higher electron temperature, but also lower electron density. This last one strongly affects the reaction rate.