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.

Gasdermin E (GSDME), also known as deafness autosomal dominant 5 (DFNA5) and previously identified to be an inducer of regulated cell death, is frequently epigenetically inactivated in different cancer types, suggesting that GSDME is a tumor suppressor gene. In this study, we aimed to evaluate the tumor-suppressive effects of GSDME in two intestinal cancer mouse models. To mimic the silencing of GSDME by methylation as observed in human cancers, a Gsdme knockout (KO) mouse was developed. The effect of GSDME on tumorigenesis was studied both in a chemically induced and in a genetic intestinal cancer mouse model, as strong evidence shows that GSDME plays a role in human colorectal cancer and representative mouse models for intestinal cancer are available. Azoxymethane (AOM) was used to induce colorectal tumors in the chemically induced intestinal cancer model (n = 100). For the genetic intestinal cancer model, Apc^1638N/+ mice were used (n = 37). In both experiments, the number of mice bearing microscopic proliferative lesions, the number and type of lesions per mouse and the histopathological features of the adenocarcinomas were compared between Gsdme KO and wild type (WT) mice. Unfortunately, we found no major differences between Gsdme KO and WT mice, neither for the number of affected mice nor for the multiplicity of proliferative lesions in the mice. However, recent breakthroughs on gasdermin function indicate that GSDME is an executioner of necrotic cell death. Therefore, it is possible that GSDME may be important for creating an inflammatory microenvironment around the tumor. This is in line with the trend towards more severe inflammation in WT compared to Gsdme KO mice, that we observed in our study. We conclude that the effect of GSDME in tumor biology is probably more subtle than previously thought.

(1) Background: Therapeutic blocking of the interaction between programmed death-1 (PD-1) with its ligand PD-L1, an immune checkpoint, is a promising approach to restore the antitumor immune response. Improved clinical outcomes have been shown in different human cancers, including non-small cell lung cancer (NSCLC). Unfortunately, still a high number of NSCLC patients are treated with immunotherapy without obtaining any clinical benefit, due to the limitations of PD-L1 protein expression as the currently sole predictive biomarker for clinical use; (2) Methods: In this study, we applied mass spectrometry imaging (MSI) to discover new protein biomarkers, and to assess the possible correlation between candidate biomarkers and a positive immunotherapy response by matrix-assisted laser desorption/ionization (MALDI) MSI in 25 formalin-fixed paraffin-embedded (FFPE) pretreatment tumor biopsies (Biobank@UZA); (3) Results: Using MALDI MSI, we revealed that the addition of neutrophil defensin 1, 2 and 3 as pretreatment biomarkers may more accurately predict the outcome of immunotherapy treatment in NSCLC. These results were verified and confirmed with immunohistochemical analyses. In addition, we provide in-vitro evidence of the immune stimulatory effect of neutrophil defensins towards cancer cells; and (4) Conclusions: With proteomic approaches, we have discovered neutrophil defensins as additional prospective biomarkers for an anti-PD-(L)1 immunotherapy response. Thereby, we also demonstrated that the neutrophil defensins contribute in the activation of the immune response towards cancer cells, which could provide a new lead towards an anticancer therapy.

Auranofin (AF) is a potent, off-patent thioredoxin reductase (TrxR) inhibitor that efficiently targets cancer via reactive oxygen species (ROS)- and DNA damage-mediated cell death. The goal of this study is to enhance the efficacy of AF as a cancer treatment by combining it with the poly(ADP-ribose) polymerase-1 (PARP) inhibitor olaparib (referred to as ‘aurola’). Firstly, we investigated whether mutant p53 can sensitize non-small cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) cancer cells to AF and olaparib treatment in p53 knock-in and knock-out models with varying p53 protein expression levels. Secondly, we determined the therapeutic range for synergistic cytotoxicity between AF and olaparib and elucidated the underlying molecular cell death mechanisms. Lastly, we evaluated the effectiveness of the combination strategy in a murine 344SQ 3D spheroid and syngeneic in vivo lung cancer model. We demonstrated that high concentrations of AF and olaparib synergistically induced cytotoxicity in NSCLC and PDAC cell lines with low levels of mutant p53 protein that were initially more resistant to AF. The aurola combination also led to the highest accumulation of ROS, which resulted in ROS-dependent cytotoxicity of mutant p53 NSCLC cells through distinct types of cell death, including caspase-3/7-dependent apoptosis, inhibited by Z-VAD-FMK, and lipid peroxidation-dependent ferroptosis, inhibited by ferrostatin-1 and alpha-tocopherol. High concentrations of both compounds were also needed to obtain a synergistic cytotoxic effect in 3D spheroids of the murine lung adenocarcinoma cell line 344SQ, which was interestingly absent in 2D. This cell line was used in a syngeneic mouse model in which the oral administration of aurola significantly delayed the growth of mutant p53 344SQ tumors in 129S2/SvPasCrl mice, while either agent alone had no effect. In addition, RNA sequencing results revealed that AF- and aurola-treated 344SQ tumors were negatively enriched for immune-related gene sets, which is in accordance with AF’s anti-inflammatory function as an anti-rheumatic drug. Only 344SQ tumors treated with aurola showed the downregulation of genes related to the cell cycle, potentially explaining the growth inhibitory effect of aurola since no apoptosis-related gene sets were enriched. Overall, this novel combination strategy of oxidative stress induction (AF) with PARP inhibition (olaparib) could be a promising treatment for mutant p53 cancers, although high concentrations of both compounds need to be reached to obtain a substantial cytotoxic effect.

In this study, we aimed to study the expression of SARS-CoV-2-related surface proteins in non-small-cell lung cancer (NSCLC) cells and identify clinicopathological characteristics that are related to increased membranous (m)ACE2 protein expression and soluble (s)ACE2 levels, with a particular focus on standard of care (SOC) therapies. ACE2 (n = 107), TMPRSS2, and FURIN (n = 38) protein expression was determined by immunohistochemical (IHC) analysis in NSCLC patients. sACE2 levels (n = 64) were determined in the serum of lung cancer patients collected before, during, or after treatment with SOC therapies. Finally, the TCGA lung adenocarcinoma (LUAD) database was consulted to study the expression of ACE2 in EGFR- and KRAS-mutant samples and ACE2 expression was correlated with EGFR/HER, RAS, BRAF, ROS1, ALK, and MET mRNA expression. Membranous (m)ACE2 was found to be co-expressed with mFURIN and/or mTMPRSS2 in 16% of the NSCLC samples and limited to the adenocarcinoma subtype. TMPRSS2 showed predominantly atypical cytoplasmic expression. mACE2 and sACE2 were more frequently expressed in mutant EGFR patients, but not mutant-KRAS patients. A significant difference was observed in sACE2 for patients treated with targeted therapies, but not for chemo- and immunotherapy. In the TCGA LUAD cohort, ACE2 expression was significantly higher in EGFR-mutant patients and significantly lower in KRAS-mutant patients. Finally, ACE2 expression was positively correlated with ERBB2-4 and ROS1 expression and inversely correlated with KRAS, NRAS, HRAS, and MET mRNA expression. We identified a role for EGFR pathway activation in the expression of mACE2 in NSCLC cells, associated with increased sACE2 levels in patients. Therefore, it is of great interest to study SARS-CoV-2-infected EGFR-mutated NSCLC patients in greater depth in order to obtain a better understanding of how mACE2, sACE2, and SOC TKIs can affect the course of COVID-19.

The antineoplastic activity of the thioredoxin reductase 1 (TrxR) inhibitor, auranofin (AF), has already been investigated in various cancer mouse models as a single drug, or in combination with other molecules. However, there are inconsistencies in the literature on the solvent, dose and administration route of AF treatment in vivo. Therefore, we investigated the solvent and administration route of AF in a syngeneic SB28 glioblastoma (GBM) C57BL/6J and a 344SQ non-small cell lung cancer 129S2/SvPasCrl (129) mouse model. Compared to daily intraperitoneal injections and subcutaneous delivery of AF via osmotic minipumps, oral gavage for 14 days was the most suitable administration route for high doses of AF (10–15 mg/kg) in both mouse models, showing no measurable weight loss or signs of toxicity. A solvent comprising 50% DMSO, 40% PEG300 and 10% ethanol improved the solubility of AF for oral administration in mice. In addition, we confirmed that AF was a potent TrxR inhibitor in SB28 GBM tumors at high doses. Taken together, our results and results in the literature indicate the therapeutic value of AF in several in vivo cancer models, and provide relevant information about AF’s optimal administration route and solvent in two syngeneic cancer mouse models.