Gas plasma-oxidized sodium chloride acts via hydrogen peroxide in a model of peritoneal carcinomatosis

Abstract

Gas plasma technology generates reactive oxygen and nitrogen species (ROS/RNS), inducing lethal oxidative damage in tumor cells. The transfer of gas plasma-derived ROS/RNS into liquids has been proposed as an innovative anti-cancer strategy targeting peritoneal carcinomatosis (PC). However, the mechanism of action is under debate. To this end, we compared gas plasma-oxidized medical-grade sodium chloride (oxNaCl) with a concentration-matched control (cmc) of NaCl enriched with equivalent concentrations of H₂O₂ and NO₃^- in several cell lines and models of PC. Strikingly, oxNaCl and cmc performed equally well in oxidation and cytotoxic activity in tumor cells in two-dimensional cultures, three-dimensional (3D) tumor spheroids, vascularized 3D tumors grown on chicken-embryo chorioallantoic membranes, and a syngeneic PC mouse model in vivo. Given the importance of immunotherapies in oncology today, we focused on immunological consequences of the treatment. Again, to a similar extent, oxNaCl and cmc increased tumor cell immunogenicity and enhanced uptake by and maturation of peripheral blood monocyte-derived dendritic cells together with an inflammatory secretion profile. Furthermore, NanoString gene expression profiling revealed immune system processes and unfolded protein response-related pathways as being linked to the observed anti-tumor effects for both oxNaCl and cmc. In conclusion, gas plasma-generated oxNaCl and cmc showed equal therapeutic efficacy in our PC-related models. In light of the many promising anti-cancer studies of gas plasma-oxidized liquids and the convenient production of corresponding cmcs in large quantities as needed in clinics, our findings may spur research lines based on low-dose oxidants in peritoneal cancer therapy.

Keywords: ICD; ROS; calreticulin; immunogenicity; plasma medicine.

Fig. 1.

Gas plasma treatment of sodium chloride (oxNaCl) deposits mainly H₂O₂ and NO₃⁻, which was mimicked in a cmc to elicit lethal oxidative stress in tumor cells. (A) Schematic overview of liquid preparation and storage for subsequent experiments. (B and C) Quantification of H₂O₂ (B) and NO₃⁻ (C). (D) Schematic overview of experimental treatment procedures in 2D and 3D cell culture models. (E) Representative fluorescence microscopy images of oxidized glutathione (GSSG) in cells exposed to either oxNaCl or cmc. (Scale bar, 20 µm.) (F) GSSG quantification. (G) Representative flow cytometry dot plots of cells. (H) Quantification of viable HT-29 (I), Panc-01 (II), and SKOV3 (III). (I) Representative brightfield images of HT-29, Panc-01, and SKOV3 tumor spheroids. (Scale bar, 200 µm.) (J and K) Kinetic evaluation of spheroid growth in HT-29 (J) and SKOV3 (K) tumor spheroids and nucleic acid–staining sytox blue. Graphs show mean ± SEM (SEM). Statistical analysis was performed using ANOVA. (*P < 0.05, **P < 0.01, ***P < 0.001; ns, nonsignificant). FACS, fluorescence activated cell sorting; HCI, high content imaging; DAPI, 4',6-Diamidino-2-phenylindol; MFI, mean fluorescence intensity.

Fig. 2.

oxNaCl and cmc equally promote tumor cell uptake by DCs and an enhanced inflammatory profile. (A) Quantification of apoptotic HT-29 (I), Panc-01 (II), and SKOV3 (III) cells normalized to NaCl. (B) Representative flow cytometry intensity histograms of CRT surface expression of viable cells. (C) CRT MFI quantification and normalization for HT-29 (I), Panc-01 (II), and SKOV3 (III) cells. (D) Schematic overview of experimental treatment procedure for coculture experiments. (E) Quantification of DC maturation markers cocultured with tumor cells cultured in medium (control [ctrl]), NaCl, oxNaCl, or cmc and normalized to controls. (F) Representative flow cytometry dot plots of CD11c+/DiD+ DCs. (G) Quantification of phagocytosis of HT-29 (I), Panc-01 (II), SKOV3 (III) tumor cells previously exposed to liquids as labeled. (H) Chemokine and cytokine profiles in supernatants of DCs cocultured with tumor cells cultured in medium (control), NaCl, oxNaCl, or cmc and normalized to controls. (I) Principal component (PC) score calculated from maturation marker and cytokine release profiles of DCs cocultured with tumor cells cultured in medium (control), NaCl, oxNaCl, and cmc. Bar graphs show mean + (SEM). Heat maps show median. Statistical analysis was performed using one-way ANOVA. (*P < 0.05, **P < 0.01, ***P < 0.001; ns, nonsignificant). MFI, mean fluorescence intensity.

Fig. 3.

oxNaCl and cmc induce comparable changes in gene expression profiling. (A) PCA of z-scored gene expression patterns in HT-29, Panc-01, and SKOV3 tumor cells 4 h after exposure to respective solutions. (B) Venn diagrams. (C) Median log fold-changes (logFCs) of 21 differentially expressed genes (DEGs) in at least two cell lines and one treatment regimen (oxNaCl or cmc). (D) String interaction network of corresponding proteins. (E) Pathway enrichment dot plots of gene ontology (GO) terms after exposure to oxNaCl (Left) and cmc solutions (Right). (F) Spearman correlation of % viability reduction against log fold-changes (logFC) of DEGs. (G) Representative Western blot images of XBP-1s expression in HT-29, Panc-01, and SKOV3 cell lines 6 h after exposure to NaCl, oxNaCl, or cmc. FDR, false discovery rate.

Fig. 4.

oxNaCl and cmc equally reduce tumor burden in ovo and in a syngeneic model of peritoneal carcinomatosis in vivo. (A) Schematic overview of experimental treatment procedure in ovo. (B) Representative images of excised in ovo tumors. (C) Assessment of tumor weight of in ovo tumor cell–DC coculture tumors. (D) Schematic overview of experimental treatment procedure in vivo. (E) Representative images of TUNEL-stained tumor nodules indicative for apoptosis with representative distance marks of penetration depths and quantification thereof (Insert: y axis shows penetration depth with regard to dead cell regions reaching within the tissues expressed in micrometers). (F) Tumor weight of excised peritoneal tumor nodules. Graphs show mean. Statistical analysis was performed using one-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001; ns, nonsignificant). (Scale bar, 1 mm.) moDC, monocyte-derived DCs.

Fig. 5.

H₂O₂ dosage correlates with immune-related and immunogenic surface marker expression profiles. (A) Schematic overview of experimental procedure. (B) Representative flow cytometry intensity histograms of CD47, CRT, HMGB1, HSP 70, and TNFR I and II. (C) PCA of z-scored marker expression profiles in HT-29 and Panc-01 cells. (D) Quantification of marker expression normalized to NaCl in HT-29 and Panc-01 cells. (E) Pearson correlation of marker MFI and H₂O₂ dosage. Heat map shows median. Graphs show mean. MFI, mean fluorescence intensity.