## 4 Discussion
Underlining the notion of ROS-induced differentiation, plasma treatment significantly spurred the release of inflammatory chemokines/cytokines (IL18, IL6, IL8, and MCP1) [71; 72; 73] in both non-PMA and PMA-pulsed cells. MCP1 leads to migration and adhesion of THP-1 [74] and peripheral blood monocytes [75] as first step of their activation. MCP1 was also reported to alter THP-1 morphology by increasing the number of cells with elongated pseudopodia [76]. In our experiments, some cells were spreading pseudopodia essential for realizing their phagocytic function in the tissue microenvironment [77]. The authors also identified other phenotypic changes in response to plasma treatment such as decreasing cell roundness as typical features of monocyte activation [78]. Another observation was made in detecting the cellular cluster area, which was reduced via PMA or plasma. Peritoneal macrophages are able to adhere and cluster via the chemokine-receptor system CCL1/CCR8 [79], and this system is mainly utilized by cells of the M2 phenotype [80], pointing to a rather pro-inflammatory ROS-induced phenotype. Plasma treatment led to intracellular oxidation in the cytosol and mitochondria. This corroborates previous findings with leucocyte populations and other cell types [81; 82; 83]. Oxidation of the cytosolic compartments seemed greater than mitochondrial oxidation. This could be due to intrinsic properties of the dyes or the cells' own antioxidant defense system scavenging ROS/RNS on their way through the cell to the mitochondria. NAC is known to act as a radical scavenger [84; 85] and has successfully abolished the oxidative effects of plasma treatment. However, other studies showed that NAC boosts the effect of anti-inflammatory drugs and leads to decreased monocytes activation via suppression of the formation of arachidonic acids [86]. This and its interference with cellular metabolism and surface marker expression demotivated its long-term use in our experiments. H\({}_{2}\)O\({}_{2}\) metabolism (likely to be altered with NAC and changes in thiol-groups of redox proteins) is important for monocyte maturation into macrophages [87]. A change of redox-protein expression was previously described in THP-1 monocytes also in response to plasma [88]. ROS-stimulated THP-1 cells exerted stronger toxic effects in tumor cells compared to non-stimulated THP-1 cells. A similar finding was made with RAW264.7 macrophages with plasma treatment and was linked to TNF\(\alpha\) release [89], which was not the case in our study, as TNF\(\alpha\) was not found in supernatants of non-PMA-treated THP-1 cells at day 4. The tumor-toxicity of murine macrophages was previously shown to be independent of nitric oxide [90], suggesting a low contribution of inducible nitric oxide synthase in killing. Although the NLRP3 inflammasome--also active in THP-1 monocytes after recognition of damage-associated molecular patterns [91]--was suggested to be important in the antitumor myeloid and immune responses [92], this was probably not the case with our results as no secretion of IL1\(\beta\) and IL18 was found in tumor toxic culture conditions (with plasma, without PMA). A potential caveat of our study is that the authors did not directly compare the effect of H\({}_{2}\)O\({}_{2}\) to plasma-derived oxidants on long-term activating effects in monocytes. However, this was done in previous studies. [PERSON] and colleagues have shown that longer plasma treatment times, but not a concentration-matched amount of H\({}_{2}\)O\({}_{2}\), induces ERK-phosphorylation in primary monocytes [93]. Plasma-mediated ERK-phosphorylation was also found in THP-1 cells [94]. However, in both primary cells and the cell line, this was only true for long plasma treatment times, coming with significant caspase 3 activation and JNK phosphorylation, and hence cell death. In addition, observations were made within 24 h and not 96 h, as in our study.