## 4 Discussion  
HA is a component of the ECM and is expressed in the tumor microenvironment formed by malignant cells, immune cells, and stroma cells [19]. In some cancers, high amounts of HA are correlated with tumor progression [20; 21]. In tumor therapy, various strategies are pursued. One is to influence the tumor microenvironment by inhibiting the HA synthesis using drugs like 4-methylumbelliferone or promoting degradation by HAdases in combination with chemotherapy [21; 25; 26; 27; 28]. Another approach is the use of physical plasma to selectively affect tumor cells alongside normal cells [57; 58; 59; 60; 61; 62; 63; 64; 65]. Both strategies could be combined by influencing the HA pericellular coat of cells through physical plasma. Since this has not been investigated so far [19], we determined the influence of PAM on the HA pericellular coat of HaCaT cells in vitro. [PERSON] et al. determined the amount of HA produced by HaCaT keratinocytes using a fluorimetric measurement [81]. In our study, using immune fluorescence staining and confocal laser scanning microscopy, we could show for the first time that HaCaT cells express the HA as a pericellular coat. We also found that the expression of HA could be reduced by incubation with PAM. Interestingly, this time-dependent effect was not comparable to HAdase treatment, where HaCaT cells completely lost their HA coat after 6 h of incubation. However, PAM had a significant effect on the adhesion of HaCaT cells during the whole observation time of 1.5 h and was much more pronounced than that of HAdase, which was only detectable after 30 min. Interestingly, HAdase treatment seems to affect the initial cell adhesion of HaCaT cells less than that of osteoblasts. In an earlier work of our group, we found a significantly reduced initial adhesion of HAdase-treated MG-63 osteoblast cells on titanium compared to untreated cells during the first 15 min [11], whereas the adhesion of HaCaT cells was only significantly affected by HAdase after an incubation time of 30 min. It would seem it is difficult to compare the behaviors of different cell types. Figure 6: Influence of hydrogen peroxide on HaCaT cells in DMEM. (**A**) Flow cytometry determination of the HA expression in non-adherent HaCaT cells after cultivation in H2O2 or DMEM for 3 h (mean \(\pm\) s.e.m., n = 2). (**B**) Online monitoring of HaCaT cell adhesion in H2O2 and DMEM up to 1.5 h. Note that both the HA pericellular coat and the adhesion of the cells were impaired by H2O2 (xCELigence Real-time Cell Analysis, normalized cell index; mean \(\pm\) s.e.m., n = 3, Mann–Whitney test, \({}^{**}\)\(p<0.01\), \({}^{***}\)\(p<0.001\) DMEM vs. H2O2). Abbr. : FL1-H: fluorescence intensity, DMEM: Dulbecco’s modified Eagle’s medium, H2O2: DMEM with H\({}_{2}\)O\({}_{2}\). Osteoblasts are bone-forming cells, which synthesize great numbers of extracellular matrix molecules, e.g., collagen I and glycosaminoglycans. HaCaT cells are human adult keratinocytes that are spontaneously immortalized and non-tumorigenic [79]. We used this cell line for the in vitro experiments described here as a model for human keratinocytes that also express relatively high amounts of HA. The HA content in different tissues varies greatly. Amounts in the skin are comparatively high and higher in fetal and young skin than in older skin [17]. Interestingly, in melanoma or other epidermal cancers, the HA content does not correlate with the tumor progression [82], and reduced expression of HA is found to be a marker for poor survival in oral squamous cell carcinoma [81; 83]. To find out more about the mechanism underlying the action of PAM on the HA pericellular coat, we tested a relatively long-lived reactive species, which usually arises from the interaction of CAP with a liquid medium, hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)). We incubated HaCaT cells in DMEM supplemented with 150 mg/L H\({}_{2}\)O\({}_{2}\).