## 4 Discussion  
The delivery of CAP-generated ROS and RNS through the liquid environment to cells changes the redox environment, which ultimately may induce cytotoxic effects [29; 30]. With respect to cancer treatment, it is widely accepted that CAP-generated ROS and RNS are the main agents to induce cell death and apoptosis in cancer cells [6; 24; 31]. After CAP application an increasing number of apoptotic cancer cells positively correlate with an increased concentration of nitric oxide (NO), ROS and lipid peroxidation products [32]. Others demonstrated that CAP induces apoptosis via ROS and dysfunction of mitochondrial membranes [23]. In addition, autophagy has been discussed as another way of cell death after CAP exposure especially in primary cells [33]. This illustrates the great potential of CAP in the therapeutic concept of tumour treatment on the basis of ROS/RNS generation. In this context eliminating cancer cells by pharmacological ROS insults has also been developed independent of CAP technology [34]. The mode of action of some chemotherapeutic agents employs partly the production of ROS and therefore enhances their cytotoxic effectiveness (e.g., cisplatin or bleomycin) [35; 36; 37; 38]. Even more so, a synergistic effect of gemcitabine and CAP in killing pancreatic tumour cells has been described recently [39]. Squamous cell carcinoma of the head and neck can grow in very close vicinity to vital structures (Figure 1), hence pose a surgical challenge for resection. Therefore, this study investigated CAP application as a potential adjuvant tool in the treatment of squamous cell carcinoma in this region. Unique advantages of the CAP application are the accessibility of localized areas and upon this time no known side effects [5; 9]. In our study the certified medical device kINPen MED was employed to generate CAP. The therapeutic concept of the kINPen is based on the generation of ROS and RNS (RONS) [40]. Recently, a beneficial effect of this argon plasma jet treatment on head and neck cancer was documented in a retrospective clinical follow-up study by [PERSON] and colleagues [41]. Several repeated cycles of CAP application for only one minute lead to partial remission of the tumour in 36% of the cases but prominent histological alterations were absent. Herein ex vivo treated tissue biopsies revealed an increased number of apoptotic cells within the tissue and elevated levels of cytochrome c in the extracellular liquid, pointing to profound apoptotic cell damage. This is in good agreement with clinical findings in head and neck squamous cell carcinoma patients [9]. In our approach, the distribution of TUNEL-positive cells was rather randomly dispersed throughout the tissue sections (data not shown), pointing to a non-cell-cell-contact-based mechanism. Contrary, results from [PERSON] et al. showed more dying cells in the outermost layers of treated pancreatic carcinoma tissue concomitant with reduced proliferation [42]. The therapeutic application of CAP in cancer treatment requires certain selectivity towards malignant cells. In our experimental setup we could show stronger induction of apoptosis in tumour tissue in situ compared to healthy tissue. In order to follow events in a 2D cell culture setup, two corresponding cell lines were selected, normal HaCaT keratinocytes and HNO97 derived from a human squamous cell carcinoma of the head and neck. However, the cell lines used revealed very little difference with respect to induction of apoptotic events after CAP exposure. Hence, the selectivity was not established in these in vitro tests. This may be due to the simple homogeneous cell population. Further the sensitivity towards CAP varies among HNSCC and non-malignant cell lines and the underlying molecular composition should be considered for future examinations. By contrast, others found that CAP selectively impairs some cell lines of head and neck squamous cell carcinoma also through non-apoptotic pathways [24; 43].