# Medical Gas Plasma Treatment in Head and Neck Cancer--Challenges and Opportunities
## 4 In Vitro and In Vivo Studies  
Several head and neck cancer cell lines have been investigated regarding the effects of physical plasma on their proliferation and cell activity (Table 1). Most of these experimental in vitro studies used the oral squamous cell carcinoma cell line SCC-15, as well as SCC-25, and highlighted their plasma-induced inactivation due to the reduction of cell growth, induction of apoptosis, and secondary DNA damage, especially through plasma-derived ROS [19; 25; 28; 34; 35; 36; 37; 38]. Other HNSCC cell lines were also tested, showing one or more of the tumor-toxic plasma-associated characteristics [6; 19; 22; 24; 26; 27; 28; 29; 37]. In addition, HNSCC has been shown to be more prone to physical plasma treatment than non-malignant cells since the viability of the latter did not significantly decrease after plasma application. The fact that plasma selectively targets cancer cells, leaving non-malignant cell types, such as human fibroblasts (e.g., human gingival fibroblast-1 (HGF-1), HS-K, and IMR-SV-90) or human keratinocytes (HaCaT) unaffected, makes plasma a promising tool for adjuvant head and neck cancer therapy [25; 27; 29]. Moreover, two studies demonstrated an enhanced selectivity of plasma after the addition of antibody-conjugated gold nanoparticles, which specifically targeted SCC-25 head and neck cancer cells [35; 36]. Cancer cell selectivity was achieved through the conjugation of anti-EGFR (epidermal growth factor receptor) or anti-TfR (transferrin-receptor) antibodies to the nanoparticles since this type of cancer usually overexpresses EGFR and TfR. As a result, an 18-fold increased efficacy of the nanoparticle treatment was found compared to the cancer cells not pretreated with plasma [35]. Therapeutic drugs often target the EGFR transmembrane protein since it plays a vital role in the regulation of the cell cycle, proliferation, differentiation, and transformation. Hence, its degradation leads to the inhibition of cell growth [66]. [PERSON] et al. showed that plasma has a similar impact on HNSCC. The plasma-derived ROS induced EGFR dysfunction and degradation in the SCC-15 and HSC-2 cell lines, thereby interrupting the EGFR downstream signaling pathway, consequently killing the squamous cell carcinoma [25]. Selectivity, however, is not necessarily a universal feature of gas plasma applications in cancer treatment. Several studies have found non-malignant cells to be more vulnerable when compared to their malignant counterparts or cancer cells in general [67; 68; 69]. This is also known from the field of redox biology, where, for instance, human leukemia cells were less prone to ROS-induced cell death compared with non-malignant myeloid cells [70]. The many studies in murine animal models and the few studies in patients (see below) nevertheless suggest that gas plasma treatment at least eradicates tumor cells while leaving the host intact. Systemic toxicity was also not observed either in mice or humans. Although it is unknown whether gas plasma treatment might be killing non-malignant cells in the tumor microenvironment, the overall decline of tumor mass will, in the end, indicate whether this technology can be usefully applied in oncology or not. Besides EGFR, studies suggested a crucial role of the p53-signaling pathway in plasma-induced head and neck cancer cell death [23; 29; 33; 37]. This protein contributes to the redox balance within the cells via regulation of the antioxidant gene expression and further senses DNA damage, initiates signaling pathways for the DNA repair, and fulfills the role of a cell-cycle regulator [20]. Subsequently, plasma-induced secondary DNA damage resulted in p53-mediated cell-cycle arrest in head and neck squamous cell carcinoma cells, ultimately impairing cell viability.