## 4 Discussion  
Exposure of human cutaneous melanoma biopsies to cold physical plasma _ex vivo_ increased the number of apoptotic cells in the treated tissue. This is in line with previous in vitro findings showing induction of cellular senescence [12] and apoptosis [13] in melanoma cells with plasma treatment. Plasma-induced apoptosis in melanoma is dependent on caspase activation and can be abrogated by prior addition of radical scavengers [14], which underscores the notion of the strong ROS-based effects of physical plasmas. Of note, apoptosis of cancer cells following plasma treatment is not restricted to melanoma cells but also includes a variety of other tumor cell types [15]. ROS, as well as cell demise, can be both the source and target of an altered inflammatory signature in the tumor microenvironment (TME). To this end, we profiled the supernatants of cultured tumor samples quantitatively for the secretion of 24 different chemokines/cytokines to understand how plasma treatment may directly or indirectly modify the TME of human melanoma samples _ex vivo_. Following exposure to plasma, markedly increased (CCL5, EGF, granulysin) or decreased (CCL2, IL10, VEGF) chemokine/cytokine secretions were observed in at least two out of six patients with a similar trend. It is well-known that VEGF and IL10 but also CCL2 support immunosuppression in the TME, leading to senescence of antitumor immunity (IL10), angiogenesis (VEGF), education of tumor-associated macrophages (CCL2), and subsequently augmented tumor growth [16]. Hence, plasma treatment may constitute a therapeutic approach to make \"cold tumors hot\", i.e., to induce a mild inflammation that is the prerequisite for antitumor immune responses. That the Nobel Prize in Medicine 2018 was awarded for the identification of checkpoint inhibition exemplifies the importance and magnitude of T-cells specifically targeting tumor cells in antitumor therapies. Yet, many patients still not benefit from such antibodies masking, e.g., PD1/2, PD-L1/2, and/or CTL4, and it is discussed that other immunosuppressive mechanisms in the TME may abrogate T-cell activity [17]. Therefore, plasma treatment may possibly add efficacy to immunotherapies in cutaneous melanoma metastases. EGF release was significantly upregulated in two out of six patient samples after plasma treatment. The activation of the EGF receptor is a therapeutic target in many cancer entities. The significant increase of CCL5, seen in three out of six patient-derived melanoma samples after plasma exposure, has been well described to support the infiltration of tumor-toxic NK cells [18]. NK cells lyse their target cells using, e.g., granulysin. Interestingly, in two of the three samples with increased CCL5 levels after plasma treatment, we also observed a 10- and 20-fold increase in granulysin. Perforin, another lytic molecule in NK cells, was, however, not increased. This could be explained by the fact that skin-resident NK cells preferentially lyse target cells via granulysin [19]. Altogether, the plasma treatment decreased immunosuppressive and increased immune-supportive chemokine/cytokine secretions in 33-50% of the patient-derived melanoma samples investigated. Despite using a highly standardized plasma treatment and sample culture protocol, the heterogeneity of our responses observed may also relate to differences in the biopsies' location and microenvironment as well as the patients' health status. In our hands, the small sample size also does not allow for correlations between changes in chemokine and cytokine secretion and the level of apoptosis. Nevertheless, the baseline chemokine/cytokine expression profile of the 24 targets presented may serve as a valued baseline resource for future studies investigating human cutaneous melanoma samples _ex vivo_.