## 4 Discussion  
Gas plasma treatment is a promising and novel avenue to target cancer cells, including breast tumors. Albeit an array of in vitro and in vivo studies, data on primary tumor material from patients is scarce, hampering the translational value of this treatment and technology in oncology. To this end, we here investigated 20 clinical breast cancer samples using matrix-microtissues along with the functional consequences of gas plasma exposure in this tumor model of primary patient material. We found an increase of apoptotic cells in our collagen-microtissue-matrices after plasma treatment accompanied by an increase of the adhesion marker E-cadherin associated with a less migratory activity of tumor cells [41]. Apoptosis and cell migration are two known effects of cold plasma in cancer therapy accompanied by consequences such as growth retardation and metastasis [42]. Tumor invasion and metastasis is the leading cause of death in cancer patients, including breast cancer [43]. The metastasis process is controlled and regulated by microenvironmental and systemic processes, including intercellular adhesion molecules. Weakening cell-cell and cell-matrix connections are essential for metastasis. Numerous families of adhesion molecules, including cadherins, integrins, adhesion molecules of the immunoglobulin family, selectins, and CD44 are identified [44]. The latter has been recently suggested to be affected by plasma treatment as well [45]. According to this hypothesis, where the lack or decreased expression of these molecules increases the rate of metastasis, these molecules can be used as markers of metastasis. E-cadherin is a transmembrane glycoprotein mediating calcium-dependent intracellular adhesion and is involved in epithelial cell-cell adhesion [46]. We had previously investigated metastasis on various pancreatic cancer cell lines by examining E-cadherin, EpCam, integrin \(\alpha\)-2, integrin \(\alpha\)-4, and ZEB-1, and concluded that the plasma treatment reduced metastasis in cancer cells [47], which is consistent with the results of cadherin expression in this study. Initiation of apoptosis is a frequently described process in plasma-treated cancer cells. In vitro, it was found for several cancer types, such as colon [48; 49; 50], pancreas [51; 52; 53], head and neck [54; 55; 56], leukemia [57; 58; 59], glioblastoma [60; 61; 62], and melanoma [63; 64; 65]. For stage IV malignant melanoma patient-derived samples, we also have previously found augmented apoptosis within tissues after gas plasma exposure with the kINPen jet [66]. It is understood that this is a consequence of supraphysiological amounts of ROS/RNS delivered by the plasma system [67]. Nevertheless, there is a sensitivity gradient, as we have previously reported across 35 different tumor cell lines that have individual capacities to cope with the ROS/RNS [68]. The tumor-toxic activity of some of the long-lived species is retained in cell culture medium and other types of liquids, which can be subsequently used for cancer therapy [69]. Our present findings in primary human breast cancer tissue underline this notion, albeit the plasma-treated medium--called \"indirect\" in this work--was slightly less active than the direct gas plasma exposure. With plasma-treated cell culture medium, it should also be kept in mind that its utilization in preclinical cancer research is of limited translational value, as these solutions have not been accredited for use in humans as certified medical products [70]. In this study, 3D tissue culture technology has been used inside collagen to monitor plasma function on the tumoral tissue growing outside the body. Gel-like substances such as agarose have been used in other studies before the monitor the plasma-mediated ROS/RNS diffusion through different matrices [71; 72; 73]. Collagen has the added value that it is part of the in vivo TME of the tumors, making our microtissue model more realistic.