_Applied_  
_sciences_  
_Article_  
**Antitumor Effects in Gas Plasma-Treated Patient-Derived**  
**Microtissues--An Adjuvant Therapy for Ulcerating**  
**Breast Cancer?**  
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nitrogen species (ROS/RNS) might be harnessed as a therapeutic adjuvant breast cancer treatment. For instance, singlet oxygen has been shown to promising tool in this regard [7], and redox-active nanomedicines are promising agents of modern oncology [8] that can be also be combined with classical therapies such as radiation [9].  
For a few years, a novel principle of generating therapeutic ROS/RNS levels has emerged in experimental oncology: plasma technology [10]. This partially ionized gas is operated at body temperature and does not convey thermal harm to cells and tissues [11]. This topical treatment modality dwells on a principle of physics for the generation of vast amounts of ROS/RNS simultaneously from reactive noble gas ions and electrons. Several in vitro studies have shown promising antitumor effects in gas plasma-treated breast cancer cells [12; 13; 14; 15; 16; 17; 18; 19; 20; 21]. Additional work has also provided evidence of reduced growth [22; 23; 24; 25] and metastasis [26; 27] in three-dimensional tumor spheroids. In addition, we have previously shown that gas plasma treatment of breast cancer in mice modulated the inflammatory milieu within the tumor microenvironment (TME) in terms of leukocyte infiltration [28]. Similar findings were made in murine melanoma models [29; 30].  
However, studies on plasma treatment of patient-derived tumor tissues are scarce. It is understood that 2D and 3D cultures of homogenous cell lines are of limited use for illustrating the complexity of the TME [31], particularly in the case of intercellular interactions and extracellular matrices reactions [32; 33]. Therefore, finding an appropriate platform for this type of culture is significant. Due to advantages, such as similarity to the extracellular matrix structure of body tissues, cell-matrix interaction, cell contacts, water, nutrient transfer, and growth and prolonged survival of encapsulated cells, microgels are suitable options for simulating the body environment [34; 35]. Besides, the third dimension of cell growth provides more spatial communication for mechanical inputs and cell adhesion, which is necessary for connecting integrins, cell shrinkage, and even intracellular signaling [36]. However, cultured 3D unicellular organisms lack immune and angiogenic cells being important for tumor growth in vivo. Hence, the direct culture of tissue extracted from the body, due to having immune cells, angiogenesis, and growth in microgels, can be a more acceptable simulator for examining the toxicity effect of drugs and treatments [37].  
To this end, we here extracted human-derived primary breast cancer tissues from 20 patients and developed a model to study the effects of gas plasma treatment of engrafted microtissues in collagen matrix and using microscopy. We found promising antitumor effects of the plasma exposure that increased cell death responses and modulated the release of several inflammatory mediators important in the TME.