## 3 Results  
In this study, direct and indirect plasma treatment was conducted (Figure 1a). The helium plasma jet emitted several reactive species, including ozone, atomic oxygen, hydroxyl radicals, and species of the second positive system of nitrogen (Figure 1b). In liquids, these species deteriorate to more stable oxidants such as hydrogen peroxide (H\({}_{2}\)O\({}_{2}\); Figure 1c) and nitrite (NO\({}_{2}\)\({}^{-}\); Figure 1d) found in plasma-treated liquid but not helium gas-treated or untreated liquids. The plasma jet was engineered to be operated at about body temperature, leading to a surface heating of about 37 \({}^{\circ}\)C (Figure 1e). Exposure of fully supplemented cell culture medium led to a degassing effect of carbon dioxide and subsequent increase of the pH (Figure 1f), which quickly normalized after incubation in the humidified and CO\({}_{2}\)-containing incubator. Human patient-derived breast cancer tissues were received after informed consent and processed in the laboratory (Figure 2). After dissection of the material, it was filtered to remove material larger than 100 \(\upmu\)m followed by filtering through a 40 \(\upmu\)m filter. The eluent of the later step including single cells and debris was discarded, and tumor microcolonies between 40-100 \(\upmu\)m in size were mixed with collagen and added to multiwell dishes. The collagen-tumor matrix was plasma-treated directly or indirectly (Figure 3a), followed by allowing the colonies to grow through the course of incubation over several days. During this time, the colonies extended their volume due to the proliferation of individual cancer cell clones within the colonies. Non-malignant cells such as fibroblasts and immune cells of the TME do not proliferate because contact inhibition provided by the matrix inhibits their growth. Colony quantification revealed a significant decline of tumor growth in the collagen matrix for both direct and indirect plasma treatment (Figure 3b). Figure 1: Characteristics of the gas plasma jet used in this study. (**a**) scheme of the indirect vs. the direct treatment regimens; (**b**) optical emission spectroscopy of the plasma jet; (**c**) hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) production of the gas plasma jet; (**d**) nitrite production of the gas plasma jet; (**e**) thermal camera image of the gas plasma jet; (**f**) pH of cell culture medium exposed to the gas plasma jet. Data are representative or mean and standard deviation (**c,d**) of three experiments. Exposure of the microtissue to helium gas alone did not give overall different results from untreated tissue in pilot experiments and hence was not included in the study. At 24 h after plasma treatment, the collagen-tumor-matrices were stained with the live/dead fluorescence dyes AO/PI (Figure 4a). Quantitative image analysis demonstrated a significantly increased presence of dead cells for both direct and indirect plasma treatment regimens (Figure 4b), showing significantly enhanced effects of the former over the latter. Next, the microtissues were fixed, and ultrathin sections were conducted. Staining was performed against active caspase or E-cadherin together with DAPI as nuclear counterstain (Figure 4c). The direct and indirect plasma treatment significantly increased the number of caspase 3-positive cells indicative of apoptosis (Figure 4d). Again, the direct treatment showed a more substantial effect compared to the indirect treatment. For the cell surface molecule E-cadherin, which is increasingly expressed in low-metastatic breast cancer tissues [40], a significantly increased presence was found for direct and indirect plasma treatment. These data suggested the indirect and especially the direct plasma treatment procedure to reduce tumor colony formation, increase apoptosis and terminal cell death, and elevated the expression of a marker associated with a more epithelial phenotype. Chemokine and cytokine release is another critical effector in the tumor microenvironment (TME).