# Cold Atmospheric Plasma Cancer Treatment, a Critical Review
## 3 Chemical-Based Direct CAP Treatment  
The earliest attempts to test the anti-cancer capacity of CAP treatment were conducted using direct treatment. Direct treatment is also the most conventional approach [51]. It is based on a simple assumption that a treatment should be performed when cells are in culture conditions or at least in an environment close to that of the standard culture conditions. A layer of medium covered cells during most studies involving direct CAP treatment [52]. Because thermal irradiation, UV irradiation, and EM emission may be blocked by this layer of medium, particularly when this layer is adequately thick, the physical effect has not been noted in most previous studies. This medium layer also plays the role of an interface between the gas phase of CAP and aqueous solutions [8]. Without such an aqueous environment, many reactions may be interfered with or fully blocked. Direct and indirect treatments share many similarities. Nearly all cellular damage can be observed both in direct and indirect treatment. Common cellular responses to direct treatment include the rise of intracellular ROS, DNA damage, mitochondrial damage, endoplasmic reticulum damage, and damage to the cytoplasmic membrane [42, 53, 54, 55, 56, 57]. These cellular changes have also been observed during indirect treatment [33, 34, 42, 58, 59]. Sensitivity of cancer cell lines to direct treatment and indirect treatment is also usually similar. The unique sensitivity of cancer cell lines to chemical-based treatment may be partially due to the specific anti-oxidant system in different cancer cell lines, which can be related to differential expression level of TP53 in various cell lines [60, 61]. Some cancer cell lines, such as melanoma cell line B16F10, are very resistant to reactive species in both direct and indirect treatments [62]. In a case like this, a long period of CAP treatment is necessary. This is a natural limitation of chemical-based CAP treatment. Apoptosis is the main cell death after chemical-based treatment; however, necrosis and autophagy-associated cell death have been also observed [56, 57, 63, 64, 65, 66, 67]. Apoptosis is  
Figure 2: Schematic illustration of the indirect CAP treatment based on CAP-activated medium in vitro. Here, the feature of apoptosis is used to represent cell death. a slow process that begins with the formation of clear blebs, representing apoptotic cells losing their pseudopodia. Cells may later detach from the substrate or may experience apoptosis on the substrate. It should be noted that cancer cells may recover to their normal shape depending on the treatment dose. Bubbling can also be observed in some cells during the cell death process, which is believed to be a feature of another form of necrotic cell death known as pyroptosis [68]. One aim of plasma medicine may be the exploration of unique cellular responses in the CAP-treated cancer cells, rather than repetitively showing the features of other methods, such as apoptosis and cell cycle arrest [69]. Quantitative comparison between direct and indirect treatment provides clues to understand the unique features of direct CAP treatment. In the studies comparing the cytotoxicity of direct and indirect CAP treatment under the same experimental conditions, the former caused a stronger effect than the latter in many cases [70; 71]. The unique features of direct treatment may contribute to these differences (Figure 3). One such feature is the activation phenomenon of CAP-treated cancer cells. The treated cancer cells quickly enter an activated state in which they become very sensitive to the cytotoxicity of reactive species, particularly H\({}_{2}\)O\({}_{2}\)[72]. In contrast, cancer cells do not enter an activation state after indirect treatment or simple long-lived reactive species treatment. Therefore, cell activation following direct treatment may be due to short-lived reactive species or physical factors. The activated cancer cells remain in this unique state for approximately 5 h [72].