# Cold Atmospheric Plasma Cancer Treatment, a Critical Review
## 5 Anti-Cancer Capability In Vivo  
The final goal of plasma medicine is its clinical application. At present, the pathway to realize this goal remains an open question. Three treatment approaches in vitro have provided some projections for potential clinical fates. In terms of animal studies, there are three main models: subcutaneous, intraperitoneal, and orthotopic tumor models [38; 81; 82]. In some cases, intracranial models have also been presented, in which PAM/PAS has been injected to affect the growth of tumors [38; 58; 83; 84]. In most cases, an anti-cancer effect was demonstrated on the subcutaneous tumor model. It should be noted that, for subcutaneous animal models, tumors are never directly exposed to bulk CAP unless surgery is performed prior to treatment to gain access to it. This is a fundamental difference between in vivo and in vitro studies. In the former case, the skin and subcutaneous tissues face the impact of bulk CAP before tumor tissue. If reactive species, particularly ROS such as H\({}_{2}\)O\({}_{2}\), cannot penetrate the barriers above the tumor tissue, the conclusions based on direct CAP treatment in vitro may not be very useful to explain in vivo treatment. In contrast, physical-based treatment may build a reasonable correlation between in vitro and in vivo studies. Because of a general conclusion that reactive species cause all observable effects, the potential roles of the EM effect and even the heating effect have not been carefully explored, particularly for the cases in which the authors used plasma with a significantly higher temperature than room temperature. Strictly speaking, these plasmas are not cold plasma. In short, it is still unknown whether physical factors play a dominant role during the studies involving direction contact between plasma and tissues/cells. Many animal studies have been performed over the past decade, and the results of these studies provide hope for the use of CAP as a clinic anti-cancer modality. The earliest animal study used a glioblastoma U87 MG xenograft tumor model to test the anti-cancer effect in mice [81]. This study was the first to demonstrate that treatment above the skin of a subcutaneous tumor can achieve a promising half decrease in tumor size [81]. A fractionated treatment resulted in a greater effect than a single, longer plasma treatment [85]. Several subsequent studies demonstrated a similar strong anti-cancer effect during treatment in subcutaneous tumor models, including bladder cancer, melanoma, pancreatic carcinoma, glioblastoma, neuroblastoma, head and neck cancer, and a breast cancer xenograft model [54; 82; 86; 87; 88; 89; 90; 91]. The safety of CAP treatment in animals has been proved in some studies, which confirmed no thermal damage or other obvious damage occurred to the surrounding normal tissues after treatment [86]. Due to the natural barrier of skin and other tissues above tumorous tissues, several attempts have been made to explain whether an inhibition effect is present during the transdermal treatment in subcutaneous tumor models. If we do not consider the potential anti-cancer effect due to physical factors, CAP-originated reactive species may need to penetrate skin and other tissues above tumorous tissues [92]. Moreover, reactive species such as ROS may just penetrate subcutaneous tissue with a limited depth but act as a second messenger to trigger other biological effect. To date, a small number of studies suggest the possible transdermal diffusion of reactive species across some skin substitutes, such as agarose gel [93; 94; 95; 96]. However, this conclusion requires more experimentation and evidence to be fully supported. The CAP-triggered immune response is a promising candidate to explain the anti-cancer effect in vivo [97]. CAP may activate the immune system to attack tumorous tissue. Such immune responses have been referred to as induction of immunogenic cancer death (ICD) [98; 99].