# Cold Atmospheric Plasma Cancer Treatment, a Critical Review
## 4 Treatment Based on Physical Factors  
To overcome the sensitivity limitation of cancer cells to reactive species, physical-based CAP treatment was recently proposed. It is different from previous approaches, not only because of the stronger anti-cancer effect in some cell lines, but also because of the novel underlying mechanism. Even this concept has not been widely accepted in academia, and the preliminary discussion relates to current in vitro studies involving six cancer cell lines. To demonstrate the physical-based biological effect, we harnessed a novel strategy. We treated the bottom of an inverted cell culture dish or multi-well plate rather than using the typical treatment approach of directly exposing cells or solutions to bulk CAP. The treated cells were immediately moved back into standard culture conditions by replenishing medium. This strategy blocked all chemical factors due to the presence of polyene material of cell culture dishes and multi-well plates, with a thickness of approximately 1 mm (Figure 4). Neither UV nor thermal irradiation caused any observable cellular change [78; 79]. EM emission generated by CAP caused the observable cellular changes. Physical-based treatment is not necessary to cause stronger cytotoxicity in cancer cells compared with chemical-based treatment. To date, six cell lines have been investigated: glioblastoma cell line U87 MG, melanoma cell line B16F10, breast cancer cell line MDA-MB-231, lung cancer cell line A549, bladder cancer cell line MB49, and MBT2 [78; 79; 80]. Among these, nearly all cell lines show a stronger sensitivity to physical-based treatment, with the exception of the bladder cancer cell line MT49 [80]. The cell death after physical-based treatment is clearly a form of necrosis, rather than apoptosis. Cellular changes can be observed approximately 1-2 min after treatment, and are characterized by a fast shrinkage of cytosol toward the nucleus and rapid leakage of cellular solutions forming bubbles on cellular surface. Bubbling can be observed on nearly all treated cells and the growth of bubbles takes place 8-10 min after treatment, after which the growth of bubbles ceases. Bubbles remain on the cellular membrane for about 1-2 h, with most of them detaching after this time. The basic cellular shape does not change for days after bubble detachment. However, DNA in the nucleus is not seen two days after treatment. Additionally, cells do not display normal cellular functions, such as duplication, movement, and apoptotic features, after treatment. It appears that cells are fixed after physical-based treatment. Unlike traditional chemical-based treatment, the efficacy of physical-based treatment may be influenced by the power of EM emission and the spatial position between CAP source and target. Physical factors can affect a significantly larger area than the contacted area of bulk CAP on the target, and even these areas are physically separated; for example, in a 96 well plate, the tip size of the CAP jet is roughly close to the diameter of a single well of the plate. At a minimum, the 10 wells close to the treated single well also experience strong growth inhibition due to physical-triggered cell death [78; 79]. These wells were physically separated by the walls of well. In contrast, the same chemical-based treatment only causes damage in the single CAP-treated well. In contrast, however, physically triggered cellular response can only be observed when the CAP source and target have a suitable gap. The direct contact of bulk CAP with the surface of the barrier (bottom of plate  
Figure 4: Schematic illustration of a physical-based CAP treatment in vitro. Here, the feature of necrosis is used to represent the cell death pathway observed in references. Quick bubbling and cytosolic shrinkage are two key features of physical-based cell death. or dish) does not always trigger cell death. Surprisingly, when the detachment of bulk plasma from the target is set to a small range, such as 8 mm, a strong physically triggered cell death can still be triggered [78].