Preclinical Cold Atmospheric Plasma Cancer Treatment: Comparison
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Cold atmospheric plasma (CAP) is generated in a rapid yet low-energy input streamer-discharge process under atmospheric pressure conditions and environment. CAP is an ionized gas with a low ionization degree and plenty of reactive species and radicals. These reactive components, and their near-room temperature nature, make CAP a powerful tool in medical applications, particularly cancer therapy. Here, wresearchers systematically introduced the research status of the preclinical application of CAP in cancer therapy, particularly in vivo studies. 

  • cold atmospheric plasma
  • cancer treatment
  • anti-tumor therapy

1. CAP and Plasma Sources

CAP has been widely used in several branches of medicine, including wound healing, microorganism sterilization, biofilm inactivation, and cancer therapy [1][2][3]. CAP is an ionized gas composed of reactive compounds such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) [4][5], and is designed to work under atmospheric pressure at near room temperature [6]. CAP sources, such as dielectric barrier discharge (DBD), atmospheric pressure plasma jet (APPJ), and plasma torch, are the foundation for plasma biomedical applications [7][8]. Six of the most commonly used CAP sources are shown in Figure 1. Alternating current (AC) is a typical power supply for these CAP sources [9]. 
Cancers 14 03461 g001
Figure 1. Typical CAP sources used in plasma medicine. Type a. Volume DBD. Type b. Surface DBD. Type c. Two-ring electrodes APPJ. Type d. Plasma torch using ring electrode. Type e. One central electrode-one ring electrode APPJ. Type f. Plasma torch using a central electrode.

2. General Picture of In Vitro Studies

To date, the promising anti-cancer performance of CAP treatment in vitro has been extensively demonstrated in dozens of cancer types, including skin, breast, colorectal, brain, lung, cervical, head and neck cancer [3][10]. Plenty of reviews and articles have been published, most of them focused on in vitro studies and corresponding conclusions [11][12][13]. Several basic cellular responses have been repeatedly observed in the publications and are listed in Table 1. These basic cellular responses build the foundation for understanding the anti-cancer effect of CAP treatment in vitro and address some in vivo observations.
Table 1. Basic cancer cellular responses of CAP treatment in vitro.
Ref Cancer Cellular Response Years
[14] Apoptosis 2004
[15] Growth Inhibition 2007
[16] Cytoskeletal Damage 2009
[17] Selective Cell Death 2010
[18] Cell Cycle Arrest 2010
[19] Nuclear and DNA Damage 2010
[19
Like most medical studies, the conclusions obtained from in vitro studies cannot be easily used to directly predict the performance of in vivo studies. For example, the relatively dry skin barrier between plasma and targeted cancerous tissues or cells under the skin is quite different from the commonly accepted experimental conditions in vitro. The in vitro environment mainly involves a relatively thick medium layer to facilitate the transition of some short-lived reactive species in the gas phase to long-lived reactive species in the liquid phase. Moreover, both long-lived and short-lived reactive species will have complex reactions at this gas/liquid interface. In vivo studies play a cornerstone role in plasma medicine before CAP can be used in clinical therapy [40][41][42]. More importantly, in vivo studies directly assess the CAP treatment’s safety on tissues and animals, such as carcinogenicity [43]. In thiRes review, ourearchers preclinical studies’ discussion will be just limited to in vivo studies.
Compared to the abundant in vitro investigations, in vivo studies have gradually become the main approach to discovering novel tissue responses to CAP treatment. Animal models’ design directly determines the use of CAP in the in vivo studies. So far, three types of animal models have been widely used to demonstrate the anti-tumor efficacy of CAP treatment: subcutaneous model, intraperitoneal model, and orthotopic model [44]. To date, most of CAP’s anti-tumor capability was demonstrated using subcutaneous models.
Subcutaneous models provided the earliest and the most apparent demonstration for the feasibility of using CAP as an anti-tumor modality. The earliest in vivo works were demonstrated by Marc Vandamme, et al. and Michael Keidar, et al. between 2010–2011. They used a glioblastoma U87MG xenograft mouse model and bladder xenograft tumor model to test a CAP treatment’s in vivo efficacy for just a few minutes, respectively. The two pioneering research articles demonstrated a drastic tumor volume reduction of more than 50% after floating electrode DBD treatment and APPJ treatment [41][45]. Correspondingly, the survival length of mice strongly increased by more than 60% in the two models [41][45]. These two works also first tested the safety of using CAP in animal studies. Results showed no toxic side effects or potential physical damage from plasma.
 
Due to the subcutaneous nature of melanomas, it has become one of the more promising candidates for CAP-based cancer therapy. Many studies have been performed on melanoma models [7]. A nanosecond pulsed DBD (nsP DBD) completely eradicated the xenografted melanoma tumor in mice after direct treatment on the skin above the melanoma. Histology of an nsP DBD treated tumor showed a typical red skin staining without tumor tissue below the epithelium. Correspondingly, the survival rate of mice increased from 0% to 66.7% 20–40 days succeeding the nsp DBD treatment [48].
Similar trends have been repeatedly observed in a series of following studies. Table 2 lists representative in vivo anti-tumor demonstrations (2010–2018) on subcutaneous xenograft tumors in mice. In the subcutaneous model, CAP treatment was mainly carried out by treating the skin above tumorous tissues. In such a setting, the effective factor, either chemical or physical factors in CAP, must penetrate the skin barrier and further trigger biological pathways to inhibit tumorous growth, therefore providing CAP treatment as a potential non-invasive anti-tumor modality. Among these studies, a general trend has been repeatedly observed. That is, a treatment just above the skin could strongly inhibit the growth of tumors and significantly extend the life length of mice [45]. 
Table 2. Representative in vivo demonstrations on subcutaneous xenografted tumor models (2010–2018).
Ref Years Tumor Types Tumor Size Survival Rate Tumor Diagnostics
[45] 2010 Glioblastoma Decreased N/A Bioluminescence imaging
[41] 2010 Bladder cancer Decreased Increased Tissue size measurement
[46] 2011 Glioblastoma Decreased Increased Bioluminescence imaging
[47] 2012 Pancreatic carcinoma Decreased N/A Bioluminescence imaging
[48] 2012 Glioblastoma Decreased N/A Bioluminescence imaging
[49] 2013 Neuroblastoma Decreased Increased Tissue size measurement
] Mitochondrial Damage 2010
[50] 2014 Melanoma Decreased N/A Tissue size measurement [20]
[51]Rise of Intracellular ROS 2011
2014 Head and neck cancer Decreased N/A Tissue size measurement [21] Chemically-based Sensitization to Drugs 2013
[40] 2015 Melanoma Decreased Increased Tissue size measurement [22] Selective Rise of Intracellular ROS 2013
[52] 2015 Endometrioid adenocarcinoma Decreased N/A Tissue size measurement [23] Senescence 2013
[53] 2016 Glioblastoma Decreased N/A Tissue size measurement [24] Immunogenic Cell Death
[2015
54] 2016 Breast cancer Decreased N/A Tissue size measurement [25] Cell-based H2O
[42]2 Generation 20172017
Melanoma Decreased N/A Bioluminescence imaging [26]
[Autophagy-associated Cell Death 2017
55] 2018 [27] Activation Phenomena 2018
[28] Physically-triggered Necrosis 2020
[29] Pyroptosis 2020
[30] Physically-based Sensitization to Drugs 2021
Together, some general conclusions can be summarized here. (1) Reactive species play a critical role in the liquid phase-based experimental setting [31]. Apoptosis, necrosis, and autophagy are the main cellular death approaches following CAP treatment with an adequately large dose [32]. (2) Physical factors, particularly electromagnetic effects from plasma, may exert a clear impact on cells, such as bacteria and mammalian cells [33][34]. (3) A noticeable rise in intracellular ROS is a pivotal cellular response to CAP treatment, which further triggers downstream cellular damage, including DNA damage, mitochondrial damage, cellular membrane damage, and cell death [1]. (4) Aqueous environment, such as a medium layer, plays a pivotal role in facilitating the transition of short-lived reactive species in the gas phase into long-lived reactive species in the liquid phase [35][36][37]. For in vitro studies, a medium layer is necessary for experimental design and is responsible for most observed cellular responses after CAP treatment, particularly for the cases involving CAP-treated solutions or media [32][38]. (5) CAP shows a selective killing effect on cancer cell lines compared to their counterpart normal cell lines in many cases [39].

3. Direct CAP Treatment In Vivo

Colorectal tumor
Decreased
N/A
Tissue size measurement

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