cancers

Review Plasma-Treated Solutions (PTS) in Cancer Therapy

Hiromasa Tanaka 1,*,† , Sander Bekeschus 2,*,† , Dayun Yan 3,*,† , Masaru Hori 1, Michael Keidar 3 and Mounir Laroussi 4

1 Center for Low-Temperature Plasma Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan; [email protected] 2 ZIK Plasmatis, Leibniz Institute for Plasma Science and Technology (INP), Felix-Hausdorff-Str. 2, 17489 Greifswald, Germany 3 Department of Mechanical and Aerospace Engineering, The George Washington University, Washington, DC 20052, USA; [email protected] 4 Plasma Engineering and Medicine Institute, Old Dominion University, Norfolk, VA 23508, USA; [email protected] * Correspondence: [email protected] (H.T.); [email protected] (S.B.); [email protected] (D.Y.) † Equally contributed as first authors.

Simple Summary: Cold physical plasma is a partially ionized gas generating various reactive oxygen and nitrogen species (ROS/RNS) simultaneously. ROS/RNS have therapeutic effects when applied to cells and tissues either directly from the plasma or via exposure to solutions that have been treated beforehand using plasma processes. This review addresses the challenges and opportunities of plasma-treated solutions (PTSs) for cancer treatment.

Abstract: Cold physical plasma is a partially ionized gas generating various reactive oxygen and



 nitrogen species (ROS/RNS) simultaneously. ROS/RNS have therapeutic effects when applied to cells and tissues either directly from the plasma or via exposure to solutions that have been treated Citation: Tanaka, H.; Bekeschus, S.; beforehand using plasma processes. This review addresses the challenges and opportunities of Yan, D.; Hori, M.; Keidar, M.; plasma-treated solutions (PTSs) for cancer treatment. These PTSs include plasma-treated cell culture Laroussi, M. Plasma-Treated Solutions (PTS) in Cancer Therapy. media in experimental research as well as clinically approved solutions such as saline and Ringer’s Cancers 2021, 13, 1737. https:// lactate, which, in principle, already qualify for testing in therapeutic settings. Several types of cancers doi.org/10.3390/cancers13071737 were found to succumb to the toxic action of PTSs, suggesting a broad mechanism of action based on the tumor-toxic activity of ROS/RNS stored in these solutions. Moreover, it is indicated that the Academic Editor: Pierre Busson PTS has immuno-stimulatory properties. Two different routes of application are currently envisaged in the clinical setting. One is direct injection into the bulk tumor, and the other is lavage in patients Received: 8 January 2021 suffering from peritoneal carcinomatosis adjuvant to standard chemotherapy. While many promising Accepted: 9 February 2021 results have been achieved so far, several obstacles, such as the standardized generation of large Published: 6 April 2021 volumes of sterile PTS, remain to be addressed.

Publisher’s Note: MDPI stays neutral Keywords: cold physical plasma; low-temperature plasma; nonthermal plasma; oncology; PAM; with regard to jurisdictional claims in plasma-activated medium; plasma medicine; reactive oxygen species; reactive nitrogen species published maps and institutional affil- iations.

1. Introduction Cancer is a devastating condition, and the second leading cause of death in Western Copyright: © 2021 by the authors. countries [1]. Despite ongoing improvements in cancer therapy, novel research lines are Licensee MDPI, Basel, Switzerland. warranted to improve the clinical efficacy of cancer treatments. A recent development This article is an open access article distributed under the terms and in oncology is increasingly focusing on combination therapies that tackle tumor cells via conditions of the Creative Commons several mechanisms, either simultaneously or consecutively. While many therapeutic ap- Attribution (CC BY) license (https:// proaches focus on targeted therapies and immuno-therapies, promising research motivated creativecommons.org/licenses/by/ continuously tackling tumors on a much broader scale involving cancer metabolism and 4.0/).

Cancers 2021, 13, 1737. https://doi.org/10.3390/cancers13071737 https://www.mdpi.com/journal/cancers Cancers 2021, 13, x FOR PEER REVIEW 2 of 20 Cancers 2021, 13, 1737 2 of 19

and reactive oxygen and nitrogen species (ROS/RNS) and their associated redox signaling pathwaysreactive oxygen[2–4]. and nitrogen species (ROS/RNS) and their associated redox signaling pathwaysCold physical [2–4]. plasma, which also goes by low-temperature plasma or nonthermal plasma,Cold is a physical partially plasma, ionized which gas generating also goes bya multitude low-temperature of ROS/RNS plasma simultaneously. or nonthermal Moreplasma, than is two a partially decades ionized ago, it was gas generatingproposed that a multitude these ROS/RNS of ROS/RNS can be used simultaneously. for thera- peuticMore purposes. than two Early decades work ago, focused it was on proposed plasma-based that these disinfection ROS/RNS of biologically can be used contam- for ther- inatedapeutic surfaces purposes. and Earlyliquids work [5,6], focused which on wa plasma-baseds later transferred disinfection to the ofmedical biologically field and con- woundtaminated decontamination surfaces and liquids[7,8]. In [ 5addition,6], which to was decontamination later transferred, it was to the soon medical realized field that and plasma-derivedwound decontamination ROS/RNS also [7,8]. targ In additionet eukaryotic to decontamination, cells by cell-intrinsic it was mechanisms soon realized [9,10]. that Moreover,plasma-derived a recent ROS/RNS randomized also targetclinical eukaryotic trial found cells that by cell-intrinsicthe disinfection mechanisms properties [9 ,10of ]. plasmaMoreover, treatment a recent are randomized negligible clinicalin promoting trial found wound that thehealing disinfection in a cohort properties of diabetic of plasma pa- tientstreatment [11]. areAs ROS/RNS negligible infollow promoting hormetic wound responses healing in inbiology a cohort [12], of diabeticit seems patients natural [11to ]. investigateAs ROS/RNS exaggerated follow hormetic ROS/RNS responses exposure in biologyfrom cold [12 physical], it seems plasmas natural as to anticancer investigate exaggerated ROS/RNS exposure from cold physical plasmas as anticancer agents. Plasma- agents. Plasma-based cancer research started more than a decade ago with promising in based cancer research started more than a decade ago with promising in vitro [13–15] and vitro [13–15] and in vivo studies [16,17] and, ever since, has generated significant interest in vivo studies [16,17] and, ever since, has generated significant interest in experimental in experimental oncology [18,19] with the first promising results being obtained in pallia- oncology [18,19] with the first promising results being obtained in palliative cancer patients tive cancer patients in recent years [20,21]. in recent years [20,21]. While the direct application of cold physical plasma to cells and tissues is promising While the direct application of cold physical plasma to cells and tissues is promising and approved in European dermatology centers [22], an increasing need has emerged to and approved in European dermatology centers [22], an increasing need has emerged utilize the plasma-derived ROS/RNS more flexibly as putative injections. One way to to utilize the plasma-derived ROS/RNS more flexibly as putative injections. One way achieve this is the treatment of liquids or solutions with plasma devices [23,24]. In this to achieve this is the treatment of liquids or solutions with plasma devices [23,24]. In process, the plasma-derived ROS/RNS are delivered from the plasma gas phase into the this process, the plasma-derived ROS/RNS are delivered from the plasma gas phase into liquid phase (Figure 1). As most ROS/RNS are short-lived by virtue of their nature, this the liquid phase (Figure1). As most ROS/RNS are short-lived by virtue of their nature, comesthis comes at the atexpense the expense of their of deterioration their deterioration [25], yet [25 leaving], yet leaving a delicate a delicate mixture mixture of long- of livedlong-lived ROS/RNS ROS/RNS that might that even might recombine even recombine or react or with react short-lived with short-lived species speciesagain when again inwhen contact in contactwith cells with [26]. cells Since [26 ].the Since idea theof investigating idea of investigating these liquids these is liquids a future is aclinical future application,clinical application, two routes two have routes been have proposed been proposed so far. The so first far. involves The first the involves injection the into injection the bulkinto tumor, the bulk while tumor, the second while therelates second to the relates lavage to of the the lavage peritoneal of the cavity peritoneal in the cavitydisease in ofthe disseminated disease of disseminated peritoneal carcinomatosis, peritoneal carcinomatosis, currently tackled currently with HIPEC tackled and with PIPEC HIPEC ther- and apyPIPEC [27]. therapy [27].

FigureFigure 1. 1. DirectDirect plasma plasma treatment treatment exposes exposes the the biological biological ta targetrget such such as as in in vitro vitro cells cells or or in in vivo vivo or or ex ex vivo tissue directly to the plasma gas phase. Plasma-treated solution (PTS), in turn, is generated vivo tissue directly to the plasma gas phase. Plasma-treated solution (PTS), in turn, is generated by by exposing a solution to the plasma gas phase and subsequently transferring this liquid to a tar- exposing a solution to the plasma gas phase and subsequently transferring this liquid to a target cell get cell culture in vitro or for injection in vivo. Reproduced from [28]. Copyright 2020 MDPI. culture in vitro or for injection in vivo. Reproduced from [28]. Copyright 2020 MDPI. 2.2. Terminology Terminology SuchSuch treated treated liquids liquids go byby severalseveral terms, terms, such such as as plasma-activated plasma-activated medium medium (PAM) (PAM) [29], [29],plasma-activated plasma-activated solution solution (PAS) (PAS) [30], plasma-stimulated[30], plasma-stimulated medium medium (PSM) [(PSM)31], plasma- [31], plasma-activatedactivated Ringer’s Ringer’s lactate lactate (PAL) (PAL) [32], plasma-treated [32], plasma-treated liquid liquid (PTL) (PTL) [33], [33], plasma-oxidized plasma-ox- idizedliquid liquid (POL) [(POL)27], plasma-treated [27], plasma-treated medium medium (PTM) [34 (PTM)], plasma-activated [34], plasma-activated water (PAW) water [35], (PAW)and plasma-activated [35], and plasma-activated acetic acid acetic Ringer’s acid solutionRinger’s (PAA)solution as (PAA) well as asplasma-activated well as plasma- activatedbicarbonate bicarbonate Ringer’s Ringer’s solution (PAB)solution [36 (PAB]. Here,) [36]. we Here, propose we harmonizingpropose harmonizing the nomenclature the no- menclaturein the field in of the plasma field of medicine plasma bymedicine using theby termusing plasma-treatedthe term plasma-treated solutions (PTSs)solutions for (PTSs)the following for the following reasons. reasons. First, the First, term the “solution” term “solution” per definitionem per definitionem includes includes all types all of typesliquids of thatliquids have that dissolved have dissolved organic or organic inorganic or inorganic compounds, compounds, which is the which case foris the all typescase forof all solutions types of used solutions in experimental used in experimental research aswell research as clinically as well accreditedas clinically solutions accredited [37 ]. Even though this is not entirely the case for plasma-treated water, the plasma treatment

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submerges reactive components generated in the ambient air such as nitrite and nitrate into the liquid, effectively generating a solution as well. Second, the term “treated” is a neutral expression describing that a plasma process modified the solution. Hence, every solution exposed to plasma is treated but not necessarily activated, as termed in many previous studies. Activated implies a biomedical response after exposure to such a solution. However, in the case of very short plasma treatment times or low energy, this is not necessarily the case, while at the biochemical level, trace amounts of modified organic or inorganic solvents and ROS/RNS might still be detectable. Third, the term medium should be avoided. Cell culture media differ to extreme extents, differences in inactivation properties with the same treatment times of about 10× were observed [38], discouraging a general utilization of the term “medium”. By contrast, we encourage specifying the type of solution immediately behind PTS—for instance, PTS-RL (Ringer’s lactate) or PTS-saline (sodium chloride). When using cell culture media PTSs, the exact chemical composition of the type of media should be given in the methods section along with the manufacturer and part number. This is the first important step of standardizing reports projected on this topic. Along similar lines, each study should contain a basic characterization of the essential types of ROS/RNS generated in the solution by the plasma source, together with information on evaporation (and its compensation) and changes in the pH. After all, the exact biochemical description of PTSs is vital in understanding and optimizing this approach towards clinical translation.

3. In Vitro Experiments of Plasma-Treated Solutions (PTSs) for Cancer Treatment It has been reported that PTSs selectively kill glioblastoma brain tumor cells against as- trocyte normal cells [23]. Antitumor effects of PAM have been demonstrated—for instance, in ovarian cancer cells [39], colon cancer cells [37,40], lung cancer cells [41,42], gastric cancer cells [43], pancreatic cancer cells [44–46], leukemia cells [47,48], melanoma cells [49], and squamous cell carcinoma [50]. Antitumor effects of plasma-treated Ringer’s lactate solution, plasma-treated acetic acid Ringer’s solution, and plasma-treated bicarbonate Ringer’s solution were investigated, and interestingly, the lactate and acetic acid solutions effectively killed ovarian cancer cells. In contrast, the plasma-treated bicarbonate solution did not [36]. These results suggest that plasma-treated lactate and acetate are important products for obtaining antitumor effects for these solutions. For cell culture media, reaction products are complex since they contain about 30 com- ponents. However, several components such as amino acids [31] and fetal bovine/calf serum, as well as other compounds such as pyruvate [51], have been investigated for their individual contributions to cytotoxic effects. In general, the drawback of using cell culture media is their complex composition, which disallows unambiguously identifying single or small groups of compounds mainly responsible for the toxicity observed. Moreover, there are many different types of standard cell culture mediums, such as RPMI and DMEM, and unfortunately, many of the differences relate to antioxidants as additives—e.g., pyruvate. If not appropriately referenced, this disallows comparison of the results obtained with plasma-treated media in the field. A comprehensive study comparing several types of media is lacking so far. One study comparing IMDM and RPMI media found a 10-fold difference in mediating cytotoxic effects [38], showing only the tip of the iceberg of how the interpretation of results across several studies may be challenging. Less drastic but still potent differences were recently suggested [52]. In terms of clinical translation, clinically approved solutions seem more promising for apparent reasons. Their composition is defined apart from standardized and quality-controlled production processes needed for medical application. In a previous study, we compared six clinically approved solutions (Table1) and their storability for their suitability as clinically relevant PTS compared to RPMI cell culture media and phosphate-buffered saline (PBS) [37]. We found sodium chloride and Ringer’s lactate to be especially promising agents for future research along more clinically relevant avenues. Cancers 2021, 13, 1737 4 of 19

Table 1. Chemical composition of six clinically approved solutions compared to phosphate-buffered saline (PBS) and RPMI cell culture medium containing 10% fetal bovine/calf serum (R10F). Reproduced from [37]. Copyright 2019 IEEE.

HES NaCl G-5 E153 Ri-Lac Gela PBS R10F 60 g/L hy- 9 g/500 mL Ringer’s Amino acids, 50 g/L 153 mval/L Gelatine 12 mM main component droxyethyl sodium solution with vitamins and glucose ions 40 g/L phosphate starch chloride 28 mmol/L 10% FCS pH-range 4.0–5.5 4.5–7.0 3.5–5.5 5.0–7.0 5.0–7.0 7.1–7.7 7.3–7.5 8.0 pH-treated 5.2 5.1 5.6 6.2 6.0 7.0 7.3 8.3 osmolarity (mOsm) 308 308 278 303 277 274 280 - acetions X amino acids X Ca X X X calcium XX hydrochloride-dihydrate calcium nitrate X carbohydrates X X X Cl X gelatine poly succinate X HCl X K X KCl X X X lactate X magnesium sulfate X magnesium X chlorid-hecyhydrat Mg X NaCl X X X X X X X phosphate XX protein X sodium acetate X X sodium hydroxide X vitamins X

3.1. Reactive Species in Plasma-Treated Solutions (PTSs) Cold physical plasma interacts with oxygen, nitrogen, and water in the air and gen- erates short-lived reactive species in the gas phase such as nitric oxide, ozone, hydroxyl radicals, singlet oxygen, and superoxide anion [53]. Long-lived species such as hydrogen peroxide, nitrite, and nitrate are major components in PTSs [25]. High concentrations of hydrogen peroxide are known to be cytotoxic [54]. For several years, Georg Bauer proposed a synergy between the different PTS components—namely, hydrogen peroxide and nitrite, especially when interacting with enzymes that have located a tumor cell membrane [55–57]. Peroxynitrite is produced from hydrogen peroxide and nitrite, followed by the primary singlet oxygen. It has been reported that the primary singlet oxygen causes inactivation of membrane-associated catalase, and hydrogen peroxide and peroxynitrite are produced continuously at the site of locally inactivated catalase, which leads to the generation of secondary singlet oxygen [26]. Other reactive species derived from solutes contribute to physiological responses in PTS-exposed cells (Figure2,[ 29]). For example, lactate in Ringer’s lactate solution is a crucial antitumor component when treated with cold physical plasma, and NMR analyses revealed that acetyl- and pyruvic acid-like groups are generated in PTSs (Ringer’s lactate) [36]. The specific cellular response in the nutrient-starved environment and the nutrient- rich environment may cause cancer cells to show drastically different responses to extracel- lular reactive species such as H2O2 [58]. The cytotoxicity of reactive species on mammalian cells was found to be dampened in one study when the PTS was made up of simple buffered solutions such as PBS [36,59]. Nevertheless, ROS/RNS are vital components in PTS. These can be monitored in living cells in vitro using fluorescent redox-sensitive reporter probes that can be analyzed by flow cytometry [54], microscopy [60], or microplate readers. The drawback of these probes is the lack of specificity towards individual types of ROS/RNS once entering the intracellular compartment, as reported many times [61,62]. Notwithstanding, they prove useful in estimating intracellular redox changes with ten- dencies towards some types of ROS over others. For instance, intracellular ROS are often detected using 5-(and-6)-chloromethyl- 20,70-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA), although it has been reported that its fluorescent product DCF Cancers 2021, 13, 1737 5 of 19

is not a direct consequence of ROS/RNS oxidation but rather an enzymatic product of intracellular oxidase. Direct plasma treatment and PTSs often induce intracellular ROS on cells, and the extent of this finding for PTS exposure depends on the type of solution used. For example, plasma-treated cell culture media were previously suggested to have stronger DCF signals compared to plasma-treated Ringer’s lactate in U251SP cells [63]. To obtain more details of intracellular ROS dynamics, 30-(p-aminophenyl) fluorescein (APF) and 30-(p-hydroxyphenyl) fluorescein (HPF) are frequently used probes in plasma medicine [46,54,60,64,65]. Intracellular hydroxyl radicals, peroxynitrite, and OCl- are de- tected using APF, while intracellular hydroxyl radicals and peroxynitrite are detected using HPF. In addition to these dyes, intracellular hydrogen peroxide, nitric oxide, peroxyni- trite, superoxide anion, and OCl- can be detected using other reagents in PTS-exposed HeLa cells to dissect putative intracellular ROS/RNS following exposure (Figure3)[ 60]). Up to 2 h after exposure, intracellular hydrogen peroxide, nitric oxide, and superoxide Cancers 2021, 13, x FOR PEER REVIEW 5 of 20 anion were dominant. Intracellular peroxynitrite was negligible. After 5 h, intracellular hydrogen peroxide, nitric oxide, and superoxide anion decreased, and intracellular perox- ynitrite increased. While these data are interesting, it nevertheless needs to be noted that directcold physical plasma exposure plasma, wasand foundNMR toanalyses be significantly revealed more that toxicacetyl- in U251and pyruvic tumor spheroids acid-like comparedgroups are to generated PTS (PBS) in treatment PTSs (Ringer’s [66]. lactate) [36].

FigureFigure 2.2.Reactive Reactive species species in in the the plasma plasma gas gas phase phase dissolve dissolve into into the the bulk bulk liquid liquid to subsequentlyto subsequently reach thereach cells the with cells potentially with potentially cytotoxic cytotoxic consequences. consequences. The example The example shows shows an in an vitro in vitrosetup. setup. Modified fromModified [29]. Copyrightfrom [29]. 2014Copyright IEEE. 2014 IEEE.

3.2. FactorsThe specific Affecting cellular the Anticancer response Efficacy in the nutr of Plasma-Treatedient-starved Solutionsenvironment (PTSs) and the nutrient- rich Theenvironment carriers ofmay these cause reactive cancer species, cells to sh suchow asdrastically cell culture different media, responses PBS, and to otherextra- solutionscellular reactive such as species lactate solutions,such as H2 areO2 [58]. important The cytotoxicity factors affecting of reactive the anticancer species on efficacy mamma- of PTSs.lian cells Specific was cancerfound to cell be lines dampened may be in vulnerable one study to when one PTS, the suchPTS was as medium, made up compared of simple tobuffered another solutions PTS, such such as PBS, as PBS as seen [36,59]. for theNevertheless, pancreatic tumorROS/RNS cells are PA-TU-8988T vital components cells and in glioblastomaPTS. These can cells be U87MGmonitored [58 ]in (Figure living4 cells). PTS in can vitro also using be combined fluorescent with redox-sensitive chemotherapy re- affectingporter probes the efficacy that can of PTS.be analyzed Using four by flow different cytometry human [54], pancreatic microscopy cancer [60], cell or lines microplate and the clinicallyreaders. The relevant drawback drugs of to these target probes those cells,is the cisplatinlack of specificity and gemcitabine, towards additiveindividual toxicity types withof ROS/RNS PTS was once found entering (Ringer’s the intracellular lactate, kINPen compartment, argon plasma as reported jet) in many both times 2D cultures [61,62]. andNotwithstanding, three-dimensional they multicellular prove useful tumors in es growntimating on intracellular the CAM of chickenredox changes embryos with (in ovo).tendencies [46]. A towards synergistic some action types of of PTS ROS (fully over supplemented others. For instance, RPMI, kINPen intracellular argon ROS plasma are jet)often and detected gemcitabine using was 5-(and-6)-chloromethyl- also confirmed in murine 2′,7′ pancreatic-dichlorodihydrofluorescein cancer cells and found diacetate, to be selectiveacetyl ester compared (CM-H2 toDCFDA), primary although murine fibroblasts it has been [67 reported]. However, that itits should fluorescent be noted product that suchDCF treatmentis not a direct also severely consequence affects of the ROS/RNS viability oxidation of human immunebut rather cells, an enzymatic such as monocytes product andof intracellular T cells [68], oxidase. while leaving Direct differentiated plasma treatment macrophages and PTSs largely often induce unaffected intracellular [69]. ROS on cells, and the extent of this finding for PTS exposure depends on the type of solution used. For example, plasma-treated cell culture media were previously suggested to have stronger DCF signals compared to plasma-treated Ringer’s lactate in U251SP cells [63]. To obtain more details of intracellular ROS dynamics, 3′-(p-aminophenyl) fluorescein (APF) and 3′-(p-hydroxyphenyl) fluorescein (HPF) are frequently used probes in plasma medi- cine [46,54,60,64,65]. Intracellular hydroxyl radicals, peroxynitrite, and OCl- are detected using APF, while intracellular hydroxyl radicals and peroxynitrite are detected using HPF. In addition to these dyes, intracellular hydrogen peroxide, nitric oxide, peroxyni- trite, superoxide anion, and OCl- can be detected using other reagents in PTS-exposed HeLa cells to dissect putative intracellular ROS/RNS following exposure (Figure 3) [60]). Up to 2 h after exposure, intracellular hydrogen peroxide, nitric oxide, and superoxide anion were dominant. Intracellular peroxynitrite was negligible. After 5 h, intracellular hydrogen peroxide, nitric oxide, and superoxide anion decreased, and intracellular per- oxynitrite increased. While these data are interesting, it nevertheless needs to be noted that direct plasma exposure was found to be significantly more toxic in U251 tumor sphe- roids compared to PTS (PBS) treatment [66].

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Figure 3.3. IntracellularIntracellular reactivereactive oxygenoxygen and and nitrogen nitrogen species species (ROS/RNS) (ROS/RNS) dynamics dynamics in in Plasma-Treated Plasma- SolutionTreated Solution (PTS)-exposed (PTS)-exposed HeLa cells. HeLa (a )cells. total (a RONS) total determinedRONS determined using CM-H2DCFDAusing CM-H2DCFDA fluorescent fluo- rescent stain reagent; (b) H2O2 determined using OxiVision fluorescent stain reagent; (c)•NO de- stain reagent; (b)H2O2 determined using OxiVision fluorescent stain reagent; (c) •NO determined termined using DAF-FM-DA fluorescent stain reagent; (d) •O2- determined using DHE fluorescent using DAF-FM-DA fluorescent stain reagent; (d) •O − determined using DHE fluorescent stain − − 2 stain reagent; (e) •OH, ONOO− , and− OCl determined using APF fluorescent stain reagent; (f) reagent; (e) •OH,− ONOO , and OCl determined using APF fluorescent stain reagent;− (f) •OH and •OH and− ONOO determined using HPF fluorescent stain reagent; (g−) ONOO determined using ONOONiSPY-3 fluorescentdetermined stain using reagent; HPF fluorescent (h) OCl− determined stain reagent; treatment (g) ONOO usingdetermined HySOx fluorescent using NiSPY-3 stain − fluorescentreagent. The stain symbols reagent; * and (h) # OCl indicatedetermined significan treatmentt differences using between HySOx PAM fluorescent and nontreatment stain reagent. and The symbolsbetween *PAM and #andindicate 24 h DMEM significant treatment. differences One betweensymbol; PAMp < 0.05, and double nontreatment symbols; and p < between0.01, triple PAM Cancers 2021, 13, x FOR PEER REVIEWandsymbols; 24 h DMEMp < 0.005. treatment. Modified One from symbol; [60]. Copyrightp < 0.05, double 2017 Wiley. symbols; p < 0.01, triple symbols; p <7 0.005.of 20

Modified from [60]. Copyright 2017 Wiley. 3.2. Factors Affecting the Anticancer Efficacy of Plasma-Treated Solutions (PTSs) The carriers of these reactive species, such as cell culture media, PBS, and other solu- tions such as lactate solutions, are important factors affecting the anticancer efficacy of PTSs. Specific cancer cell lines may be vulnerable to one PTS, such as medium, compared to another PTS, such as PBS, as seen for the pancreatic tumor cells PA-TU-8988T cells and glioblastoma cells U87MG [58] (Figure 4). PTS can also be combined with chemotherapy affecting the efficacy of PTS. Using four different human pancreatic cancer cell lines and the clinically relevant drugs to target those cells, cisplatin and gemcitabine, additive tox- icity with PTS was found (Ringer’s lactate, kINPen argon plasma jet) in both 2D cultures and three-dimensional multicellular tumors grown on the CAM of chicken embryos (in ovo). [46]. A synergistic action of PTS (fully supplemented RPMI, kINPen argon plasma jet) and gemcitabine was also confirmed in murine pancreatic cancer cells and found to FigurebeFigure selective 4.4. The comparedPTS composition composition to primary affects affects itsmurine its extent extent fibroblastsof of cytotoxicity. cytotoxicity. [67]. (a ( )a However,Pancreatic) Pancreatic cancerit cancer should cell cell linebe line notedPA- PA- TU-8988T.thatTU-8988T. such ((btreatmentb)) GlioblastomaGlioblastoma also cellcellseverely lineline U87MG.U87MG. affects Student’sStudent’s the viabilityt t-test-test was wasof human performed performed immune and and the the cells, significance significance such as is indicatedmonocytesis indicated as as ***and ***p < Tp 0.005. oxidative stress and induces apoptosis. The dynamics of gene expression analyses related to GADD45 signaling between PTS- (me- dium) and PTS-exposed (Ringer’s lactate) U251SP cells revealed that the latter only showed minimal effects in GADD45 signaling (Figure 5, [63]). These results are consistent with the results that PTS (medium) induced more ROS than PTS (Ringer’s lactate). Sur- vival and proliferation signaling pathways such as the PI3K–AKT pathway and the RAS– MAPK pathway were downregulated in PTS-exposed U251SP cells [73]. These results are also consistent with the fact that AKT inhibition induces GADD45α gene expression. GAD45α/p38 signaling pathway increases the activation of ATF3 and c-JUN transcription factors, which form the AP-1 complex.

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3.3. Intracellular Molecular Mechanism of Cancer Cell Death Induced by Plasma-Treated Solution (PTS) The transmembrane diffusion of reactive species may affect the cytotoxicity of PTS (cell culture medium) on cancer cells. One example is the study on aquaporins (AQPs) [70]. Knocking down of the gene encoding AQP8, one type of H2O2 channel in glioblastoma cells, reduced the cytotoxicity of PTS. To identify intracellular molecular mechanisms of cell death of PTS-exposed cells, many cell lines have been used. For example, U251SP, U87MG, LN229, and T98G are glioblastoma cell lines, and their genetic backgrounds are different. The TP53 gene is the wildtype in the U87MG cell line, and the PTEN gene is the wildtype in the LN229 cell line. Sensitivity against PTS is different among those cell lines [71]. Further studies are needed to elucidate what the effectiveness of specific types of PTS depends on. The ovarian cancer cell lines SKOV3, ES2, NOS2, and TOV21G have also been investigated [72]. Ovarian clear-cell carcinoma (CCC) is a subtype of epithelial ovarian carcinoma (EOC), and it is naturally chemoresistant and often associated with a poor prognosis. TOV21G was used as a CCC cell line. Direct plasma exposure and PTS induce apoptosis in cells. For example, cleaved Caspases 3 and 7 were detected in PTS-exposed (cell culture medium) U251SP cells [23] and PTS-exposed (Ringer’s lactate) U251SP cells [36]. However, intracellular molecular mechanisms of cell death are different between both exposure types [63]. Transcriptome microarray analyses of PTS-exposed U251SP suggested that PTS-induced gene expres- sion related to GADD45 signaling is activated by oxidative stress and induces apoptosis. The dynamics of gene expression analyses related to GADD45 signaling between PTS- (medium) and PTS-exposed (Ringer’s lactate) U251SP cells revealed that the latter only showed minimal effects in GADD45 signaling (Figure5,[ 63]). These results are consistent with the results that PTS (medium) induced more ROS than PTS (Ringer’s lactate). Survival and proliferation signaling pathways such as the PI3K–AKT pathway and the RAS–MAPK pathway were downregulated in PTS-exposed U251SP cells [73]. These results are also con- α Cancers 2021, 13, x FOR PEER REVIEWsistent with the fact that AKT inhibition induces GADD45α gene expression. GAD45 8/p38 of 20 signaling pathway increases the activation of ATF3 and c-JUN transcription factors, which form the AP-1 complex.

FigureFigure 5. 5.Differences Differences in in gene gene expression expression dynamics dynamics between between PTS- PTS- (medium, (medium, plasma-activated plasma-activated medium me- (“PAM”))dium (“PAM”)) and PTS-exposed and PTS-exposed (Ringer’s (Ringer’s lactate, plasma-activatedlactate, plasma-activated Ringer’s Ringer’s lactate (“PAL”)) lactate (“PAL”)) U251SP cells. RelativeU251SP mRNAcells. Relative expression mRNA of GADD45 expressionα ( aof), GADD45 βα (ba),), GADD45 ATF3 (c),β and (b), c-JUN ATF3 ((dc)), was and calculatedc-JUN (d) usingwas calculated qRT-PCR. using Reproduced qRT-PCR. from Reproduced [63]. from [63].

PTSPTS (medium)(medium) alsoalso inducesinduces caspase-independentcaspase-independent cellcell deathdeath inin A549 A549 nonsmall nonsmall lung lung cancercancer cellscells [41[41].]. InjuredInjured mitochondriamitochondria butbut not not caspase caspase 3/7 3/7 activationactivation werewere observedobserved inin PTS-exposed A549 cells. Bcl2 mRNA was significantly decreased and CHOP mRNA was induced in PTS-exposed A549 cells. The formation of poly ADPR was detected in the nu- clei of PTS-exposed A549 cells, and the accumulation of AIF surrounding the nucleus was detected in PTS-exposed A549 cells. PTS exposure elevated intracellular calcium ion con- centration by activating the nonselective cation channel TRPM2 in A549 cells. Based on these results, intracellular molecular mechanisms of cell death have been proposed (Fig- ure 6, [41]).

Figure 6. Intracellular molecular mechanisms of cell death in PTS-exposed (cell culture medium) A549 cells. Reproduced from [41]. Copyright 2014 S Elsevier.

PTS affects gene expression, signal transduction, and metabolic networks (Figure 7). The interface between PTS and the cell is the cell membrane, and PTS first affects proteins

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Figure 5. Differences in gene expression dynamics between PTS- (medium, plasma-activated me- dium (“PAM”)) and PTS-exposed (Ringer’s lactate, plasma-activated Ringer’s lactate (“PAL”)) Cancers 2021, 13, 1737 U251SP cells. Relative mRNA expression of GADD45α (a), GADD45β (b), ATF3 (c), and c-JUN (d) 8 of 19 was calculated using qRT-PCR. Reproduced from [63].

PTS (medium) also induces caspase-independent cell death in A549 nonsmall lung cancerPTS-exposed cells [41]. A549 Injured cells. mitochondria Bcl2 mRNA but was not significantly caspase 3/7 activation decreased were and observed CHOPmRNA in was PTS-exposedinduced in PTS-exposedA549 cells. Bcl2 A549mRNA cells. was significantly The formation decreased of poly and ADPR CHOP wasmRNA detected was in the inducednuclei of in PTS-exposedPTS-exposed A549 A549 cells. cells, The and formation the accumulation of poly ADPR of was AIF detected surrounding in the nu- the nucleus cleiwas of detected PTS-exposed in PTS-exposed A549 cells, and A549 the accu cells.mulation PTS exposure of AIF surrounding elevated intracellular the nucleus was calcium ion detected in PTS-exposed A549 cells. PTS exposure elevated intracellular calcium ion con- concentration by activating the nonselective cation channel TRPM2 in A549 cells. Based centration by activating the nonselective cation channel TRPM2 in A549 cells. Based on theseon these results, results, intracellular intracellular molecular molecular mechanisms mechanisms of cell death of have cell been death proposed have been (Fig- proposed ure(Figure 6, [41]).6,[ 41]).

FigureFigure 6. 6. IntracellularIntracellular molecular molecular mechanisms mechanisms of cell death of cell in death PTS-exposed in PTS-exposed (cell culture (cell medium) culture medium) A549A549 cells. cells. Reproduced Reproduced from from [41] [.41 Copyright]. Copyright 2014 2014S Elsevier. S Elsevier. Cancers 2021, 13, x FOR PEER REVIEW 9 of 20 PTSPTS affects affects gene gene expression, expression, signal signal transd transduction,uction, and metabolic and metabolic networks networks (Figure 7). (Figure 7). TheThe interface interface between between PTS PTS and and the cell the is cell the is cell the membrane, cell membrane, and PTS and first PTS affects first proteins affects proteins suchsuch as as receptors, receptors, ion ion channels, channels, transporte transporters,rs, and lipids and lipidson the oncell the membrane. cell membrane. Then, PTS- Then, PTS- affectedaffected proteins proteins and and lipids lipids alter alter the thesignal signal transduction transduction network. network. Such alterations Such alterations affect affect genegene expression expression and and metabolic metabolic networks. networks. Systematic Systematic analyses analyses such as suchtranscriptomics, as transcriptomics, proteomics, and and metabolomics metabolomics are arepowerful powerful tools toolsto understand to understand intracellular intracellular molecular molecular mechanismsmechanisms of of PTS-exposed PTS-exposed cells. cells.

FigureFigure 7. 7. GeneGene expression expression network, network, signal signal transducti transductionon network, network, and metabolic and metabolic network affected network affected byby PTS. PTS.

Moreover, PTS (fully supplemented cell culture medium, kINPen argon plasma jet) was shown to affect mitochondrial dynamics in melanoma cells, critically synergizing with complex I inhibitors to augment cancer cell death (Figure 8) [74].

Figure 8. Inhibition of cytochrome C oxidase and the addition of PTS (fully supplemented cell culture medium, kINPen argon plasma jet) leads to an increase in superoxide anions (red) in the mitochondrial matrix that results in loss of MMP and subsequent ATP depletion. This finally leads to an energy crisis and cell death. Reproduced from [74]. Copyright 2018 Springer Nature.

3.4. Some Guidelines to Make PTS Extending the plasma treatment time (dose) is the most straightforward strategy to enhance the anticancer efficacy of PTSs [23,36,75–77]. Several general principles to im- prove the efficacy of PTS (cell culture medium) have been demonstrated by regulating

Cancers 2021, 13, x FOR PEER REVIEW 9 of 20

such as receptors, ion channels, transporters, and lipids on the cell membrane. Then, PTS- affected proteins and lipids alter the signal transduction network. Such alterations affect gene expression and metabolic networks. Systematic analyses such as transcriptomics, proteomics, and metabolomics are powerful tools to understand intracellular molecular mechanisms of PTS-exposed cells.

Cancers 2021, 13, 1737 9 of 19 Figure 7. Gene expression network, signal transduction network, and metabolic network affected by PTS.

Moreover, PTSPTS (fully(fully supplementedsupplemented cellcell cultureculture medium,medium, kINPenkINPen argon plasmaplasma jet) waswas shownshown to to affect affect mitochondrial mitochondrial dynamics dynamics in melanomain melanoma cells, cells, critically critically synergizing synergizing with complexwith complex I inhibitors I inhibitors to augment to augment cancer cancer cell death cell death (Figure (Figure8)[74]. 8) [74].

Figure 8. InhibitionInhibition of of cytochrome cytochrome C Coxidase oxidase and and the the addition addition of PTS of PTS (fully (fully supplemented supplemented cell cell cultureculture medium, kINPen argon argon plasma plasma jet) jet) leads leads to to an an increase increase in in superoxi superoxidede anions anions (red) (red) in inthe the mitochondrial matrixmatrix thatthat results results in in loss loss of of MMP MMP and and subsequent subsequent ATP ATP depletion. depletion. This This finally finally leads leads to anto an energy energy crisis crisis and and cell cell death. death. Reproduced Reproduced from from [74 [74]]. Copyright. Copyright 2018 2018 Springer Springer Nature. Nature.

3.4. Some Guidelines to Make PTS Cancers 2021, 13, x FOR PEER REVIEW 10 of 20 Extending the plasma plasma treatment treatment time time (dose) (dose) is is the the most most straightforward straightforward strategy strategy to toenhance enhance the the anticancer anticancer efficacy efficacy of ofPTSs PTSs [23,36,75–77]. [23,36,75–77 Several]. Several general general principles principles to im- to improveprove the the efficacy efficacy of of PTS PTS (cell (cell culture culture medi medium)um) have have been been demonstrated by regulatingregulating threethree treatmenttreatment factors:factors: thethe gapgap betweenbetween thethe plasmaplasma sourcesource andand thethe solutionsolution surface,surface, thethe contactingcontacting areaarea betweenbetween bulk bulk plasma plasma and and the the surface surface (Figure (Figure9), and9), and the the volume volume of solution. of solu- Thesetion. These principles principles have have recently recently been testedbeen tested in other in other PTSs, PTSs, such such as PBS as byPBS other by other groups groups [78]. A[78]. larger A larger containing containing area area between between the bulkthe bulk plasma plasma and and the the solution solution surface surface will will result result in ain higher a higher reactive reactive species species concentration concentration in in the the PTS PTS [75 [75].]. For For example, example, the the PTS PTS mademade inin aa wellwell of a 6-well plate is much more more toxic toxic to to cancer cancer cells cells than than the the PTS PTS made made in in a awell well of of a a96-well 96-well plate. plate. Additionally, Additionally, a shorter a shorter gap gap betw betweeneen the theplasma plasma source source and and the solution the solution also alsogenerates generates a more a more toxic toxic PTS PTSwith with a higher a higher reactive reactive species species concentration. concentration. The The stronger stronger ca- capacitypacity of ofPTS PTS can can also also be beachieved achieved by bydecr decreasingeasing the the solution solution volume. volume. Furthermore, Furthermore, the thebasic basic operational operational parameters parameters of plasma of plasma can canalso also modulate modulate the theefficacy efficacy of PTSs. of PTSs. For For in- instance, the discharge voltage can noticeably affect the H2O2 generation in PTS, which stance, the discharge voltage can noticeably affect the H2O2 generation in PTS, which fur- further affects the anticancer performance [79]. ther affects the anticancer performance [79].

Figure 9.9. The well size-dependent ROS/RNS accumulation accumulation in in PTS. PTS. ( (aa)) Relative Relative RNS RNS concentration concentration in 1 mL of PTS. (b) Relative H2O2 concentration in 1 mL of PTS. Student’s t-tests were performed in 1 mL of PTS. (b) Relative H2O2 concentration in 1 mL of PTS. Student’s t-tests were performed and the significance compared with the first bar is indicated as *** p < 0.005. Reproduced from and the significance compared with the first bar is indicated as *** p < 0.005. Reproduced from [75]. [75]. Copyright 2015 Springer Nature. Copyright 2015 Springer Nature. It is necessary to emphasize that some media components are very reactive to the plasma-derived ROS/RNS. For example, fetal bovine/calf serum (FBS/FCS), a standard component of a typical cell culture media, is highly reactive to ROS. FBS/FCS weakens the efficacy of PTS on glioblastoma cells [80]. In addition, pyruvate is also reactive with H2O2 in PTS [41]. Cysteine is a common component in nearly all standard cell culture media. Among all 20 amino acids, however, cysteine is most reactive with ROS [80]. Shortly, an ideal PTS should avoid containing FBS, pyruvate, and cysteine if maximizing ROS/RNS concentrations is desired.

3.5. The Storage of Plasma-Treated Solutions (PTSs) Most media used for cell culture purposes have ideal storage temperatures ranging between 2 and 8 °C. Therefore, an ideal PTS made of cell culture media should also be stably stored in such a temperature range. However, the degradation of PTSs (cell culture media) after the storage has been investigated across a broader range of temperatures. The anti- cancer species and corresponding efficacy of such PTSs will gradually degrade during the storage over a wide temperature range for less than just 1 day [41,75,80–82]. For H2O2, its concentration also drastically decreased after storage, which was just proportional to the decreased efficacy of such PTSs [82]. As a typical example, Mohades et al. observed that a stored PTS (cell culture medium) lost some of its potency in killing cancer cells at 12 h post- PTS exposure (Figure 10) [82]. PTS was stored at room temperature for 1, 8 and 12 h before its use. Comparing the cell viability outcome of SCaBER treated with stored PTS indicated that toxic efficacy of PTS decreased with increasing storage time [82]. However, the plasma exposure time used to generate such PTSs also plays an important role. Reduction in the efficacy of PTS is more significant for shorter compared to longer exposure times, suggesting that ROS/RNS concentrations remain high enough after storage for PTS gen- erated with longer plasma exposure times. PTS (PBS, kINPen argon plasma jet) was also found to be storable for up to three weeks at −20 °C without a decline of activity [40]. In a structured comparison of 8 different

Cancers 2021, 13, 1737 10 of 19

It is necessary to emphasize that some media components are very reactive to the plasma-derived ROS/RNS. For example, fetal bovine/calf serum (FBS/FCS), a standard component of a typical cell culture media, is highly reactive to ROS. FBS/FCS weakens the efficacy of PTS on glioblastoma cells [80]. In addition, pyruvate is also reactive with H2O2 in PTS [41]. Cysteine is a common component in nearly all standard cell culture media. Among all 20 amino acids, however, cysteine is most reactive with ROS [80]. Shortly, an ideal PTS should avoid containing FBS, pyruvate, and cysteine if maximizing ROS/RNS concentrations is desired.

3.5. The Storage of Plasma-Treated Solutions (PTSs) Most media used for cell culture purposes have ideal storage temperatures ranging between 2 and 8 ◦C. Therefore, an ideal PTS made of cell culture media should also be stably stored in such a temperature range. However, the degradation of PTSs (cell culture media) after the storage has been investigated across a broader range of temperatures. The anticancer species and corresponding efficacy of such PTSs will gradually degrade during the storage over a wide temperature range for less than just 1 day [41,75,80–82]. For H2O2, its concentration also drastically decreased after storage, which was just proportional to the decreased efficacy of such PTSs [82]. As a typical example, Mohades et al. observed Cancers 2021, 13, x FOR PEER REVIEW 11 of 20 that a stored PTS (cell culture medium) lost some of its potency in killing cancer cells at 12 h post-PTS exposure (Figure 10)[82]. PTS was stored at room temperature for 1, 8 and 12 h before its use. Comparing the cell viability outcome of SCaBER treated with typesstored of PTSsolutions, indicated including that toxic 6 clinically efficacy ofapproved PTS decreased variants, with several increasing solutions storage were time dis- [82]. qualifiedHowever, in terms the plasma of storage exposure at −20 time °C [37]. used Sodium to generate chloride such (saline) PTSs also and plays Ringer’s an important lactate at role.0.9% Reductiongave the best in theresults efficacy in term of PTSs of storability. is more significant The temperature for shorter of − compared20 °C was tochosen longer asexposure this is most times, realistic suggesting for a quick that ROS/RNS on-site-surgery concentrations use-on-demand remain highsetting. enough after storage for PTS generated with longer plasma exposure times.

FigureFigure 10. 10. EffectivenessEffectiveness of ofstored/aged stored/aged PTS PTS (cell (cell cultur culturee medium) medium) to induce to induce cell celldeath death in SCaBER in SCaBER cellscells for for storage storage times times of 1, of 8, 1,and 8, 12 and h, 12when h, whenmeasuring measuring metabolic metabolic activity activity another another 12 h later. 12 Re- h later. producedReproduced from from[82].[ 82].

WithoutPTS (PBS, knowing kINPen the argon exact plasma degradation jet) was mechanisms, also found to beonly storable storage for in up low-tempera- to three weeks ◦ tureat −environments20 C without (e.g., a decline −80 °C of in activity a freezer [40 or]. In−150 a structured °C in liquid comparison nitrogen) ofcan 8 inhibit different PTSs’ types degradationof solutions, over including long periods 6 clinically [36,41,81]. approved For cell variants, culture several medium, solutions a degradation were disqualified mecha- ◦ nismin terms has been of storage recently at proposed.−20 C[37 Comparing]. Sodium chloridethe H2O2 (saline)concentrations and Ringer’s in PTS lactate (cell culture at 0.9% medium vs. PBS) after storage at −25, 8 and 22 °C, medium components and some amino acids were found to be responsible for the observations [31]. Cysteine and methionine mainly cause the degradation of H2O2 PTS (cell culture medium) (Figure 11). The PTS without cysteine and methionine was much more stable than the PTS that included both amino acids during storage at these temperatures [31]. Moreover, it was found that PTS (PBS, kINPen argon plasma jet) could induce apop- tosis in human [83] and murine pancreatic cancer cells in vitro and in vivo [45]. Interest- ingly, murine primary fibroblasts were less affected. The toxic effects on the cancer cells were not affected by adding several amino acids (alanine, leucine, tryptophan, tyrosine, valine; all 20 mM), suggesting their scavenging capability to be minor when added after plasma treatment in this particular setting. By contrast, cysteine added to PBS before plasma treatment did affect cells [84], suggesting that the oxidation generated by the plasma treatment on this amino acid conserves some of the initial toxicity of the short- lived ROS/RNS. Stability and effects of the gap and gas flow rates on ROS/RNS generation in PTS (PBS, kINPen argon plasma) have been investigated previously (Figure 12) [85].

Cancers 2021, 13, 1737 11 of 19

gave the best results in terms of storability. The temperature of −20 ◦C was chosen as this is most realistic for a quick on-site-surgery use-on-demand setting. Without knowing the exact degradation mechanisms, only storage in low-temperature environments (e.g., −80 ◦C in a freezer or −150 ◦C in liquid nitrogen) can inhibit PTSs’ degradation over long periods [36,41,81]. For cell culture medium, a degradation mecha- nism has been recently proposed. Comparing the H2O2 concentrations in PTS (cell culture medium vs. PBS) after storage at −25, 8 and 22 ◦C, medium components and some amino acids were found to be responsible for the observations [31]. Cysteine and methionine mainly cause the degradation of H2O2 PTS (cell culture medium) (Figure 11). The PTS Cancers 2021, 13, x FOR PEER REVIEWwithout cysteine and methionine was much more stable than the PTS that included12 both of 20

amino acids during storage at these temperatures [31].

FigureFigure 11. 11.Cysteine Cysteine and and methionine methionine mainly mainly cause cause the the degradation degradation of of PTS PTS (cell(cell cultureculture medium)medium) atat ◦ ◦ 2222°CC and and 8 8 °C.C. ((aa)) TheThe HH22O2 concentrationconcentration in in PTS PTS (PBS) (PBS) contai containingning a aspecific specific component component during during the storage at 22◦ °C. (b) The H2O2 concentration in the PTS (cys/met-free DMEM, cys-free DMEM, the storage at 22 C. (b) The H2O2 concentration in the PTS (cys/met-free DMEM, cys-free DMEM, met-freemet-free DMEM, DMEM, and and standard standard DMEM) DMEM) during during thethe storagestorage atat 88 ◦°C.C. ((cc)) TheThe anticanceranticancer effect of the PTS (cys/met-free DMEM, cys-free DMEM, met-free DMEM, and standard DMEM) after storage at PTS (cys/met-free DMEM, cys-free DMEM, met-free DMEM, and standard DMEM) after storage at 8 °C. Student’s t-tests were performed and the significance is indicated as * p < 0.05 and *** p < 8 ◦C. Student’s t-tests were performed and the significance is indicated as * p < 0.05 and *** p < 0.005. 0.005. Reproduced from [31]. Copyright 2016 Springer Nature. Reproduced from [31]. Copyright 2016 Springer Nature.

Moreover, it was found that PTS (PBS, kINPen argon plasma jet) could induce apopto- sis in human [83] and murine pancreatic cancer cells in vitro and in vivo [45]. Interestingly, murine primary fibroblasts were less affected. The toxic effects on the cancer cells were not affected by adding several amino acids (alanine, leucine, tryptophan, tyrosine, valine; all 20 mM), suggesting their scavenging capability to be minor when added after plasma treat- ment in this particular setting. By contrast, cysteine added to PBS before plasma treatment did affect cells [84], suggesting that the oxidation generated by the plasma treatment on this amino acid conserves some of the initial toxicity of the short-lived ROS/RNS. Stability and effects of the gap and gas flow rates on ROS/RNS generation in PTS (PBS, kINPen argon plasma) have been investigated previously (Figure 12)[85]. Figure 12. The effect of the gap, gas flow rate, plasma treatment time, the occurrence of discharges at the liquid surface, and the stability of a PTS (PBS, kINPen argon plasma jet) on the concentra- tions of NO2- and H2O2 in PTS and on the anticancer capacity of PTSs for three different cancer cell lines. Reproduced from [85]. Copyright 2017 Springer Nature.

4. In Vivo Experiments of Plasma-Treated Solutions (PTSs) for Cancer Treatment 4.1. The Anticancer Efficacy of Plasma-Treated Solutions (PTSs) The effectiveness of PTSs (RPMI cell culture medium) has been tested using xenograft mouse models and animal disease models. For example, chemoresistant ovarian cancer cells (NOS2TR) and its parental ovarian cancer cells (NOS2) were subcutaneously injected into the bilateral flank of mice [39]. The PTS-exposed group of mice received PTS by sub- cutaneous injection in each side, three times a week. The control group of mice received an untreated medium in the same way. PTS significantly reduced the growth of both

Cancers 2021, 13, x FOR PEER REVIEW 12 of 20

Figure 11. Cysteine and methionine mainly cause the degradation of PTS (cell culture medium) at 22°C and 8 °C. (a) The H2O2 concentration in PTS (PBS) containing a specific component during the storage at 22 °C. (b) The H2O2 concentration in the PTS (cys/met-free DMEM, cys-free DMEM, met-free DMEM, and standard DMEM) during the storage at 8 °C. (c) The anticancer effect of the PTS (cys/met-free DMEM, cys-free DMEM, met-free DMEM, and standard DMEM) after storage at Cancers 2021, 13, 1737 12 of 19 8 °C. Student’s t-tests were performed and the significance is indicated as * p < 0.05 and *** p < 0.005. Reproduced from [31]. Copyright 2016 Springer Nature.

Figure 12. The effect of the gap, gas flow rate, plasma treatment time, the occurrence of discharges at Figure 12. The effect of the gap, gas flow rate, plasma treatment time, the occurrence of discharges the liquid surface, and the stability of a PTS (PBS, kINPen argon plasma jet) on the concentrations of at the- liquid surface, and the stability of a PTS (PBS, kINPen argon plasma jet) on the concentra- NO2 and H2O2 in PTS and on the anticancer capacity of PTSs for three different cancer cell lines. tions of NO2- and H2O2 in PTS and on the anticancer capacity of PTSs for three different cancer cell Reproduced from [85]. Copyright 2017 Springer Nature. lines. Reproduced from [85]. Copyright 2017 Springer Nature. 4. In Vivo Experiments of Plasma-Treated Solutions (PTSs) for Cancer Treatment 4.4.1. In TheVivo Anticancer Experiments Efficacy of ofPlasma-Treated Plasma-Treated Solutions (PTSs) (PTSs) for Cancer Treatment 4.1. TheThe Anticancer effectiveness Efficacy of PTSs of Plasma-Treated (RPMI cell culture Solutions medium) (PTSs) has been tested using xenograft mouseThe models effectiveness and animal of PTSs disease (RPMI models. cell cultur Fore medium) example, has chemoresistant been tested using ovarian xenograft cancer mousecells (NOS2TR) models and and animal its parental disease ovarian models. cancer For cells example, (NOS2) chemoresistant were subcutaneously ovarian injected cancer Cancers 2021, 13, x FOR PEER REVIEW 13 of 20 cellsinto (NOS2TR) the bilateral and flank its parental of mice ovarian [39]. The cancer PTS-exposed cells (NOS2) group were of subcutaneously mice received injected PTS by intosubcutaneous the bilateral injection flank of in mice each [39]. side, The three PTS-exposed times a week. group The control of mice group received of mice PTSreceived by sub- cutaneousan untreated injection medium in each in the side, same three way. times PTS a week. significantly The control reduced group the of growth mice received of both parentalanparental untreated and and chemoresistant chemoresistantmedium in the ovarian ovarian same cancerway. cancer PTS cells cells significantly (Figure (Figure 13,13,[ [39]). 39reduced]). Similar Similar the results resultsgrowth were were of alsoboth also observedobserved in in pancreatic pancreatic cancer cancer cells. cells.

FigureFigure 13. 13. AntitumorAntitumor effect effect of PTS of PTS (RPMI, (RPMI, “PAM”) “PAM”) in mice in mice with withNOS2 NOS2 and NOS2TR and NOS2TR (paclitaxel-resistant) (paclitaxel-resistant) cell lines. cell lines. ((AA,B,B):): The The macroscopic macroscopic observation observation of of nude nude mice mice bearing subcutaneous NOS2 ((A)) andand NOS2TRNOS2TR ((BB)) tumorstumors onon bothboth flanks. flanks.Mice were Miceinjected were injected with NOS2 with NOS2 and NOS2TR and NOS2TR cells andcells then and receivedthen received medium medium alone alone or NEAPP-AM. or NEAPP-AM. A total A total of 0.4 of mL 0.4 of mLmedium of medium or NEAPP-AM or NEAPP-AM was administered was administered locally locally into bothinto sidesboth sides of mice of mice three three times times a week. a week. All mice All mice were were sacrificed sacri- at ficed29 days at 29 after days implantation. after implantation. Green Green arrowheads arrowheads indicate indicate tumor tumor formation. formation. (C,D ):(C Time-dependent,D): Time-dependent changes changes in the in tumorthe tumor volume in xenografted models are shown, medium alone or NEAPP-AM. Each point on the line graph represents volume in xenografted models are shown, medium alone or NEAPP-AM. Each point on the line graph represents the mean the mean tumor volume (mm3) for each group on a particular day after implantation, and the bars represent SD. * p < tumor volume (mm3) for each group on a particular day after implantation, and the bars represent SD. * p < 0.05, ** p < 0.01 0.05, ** p < 0.01 versus control. Reproduced from [39]. Copyright 2013 PLOS. versus control. Reproduced from [39]. Copyright 2013 PLOS.

ToTo examine examine the the effectiveness effectiveness of of the the antitu antitumormor effects effects of of PTS PTS (cell (cell culture culture medium), medium), enhancedenhanced green green fluorescentfluorescent protein-taggedprotein-tagged GCIY GCIY gastric gastric cancer cancer (GCIY-EGFP) (GCIY-EGFP) cell-induced cell-in- ducedperitoneal peritoneal carcinomatosis carcinomatosis was investigated was investig [78ated]. The [78]. intraperitoneal The intraperit administrationoneal administration of PTS ofor PTS untreated or untreated medium medium (control) (control) was performed was performed on days on 1 days to 4 and 1 to days 4 and 8 days to 11. 8 The to 11.in The vivo inimaging vivo imaging assays assays were performedwere performed on days on 1,da 8ys and 1, 8 15and and 15 itand was it was demonstrated demonstrated that that PTS PTStreatment treatment inhibited inhibited the formationthe formation of peritoneal of peritoneal metastases metastases (Figure (Figure 14,[ 14,78]). [78]).

Figure 14. Efficacy of the intraperitoneal administration of PTS (“PAM”) in vivo. Reproduced from [78]. Copyright 2017 Springer Nature.

In another study, RPMI cell culture medium (5 mL) was treated with plasma of the clinically accredited argon plasma jet kINPen MED for 10 min. Although a large number of

Cancers 2021, 13, x FOR PEER REVIEW 13 of 20

parental and chemoresistant ovarian cancer cells (Figure 13, [39]). Similar results were also observed in pancreatic cancer cells.

Figure 13. Antitumor effect of PTS (RPMI, “PAM”) in mice with NOS2 and NOS2TR (paclitaxel-resistant) cell lines. (A,B): The macroscopic observation of nude mice bearing subcutaneous NOS2 (A) and NOS2TR (B) tumors on both flanks. Mice were injected with NOS2 and NOS2TR cells and then received medium alone or NEAPP-AM. A total of 0.4 mL of medium or NEAPP-AM was administered locally into both sides of mice three times a week. All mice were sacri- ficed at 29 days after implantation. Green arrowheads indicate tumor formation. (C,D): Time-dependent changes in the tumor volume in xenografted models are shown, medium alone or NEAPP-AM. Each point on the line graph represents the mean tumor volume (mm3) for each group on a particular day after implantation, and the bars represent SD. * p < 0.05, ** p < 0.01 versus control. Reproduced from [39]. Copyright 2013 PLOS.

To examine the effectiveness of the antitumor effects of PTS (cell culture medium), enhanced green fluorescent protein-tagged GCIY gastric cancer (GCIY-EGFP) cell-in- duced peritoneal carcinomatosis was investigated [78]. The intraperitoneal administration Cancers 2021, 13, 1737 of PTS or untreated medium (control) was performed on days 1 to 4 and days 8 to 11.13 The of 19 in vivo imaging assays were performed on days 1, 8 and 15 and it was demonstrated that PTS treatment inhibited the formation of peritoneal metastases (Figure 14, [78]).

FigureFigure 14. 14. EfficacyEfficacy of the of theintraperitoneal intraperitoneal administration administration of PTS of(“PAM”) PTS (“PAM”) in vivo. Reproducedin vivo. Reproduced from [78]from. Copyright [78]. Copyright 2017 Springer 2017 Springer Nature. Nature.

InIn another another study, study, RPMI cell cell culture medium medium (5 (5 mL) mL) was treated with plasma of the clinicallyclinically accredited accredited argon argon plasma plasma jet jet kINPen kINPen MED MED for for 10 10 min. min. Although Although a large a large number number of of injections were needed (1–2 dozens), an apparent reduction in peritoneal carcinomatosis was identified in a syngeneic orthotopic model of pancreatic cancer using such a PTS (Figure 15)[86]. The model has the benefit that the immune system is not neglected as the tumor cells are of the same genetic background as the mouse strain into which the tumors were inoculated. The findings were accompanied by intratumoral apoptosis (qualitatively and quantitatively shown using TUNEL staining), as well as a decreased intratumoral proliferation (qualitatively and quantitatively showed using Ki67 staining) in the metastatic nodules. PTS exposure led to a significantly improved overall survival of the mice in the PTS group compared to the vehicle medium control. Detailed analysis of the tumor microenvironment (TME) revealed an increased influx of macrophages into the tumor nodules that were repeatedly exposed to PTS in vivo, along with more T cells as well as an increase in calreticulin (CRT) [87], a marker of the immunogenic cancer cell death [88]. In vitro experiments confirmed the selectivity of this approach when comparing the PDA6606 pancreatic cancer cells against murine primary fibroblasts. In a recent report, we used a PTS (PBS, kINPen argon plasma jet) to investigate its toxicity in four different gastrointestinal tumor cell lines in vitro. In addition to analyzing the liquid chemistry, we observed the onset of ICD in CT26 colorectal cancer cells in vitro. Strikingly, this was accompanied by a massive antitumor action of PTS (NaCl, kINPen argon plasma jet) in vivo and an increase in intratumoral macrophages as well as T cells (Figure 16), which corroborates findings with direct plasma exposure in vivo [89]. This is the first study using a clinically approved solution (NaCl) together with a clinically approved plasma source (kINPen) in a clinically realistic setting (only PTS stored at −20 ◦C was used to mimic the clinical situation in which PTS likely would be generated before application as the generation time of the PTS of 1 h was too long to be generated ad hoc during surgical procedures) in an immune-competent (syngeneic, immuno-competent) animal model with the tumors growing at the location where they also would appear clinically (orthotopic, peritoneal cavity of metastatic colon cancer) [40]. Cancers 2021, 13, x FOR PEER REVIEW 14 of 20

injections were needed (1–2 dozens), an apparent reduction in peritoneal carcinomatosis was identified in a syngeneic orthotopic model of pancreatic cancer using such a PTS (Figure 15) [86]. The model has the benefit that the immune system is not neglected as the tumor cells are of the same genetic background as the mouse strain into which the tumors were inoculated. The findings were accompanied by intratumoral apoptosis (qualitatively and quantitatively shown using TUNEL staining), as well as a decreased intratumoral prolifer- ation (qualitatively and quantitatively showed using Ki67 staining) in the metastatic nod- ules. PTS exposure led to a significantly improved overall survival of the mice in the PTS group compared to the vehicle medium control. Detailed analysis of the tumor microen- vironment (TME) revealed an increased influx of macrophages into the tumor nodules that were repeatedly exposed to PTS in vivo, along with more T cells as well as an increase Cancers 2021, 13, 1737 in calreticulin (CRT) [87], a marker of the immunogenic cancer cell death [88]. In14 vitro of 19 experiments confirmed the selectivity of this approach when comparing the PDA6606 pancreatic cancer cells against murine primary fibroblasts.

Cancers 2021, 13, x FOR PEER REVIEWFigureFigure 15.15. EfficacyEfficacy ofof thethe repeatedrepeated intraperitoneal intraperitoneal administration administration (a ()a of) of PTS PTS (RPMI (RPMI medium medium15 without with-of 20

supplements)out supplements) against against syngeneic, syngeneic, orthotopic, orthotopic, disseminated disseminated pancreatic pancreatic cancer cancer analyzed analyzed using using MR- ImagingMR-Imaging (I–II) (I–II) and macroscopicand macroscopic imaging imaging (III–IV) (III–IV) (b) and(b) and its quantification its quantification (c) as(c)well as well the the absolute abso- tumorlute tumor weights weights (d). ( Eachd). Each triangle triangle represents represents one one mouse. mouse. * *p p< < 0.05,0.05, **** p < 0.01. 0.01. Reprinted Reprinted with with animalpermission model from with [86] the. Copyright tumors growing2017 Springer at the Nature. location where they also would appear clin- icallypermission (orthotopic, from [86 peritoneal]. Copyright cavity 2017 Springer of metastatic Nature. colon cancer) [40]. In a recent report, we used a PTS (PBS, kINPen argon plasma jet) to investigate its toxicity in four different gastrointestinal tumor cell lines in vitro. In addition to analyzing the liquid chemistry, we observed the onset of ICD in CT26 colorectal cancer cells in vitro. Strikingly, this was accompanied by a massive antitumor action of PTS (NaCl, kINPen argon plasma jet) in vivo and an increase in intratumoral macrophages as well as T cells (Figure 16), which corroborates findings with direct plasma exposure in vivo [89]. This is the first study using a clinically approved solution (NaCl) together with a clinically ap- proved plasma source (kINPen) in a clinically realistic setting (only PTS stored at −20 °C was used to mimic the clinical situation in which PTS likely would be generated before application as the generation time of the PTS of 1 h was too long to be generated ad hoc during surgical procedures) in an immune-competent (syngeneic, immuno-competent)

FigureFigure 16. 16. EfficacyEfficacy of the repeated intraperitonealintraperitoneal administration administration of of PTS PTS (NaCl, (NaCl, kINPen kINPen argon argon plasma plasmajet) against jet) against syngeneic, syngeneic, orthotopic orthotopic and disseminated and disseminated colorectal colorectal in mice in mice (a) and (a) itsand absolute its absolute tumor tumorweights weights (b). Each (b). triangleEach triangle represents represents one mouse. one mouse. *** p ***< 0.001. p < 0.001. Reproduced Reproduced from from [40]. [40] Copyright. Copy- right2019 2019 Springer Springer Nature. Nature.

AsAs concluded concluded from from above, above, the the oxidative oxidative stre stressss inflicted inflicted upon upon tumor tumor cells cells using using PTSs PTSs hashas an an immuno-modulatory immuno-modulatory dimension. dimension. The The exte extentnt of ofthis this dimension dimension is underexplored is underexplored at theat themoment. moment. The The main main idea idea is isthat that the the PTS-induced PTS-induced tumor tumor cell cell death death is is immunogenic, immunogenic, therebythereby promoting promoting the the immune immune system system to to recognize recognize dying dying tumor tumor cells cells in in an an inflammatory inflammatory contextcontext with with subsequent subsequent induction induction and and prom promotionotion of ofantitumor antitumor immunity. immunity. The The exact exact re- quirementsrequirements for for PTS PTS to toinduce induce such such an an effect effect remain remain to to be be elucidated. elucidated. However, However, from from the the inin vivo vivo studiesstudies mentioned mentioned above, above, it it is is clear clear that that PTS PTS exposure exposure of of gastrointestinal tumor tumor burdenburden increases increases influx influx of of professional professional antige antigen-presentingn-presenting cells cells such such as dendritic as dendritic cells cellsand macrophages. It is also possible that the immune cells present within the TME will be directly by PTS. As mentioned above, T cells severely succumb to oxidation-induced tox- icity. Data are intratumoral immune cells and their cell death after PTS exposure has not been reported so far. Another point is that the anticancer mechanisms of PTSs are of a general nature—it is uncertain as to whether they are more potent in some tumor entities over others. Along similar lines, great heterogeneity is observed within individual tumor entities, which is related to different mutational loads, variation in cell types within the TME, and signaling pathway deregulation. The current response rates to conventional drugs and immuno- therapy show that responders and nonresponders can be stratified according to these fac- tors, and it seems likely that PTS therapy will face similar drawbacks. However, progress of the PTS regimen to clinics is absent at the moment, leaving such aspects to be debated in the future. Comparative preclinical studies that incorporate different tumor entities or cell types with different traits of the same entity would greatly facilitate a much-needed gain in the knowledge in the field of PTSs. Such studies should also elaborate on the “dos- ing” of PTS—i.e., whether hormetic effects are observed that might show subtoxic effects at low doses.

4.2. The Safety of Plasma-Treated Solutions (PTSs) The safety of a PTS (water) in immune-deficient nude mice was investigated using this PTS by oral lavage treatment [35]. The acute toxicity test results showed that the PTS had no lethal effect or other acute toxicity, even when it was made with a 15 min plasma treatment time. After two weeks of exposure, the PTS did not cause significant changes in mice’s body weights and survival status. The major organs, including the heart, liver, spleen, lung and kidney, did not show observable changes. Liver function, renal function, electrolytes, glucose metabolism and lipid metabolism were also not affected by PTS

Cancers 2021, 13, 1737 15 of 19

and macrophages. It is also possible that the immune cells present within the TME will be directly by PTS. As mentioned above, T cells severely succumb to oxidation-induced toxicity. Data are intratumoral immune cells and their cell death after PTS exposure has not been reported so far. Another point is that the anticancer mechanisms of PTSs are of a general nature— it is uncertain as to whether they are more potent in some tumor entities over others. Along similar lines, great heterogeneity is observed within individual tumor entities, which is related to different mutational loads, variation in cell types within the TME, and signaling pathway deregulation. The current response rates to conventional drugs and immunotherapy show that responders and nonresponders can be stratified according to these factors, and it seems likely that PTS therapy will face similar drawbacks. However, progress of the PTS regimen to clinics is absent at the moment, leaving such aspects to be debated in the future. Comparative preclinical studies that incorporate different tumor entities or cell types with different traits of the same entity would greatly facilitate a much- needed gain in the knowledge in the field of PTSs. Such studies should also elaborate on the “dosing” of PTS—i.e., whether hormetic effects are observed that might show subtoxic effects at low doses.

4.2. The Safety of Plasma-Treated Solutions (PTSs) The safety of a PTS (water) in immune-deficient nude mice was investigated using this PTS by oral lavage treatment [35]. The acute toxicity test results showed that the PTS had no lethal effect or other acute toxicity, even when it was made with a 15 min plasma treatment time. After two weeks of exposure, the PTS did not cause significant changes in mice’s body weights and survival status. The major organs, including the heart, liver, spleen, lung and kidney, did not show observable changes. Liver function, renal function, electrolytes, glucose metabolism and lipid metabolism were also not affected by PTS treatment. Only blood neutrophils and mononuclear cells were found to be slightly increased [35]. The repeated application of PTSs (cell culture medium, kINPen argon plasma jet) in the peritoneal cavity of tumor-bearing mice suffering from peritoneal pancreatic carcinomatosis was tolerated well without visible side effects. The relative frequencies of several blood parameters were not affected in the PTS group compared to the vehicle group. This was the case for neutrophils, monocytes, lymphocytes, T helper cells, cytotoxic T cells, B cells, and NK cells. For the non-nucleated blood cells (erythrocytes and thrombocytes), similar counts and volumes were determined in both groups. The quantification of six different chemokines and cytokines from nonstimulated and LPS-stimulated splenocytes of both groups also did not show any differences [86]. Hence, it can be concluded that the repeated application of a PTS (RPMI cell culture medium) does not cause any apparent harm in C57BL/6 mice in vivo.

5. Conclusions Plasma-treated solutions (PTSs) show potent antitumor efficacy in many cancer cell lines in vitro and in vivo. Cutting-edge research currently addresses the immuno- modulating effects of the exposure of PTS to cancer cells and immune cells of the tumor microenvironment. A future step is to increase the translation of this promising field into clinics using solutions accredited for administration in humans. Along those lines, large bulk plasma-generators and ISO-standardized processes are needed to generate sufficient amounts of PTS in a reproducible and quality-controlled manner.

Author Contributions: All authors contributed to conceptualization, writing—original draft prepa- ration, writing—review and editing, and visualization. All authors have read and agreed to the published version of the manuscript. Funding: The research of SB is funded by the German Federal Ministry of Education and Research (grant numbers 03Z22DN11, 03Z22Di1, 03COV06A, and 03Z22D511), the European Social Fund (ESF, Cancers 2021, 13, 1737 16 of 19

grant number ESF/14-BM-A55-0006/18), the German Research Foundation (DFG, grant number AOBJ 669606), the Comprehensive Cancer Center (CCC) Mecklenburg-Western-Pomerania (Ger- many), and the Stiftung Tumorforschung Kopf-Hals (Germany). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest.

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