Author's Accepted Manuscript

Plasma in Cancer Treatment

Jürgen Schlegel, Julia Köritzer, Veronika Box-hammer

PII: S2212-8166(13)00020-6DOI: http://dx.doi.org/10.1016/j.cpme.2013.08.001Reference: CPME11

To appear in: Clinical Plasma Medicine

Received date: 17 June 2013Revised date: 19 August 2013Accepted date: 28 August 2013

Cite this article as: Jürgen Schlegel, Julia Köritzer, Veronika Boxhammer,Plasma in Cancer Treatment, Clinical Plasma Medicine, http://dx.doi.org/10.1016/j.cpme.2013.08.001

This is a PDF file of an unedited manuscript that has been accepted forpublication. As a service to our customers we are providing this early version ofthe manuscript. The manuscript will undergo copyediting, typesetting, andreview of the resulting galley proof before it is published in its final citable form.Please note that during the production process errors may be discovered whichcould affect the content, and all legal disclaimers that apply to the journalpertain.

www.elsevier.com/locate/cpme

Title:

Plasma in Cancer Treatment

Authors Names and Affiliation:

Jürgen Schlegel, Julia Köritzer and Veronika Boxhammer

Division of Neuropathology

Institute of Pathology

Technische Universität München

Ismaninger Str. 22

D-81675 München

E-Mail-Adresses:

Jürgen Schlegel <schlegel@tum.de>

Julia Köritzer <julia.koeritzer@googlemail.com>

Veronika Boxhammer <boxhammer@mpe.mpg.de>

Corresponding Author:

Jürgen Schlegel, Phone +49-89-4140 6112, E-Mail: schlegel@tum.de

Abstract

Plasma oncology, i.e. the use of cold atmospheric plasma (CAP) for the treatment of tumours

is a new field in plasma medicine. The results of several studies that are summarized within

this review show that CAP is effective against tumour cells both in vitro and in vivo. It has

been shown, that CAP in low concentration is able to stop tumour cells growing, to induce

cell death in higher concentrations and that this is more effective than standard therapies.

Moreover, first results indicate that CAP seems to be selective for cancer cells since it is more

effective in tumour cells than in normal non-neoplastic cells. The current developments in this

field have fuelled the hope that CAP could be an interesting new therapeutic approach in the

treatment of cancer.

Keywords

plasma medicine, plasma oncology, cold atmospheric plasma, cancer therapy, tumour cells,

selectivity

1. Introduction

Although a relatively new field in plasma medicine, the use of cold atmospheric plasma

(CAP) on tumour cells has attracted a great deal of attention. The results of several groups

have fuelled the hope that CAP could be an interesting new therapy opportunity in the

treatment of cancer. It has been shown in vitro, that CAP in low concentration was able to

stop tumour cells growing, to induce cell death in higher concentrations and that this was

more effective than some standard treatments including radiation and chemotherapy.

Moreover, first results indicated that CAP seemed to be selective for cancer cells since it was

more effective in tumour cells than in normal non-neoplastic cells.

There are some difficulties to review plasma in oncology, too. The different groups working

in this field used different plasma devices with completely different plasma chemistries and

cell lines derived from different tumour tissues. Furthermore, most results were obtained in

cell culture. To this day there are only the very first in vivo studies. We will therefore

summarize the data available in this new field of plasma medicine in a chronological order.

2. The early years

The first results of plasma treatment of mammalian cells were reported by the group of Eva

Stoffels almost 10 years ago. Although not obtained in tumorigenic cells their results are

important since they established the basic effects of cold plasma on mammalian cells. In

addition, immortalized non-neoplastic cells share many aspects in their cellular biology and

growth behaviour with tumour cell lines. Using a plasma needle device the Stoffels group

showed that fibroblasts and vascular cells detached from the surface of culture dishes in a

time dependent manner. The detachment occurred in a circumscribed area near to the plasma

impact. Applying higher powers they observed decreased viability of the cells [1,2]. Similar

results were obtained in human tumour cells derived from non-small cell lung cancer

(NSCLC) [3]. In a subsequent study, Stoffels and co-workers confirmed their results and

showed the delayed onset of cell death in vascular cells, and that the mode of cell death was

dose dependent: low doses of plasma resulted in apoptosis and high doses led to necrosis [4].

Interestingly, the presence of the cell culture medium as a liquid overlay inhibited the

detachment of the cells. The authors concluded, that the detachment is a physical process

caused by the short-living components in the plasma.

These results were corroborated in tumour cells by many others. Yonson et al. [5] used cells

derived from human liver cancer and demonstrated that detached cells were viable and

reattached after a period of time, indicating that cell death mechanisms initiated by plasma

treatment were different from the effects on cell adhesion. In human melanoma cells down-

regulation of integrins and focal adhesion kinase (FAK) following plasma treatment and

preceded detachment [6] were shown. In a more recent study the down-regulation of E-

cadherin and the epidermal growth factor (EGF) receptor has been observed in human

keratinocytes [7]. These plasma properties also seemed to affect cellular motility [8].

Although this effect was observed in primary fibroblasts, the results could also be important

in cancer research, since migration and infiltration are critical issues in tumour growth and

metastasis.

The cell death mechanisms induced by treatment with CAP were investigated in more detail

in different cell lines derived from human melanoma [6,9], liver cancer [10], breast cancer

[11], and more recently in cell lines derived from brain tumours [12] and leukemia [13]. It

could be confirmed that apoptosis is the leading mechanism at lower doses. Apoptosis in

pathological conditions, including intended therapy effects, shows some similarities with

programmed cell death during development [14]. It consists of characteristic morphological

changes, including cell shrinkage, nuclear fragmentation and chromosomal condensation.

Most importantly, apoptotic cell fragments are engulfed and removed by phagocytosis.

Therefore, the release of cellular fragments toxic to the surrounding tissue and inflammatory

changes that are common in necrosis, do not occur.

Several studies addressed the molecular mechanisms caused by plasma. Leduc and co-

workers [15] investigated the uptake of dextrans of different sizes (3 - 70 kDa) into HeLa

cells after plasma treatment. Using this approach they concluded that plasma induced

generation of pores of a maximum radius below 6.5 nm in the cell membranes. They also

investigated the direct effects of plasma on DNA using plasmid DNA that was transfected

into the cells after the treatment. In another approach Lupu et al. [16,17] showed the

importance of oxygen and postulated that reactive oxygen species (ROS) mediate the cellular

effects of plasma by blocking the outward channels in multi drug resistance protein-

overexpressing human colon cancer cells.

In accordance with these data the activation of intracellular stress pathways, that are involved

in the cellular reactions to different adverse conditions, could be demonstrated in another

panel of colon cancer cell lines [18].

Almost all of the results from the first period of mammalian cell research in plasma medicine

were published in physical journals. Although the medical aspects of this new therapy option

were also highlighted by some review articles [19,20] it was just within the last 2 years that

plasma treatment was brought to a broader readership of medical and specialized oncological

journals.

3. The current developments in "plasma oncology"

The first in vivo anti-cancer treatment experiments were reported by Vandamme et al. [21,22]

using human U87 glioblastoma cells as heterotopic subcutaneous xenotransplants in nude

mice. Glioblastoma is the most common and most aggressive human brain tumour. It is

highly resistant against standard treatment including radiotherapy and chemotherapy. CAP

treatment for 5 consecutive days showed a marked anti-tumour effect resulting from a

significant reduction in tumour volume and a consequent elongation of survival times. In a

subsequent study [23] the authors showed that these effects were mainly due to induction of

apoptosis and to a lesser extent to cell cycle arrest. They implicated ROS as the major

mediators of the therapeutic effects by fluorescence probes and by pre-treatment with ROS-

scavengers. In another in vivo experiment Keidar and co-workers demonstrated the effects of

CAP therapy in a syngenic mouse melanoma model and a heterotopic human bladder cancer

xenograft model. In both circumstances a single treatment led to a significant reduction of

tumour size and a consecutive elongation of survival time [24]. These results were

corroborated recently in a heterotopic mouse model using neuroblastoma cells [25] and in an

orthotopic pancreas carcinoma model [26]. In a different approach, Pertecke et al. [27] used a

tumour chorio-allantoic model for the investigation of CAP effects in pancreas carcinoma

cells. This organoid model, in which tumour cells are implanted into eggs, addresses several

aspects of in vivo tumour growth e.g. angiogenesis. All in vivo experiments showed beneficial

effects of CAP treatment on different tumour cell types and that these effects are mainly

mediated by apoptosis. The cellular response seemed to be mediated by the free radicals

generated by the plasma including ROS and RNS.

These results are in accordance with in vitro data obtained in other tumours including human

breast, cervical and liver cancer [28-30]. In an effort to further elucidate the cascade of

molecular events that follow plasma treatment, Kalghatgi and co-workers analysed human

breast cancer cells, and showed dose-dependent apoptosis upon CAP treatment. The amount

of DNA damage detected by the phosphorylated histone variant H2AX, that is recruited to

DNA damage foci, was neither significantly affected by the exclusion of charged particles nor

mediated by the UV content. Using dilution-experiments they postulated that the cellular

effects are mediated by peroxidation of amino acids in the cell culture medium [29]. Next, the

authors demonstrated that DNA damage is induced by intracellular reactive species. The

phosphorylation of H2AX seemed mainly mediated by the ataxia teleangiectatica related

protein ATR and not by ataxia teleangiectatica mutated (ATM) that is primarily involved in

the reaction to IR and H2O2. ROS also mediated the plasma effects in human liver cancer cells,

where a significant increase of lipid peroxidation products were detected [30]. Further, it has

been shown that intracellular ROS may lead to mitochondrial dysfunction [28] [31]. Plasma

treatment induced a decrease in mitochondrial transmembrane potential (MMP) and

consequently mitochondrial enzymatic dysfunction and morphological alterations. In a

discovery driven approach, Keidar and Schmidt and their co-workers investigated alterations

in the gene expression patterns after plasma treatment and observed a broad range of

differentially expressed genes in response to the therapy [24,32].

Recently, an issue of plasma therapy evolved that could be particularly interesting for

oncologists. It has been shown, that CAP treatment seems to be more effective in cancer cells

than in non-neoplastic cells providing the basis for a selective treatment. Using a co-culture

approach of melanoma cells and normal keratinocytes it has been shown that plasma

treatment induced a growth arrest after short treatment and a significant cell death after longer

treatment and that these effects were much more pronounced in the tumour cells [33]. In a

separate treatment approach CAP induced apoptosis in melanoma and colon cancer cells to a

much greater extend when compared with normal macrophages treated in the same schedule

[34]. In a complex analysis Keidar et al. analysed different tumor types and compared their

response with normal non-neoplastic cells. Again, plasma effects were much more

pronounced in the tumour cells [24]. These data were corroborated using tumour cells derived

from ovarian cancer, lung cancer, melanoma and glioblastoma [31,35-37]. The results of these

experiments also shed new light on the initial experiments obtained on non-neoplastic cells

and might explain some of the differences in the reactions to the plasma treatment. These

differences are, however, solely quantitative and not qualitative.

4. Where do we stand?

After a decade of "plasma oncology" a broad spectrum of different tumours cells has been

treated, including carcinomas, skin cancer and brain tumours. In all different tumour types

CAP was effective indicating that the effects of plasma seem to be uniform and are not

restricted to a particular tumour type. This is remarkable since normal non-neoplastic cells

seem to react in a more restrictive way, indicating that there are cell type-specific differences

in the cellular reaction to CAP. Therefore, it is most important to elucidate the molecular

basis of the plasma effects. So far, the different cellular mechanisms that are involved in this

process are not fully understood. Because of the uniform reaction of the tumour cell types it

seems likely that the same pathways are involved in the different tumour types.

It has been shown by the vast majority of the experiments summarized in this review that the

mechanism of cell death in mid-range plasma doses is apoptosis and necrosis at high doses.

However, the reactions to low plasma doses are less clear. This point is important since the

distribution of the therapeutic plasma concentrations within a tumour tissue could not be

expected to be maximal in all regions. Lower doses will reach individual cells at the edges of

the treatment zone and in the depth of the tissue. The data of two recent articles highlight this

particular important issue in cancer treatment. Arndt and co-workers showed the induction of

cellular senescence in human melanoma cells after CAP treatment in low doses [38] and our

own group demonstrated a significant cell cycle arrest in human glioblastoma cells [39].

These observations could also explain the tumour specific effects of CAP. Due to their

enhanced proliferation, tumour cells show a different cell cycle distribution than normal cells.

In accordance with this aspect it has been shown that tumour cells seem to be most vulnerable

to plasma treatment in the S-phase of the cell cycle [40].

Another issue involves the cellular effects induced by plasma that lead to cell cycle arrest and

apoptosis. The majority of reports showed intracellular accumulation of ROS using

fluorescence probes or direct measures after plasma treatment. Moreover, plasma effects

could be enhanced by blocking detoxifying membrane channels [16] and could be inhibited

by ROS scavangers [28,41]. Several groups used the phosphorylation of the histone variant

H2AX to detect the accumulation of DNA damage. H2AX is phosphorylated by the

Phosphatidyl inositol-3' kinases ATM, ATR and DNA dependent protein kinase (DNA-PK) at

places of DNA double strand breaks and stalled replication forks. In one report [29] it was

also demonstrated that H2AX phosphorylation seemed to be mediated by ATR and not by

ATM indicating that plasma effects might act primarily on DNA replication, a point of view

that would be in accordance with its effects on S-phase cells [40]. It is less clear if ROS affect

the DNA by oxidative damage or other mechanisms. Although an accumulation of lipid

peroxidation products has been documented after plasma treatment [30] this seems to be

unlikely to be the cause of DNA damage since blockade of lipid peroxidation had no effect on

H2AX activation [29]. Oncologically important, plasma effects were more pronounced in

several tumour types than established chemotherapy [38,39]. Therefore, the accumulation of

ROS directly induced by plasma or other mechanisms, might open a novel rationale of tumour

therapy.

Another scientific field is the clarification of the sources of ROS. Plasma effects including

ROS generation and activation of H2AX could also be achieved by the separate treatment of

culture media without cells and subsequent transfer onto the cells [29]. This scenario would

indicate that CAP generates toxic intermediates in the liquid phase around the cells in vitro.

However, this experimental setting is highly artificial, since solid tumours are not distributed

in a liquid phase. In other experiments a direct treatment in the absence of culture media was

utilized with comparable results [39]. In both settings the cellular surface takes the centre

stage in mediating plasma effects. Although lipid peroxidation doesn't seem to play a role in

CAP effects to DNA damage, short-term ROS effects at the cell membrane by peroxidation of

membrane lipids could mediate the plasma effects from the surface to the cytoplasm of

tumour cells. Therefore, ROS present in the microenvironment of the cell must play an

important role. The demonstration of nanoholes in the cell membrane after plasma treatment

would present another hypothesis in such a model [15]. So far, the component(s) of the

plasma cocktail responsible for the cellular effects are not completely defined. Charged

particles and UV have been excluded by several experiments. Another critical issue is the

penetration depth of plasma. It has been shown that the penetration into tissue is approx.

50�m [27]. It is not clear, how the CAP treatment could then reach the tumours in the

subcutaneous transplantation experiments [22,24]. The successful therapy might indicate

effects to the microenvironment or bystander effects that demand further elucidation.

5. Prerequisites for further development

Plasma oncology is a growing scientific field with several important open questions. The

answers will be the prerequisite for the further development to a serious medical treatment of

cancer. A standardized measurement of the capacities of the different plasma devices and the

resulting plasma chemistry would be helpful for a better comparability of the different

approaches [42]. It is absolutely necessary to develop orthotopic approaches as experimental

tools for the further investigations since the in vitro models are highly artificial, including the

unresolved question of the importance of the liquid phase for the plasma effects. To date there

is no "ideal tumour" for CAP treatment, although surface tumours including skin cancer or

colon tumours seem to be good candidates for a medical application in oncology. However,

other tumour types should not be excluded from further experiments because the scientific

field is in an early phase and the complete resolution of the biological effects of CAP

treatment might open new approaches to the treatment of "untypical" tumours. A very

important issue is the development of appropriate devices for medical use. First experiments

with endoscopic devices have been successfully performed [26,43] but many novel

applications could be imaginable highlighting the importance of a close interdisciplinary

cooperation between oncologists, biologists, physicists, chemists and engineers. It is most

likely that plasma could enter a place in multimodal adjuvant tumour treatment as part of a

combination therapy. First results indicate, that plasma could enhance the effects of standard

chemotherapy regimens even in resistant tumour cells and may restore chemosensitivity

[26,37,39]. These data highlight another interesting field in plasma oncology: Plasma could

be used to mediate drug delivery and drug uptake by tumour cells. The generation of

nanoholes in the cell membrane and/or other biochemical reactions at the cell surface could

enhance the uptake and the drug action of chemotherapeutical compounds.

6. Conclusion

Plasma oncology, i.e. the use of cold atmospheric plasma (CAP) for the treatment of tumours

is a novel field in plasma medicine. The results of several studies that are summarized within

this review show that CAP is effective against tumour cells both in vitro and in vivo. Plasma

treatment induces cell cycle arrest in low doses and apoptosis in medium doses. These effects

seem to be mediated by reactive species, mainly reactive oxygen species (ROS). The current

developments in this field show the prospects of this new approach by the construction of new

plasma devices for medical applications including endoscopic devices and combined

approaches in the adjuvant tumour therapy including plasma mediated drug delivery and drug

action.

Conflict of interest statement

The authors disclose any financial and personal relationships with other people or

organisations that could inappropriately influence their work.

References [1] Kieft IE, Broers JL, Caubet-Hilloutou V, Slaaf DW, Ramaekers FC, et al. Electric

discharge plasmas influence attachment of cultured CHO K1 cells. Bioelectromagnetics 2004; 25: 362-368

[2] Kieft IE, Darios D, Roks AJM, Stoffels E. Plasma treatment of mammalian vascular cells: a quantitative description. IEEE Transactions on Plasma Science 2005; 33: 771-775

[3] Stoffels E, Kieft IE, Sladek REJ, Bedem LJMvd, Laan EPvd, et al. Plasma needle for in vivomedical treatment: recent developments and perspectives. Plasma Sources Science and Technology 2006; 15: S169-S180

[4] Stoffels E, Roks AJM, Deelman LE Delayed Effects of Cold Atmospheric Plasma on Vascular Cells. Plasma Process Polym 2008; 5: 599-605

[5] Yonson S, Coulombe S, Leveille V, Leask RL. Cell treatment and surface functionalization using a miniature atmospheric pressure glow discharge plasma torch. J Phys D: Appl Phys 2006; 39: 3508-3513

[6] Kim G-C, Lee HJ, Shon C-H. The Effects of Micro plasma on Melanoma (G361) Cancer Cells. J Korean Phys Soc 2009; 54: 625-632

[7] Haertel B, Hahnel M, Blackert S, Wende K, von Woedtke T, et al. Surface molecules on HaCaT keratinocytes after interaction with non-thermal atmospheric pressure plasma. Cell Biol Int 2012; 36: 1217-1222

[8] Shashurin A, Keidar M, Bronnikov S, Jurjus RA, Stepp MA. Living tissue under treatment of cold plasma atmospheric jet. Appl Phys Lett 2008; 93: 181501

[9] Fridman G, Shereshevsky A, Jost MM, Brooks AD, Fridman A, et al. Floating Electrode Dielectric Barrier Discharge Plasma in Air Promoting Apoptotic Behavior in Melanoma Skin Cancer Cell Lines. Plasma Chem Plasma Process 2007; 27: 163-176

[10] Zhang X, Li M, Zhou R, Feng K, Yang S. Ablation of liver cancer cells in vitro by a plasma needle. Appl Phys Lett 2008; 93: 021502

[11] Kim SJ, Chung TH, Bae SH, Leem SH. Induction of apoptosis in human breast cancer cells by a pulsed atmospheric pressure plasma jet. Appl Phys Lett 2010; 97: 023702

[12] Kaushik NK, Uhm H, Choi EH. Micronucleus formation induced by dielectric barrier discharge plasma exposure in brain cancer cells. Appl Phys Lett 2012; 100: 84102

[13] Barekzi N, Laroussi M. Dose-dependent killing of leukemia cells by low-temperature plasma. J Phys D: Appl Phys 2012; 45: 422002

[14] Wyllie AH. "Where, O death, is thy sting?" A brief review of apoptosis biology. Mol Neurobiol 2010; 42: 4-9

[15] Leduc M, Guay D, Leask RL, Coulombe S. Cell permeabilization using a non-thermal plasma. New J Phys 2009; 11: 115021

[16] Lupu AR, Georgescu N, Calugaru A, Cremer L, Szegli G, et al. The effects of cold atmospheric plasma jets on B16 and COLO320 tumoral cells. Roum Arch Microbiol Immunol 2009; 68: 136-144

[17] Lupu AR, Georgescu N. Cold atmospheric plasma jet effects on V79-4 cells. Roum Arch Microbiol Immunol 2010; 69: 67-74

[18] Kim CH, Bahn JH, Lee SH, Kim GY, Jun SI, et al. Induction of cell growth arrest by atmospheric non-thermal plasma in colorectal cancer cells. J Biotechnol 2010; 150: 530-538

[19] Laroussi M. Low-temperature plasmas for medicine? IEEE Trans Plasma Sci 2009; 37: 714-725

[20] Fridman G, Friedman G, Gutsol A, Shekhter AB, Vasilets VN, et al. Applied Plasma Medicine. Plasma Process Polymer 2008; 5: 503-533

[21] Vandamme M, Robert E, Pesnel S, Barbosa E, Dozias S, et al. Antitumor Effect of Plasma Treatment on U87 Glioma Xenografts: Preliminary Results. Plasma Process Polymers 2010; 7: 264-273

[22] Vandamme M, Robert E, Dozias S, Sobilo J, Lerondel S, et al. Response of Human Glioma U87 Xenografted on Mice to Non Thermal Plasma Treatment. Plasma Medicine, 2011; 1: 27–43

[23] Vandamme M, Robert E, Lerondel S, Sarron V, Ries D, et al. ROS implication in a new antitumor strategy based on non-thermal plasma. Int J Cancer 2012; 130: 2185-2194

[24] Keidar M, Walk R, Shashurin A, Srinivasan P, Sandler A, et al. Cold plasma selectivity and the possibility of a paradigm shift in cancer therapy. Br J Cancer 2011; 105: 1295-1301

[25] Walk RM, Snyder JA, Srinivasan P, Kirsch J, Diaz SO, et al. Cold atmospheric plasma for the ablative treatment of neuroblastoma. J Pediatr Surg 2013; 48: 67-73

[26] Brulle L, Vandamme M, Ries D, Martel E, Robert E, et al. Effects of a non thermal plasma treatment alone or in combination with gemcitabine in a MIA PaCa2-luc orthotopic pancreatic carcinoma model. PLoS One 2012; 7: e52653

[27] Partecke LI, Evert K, Haugk J, Doring F, Normann L, et al. Tissue Tolerable Plasma (TTP) induces apoptosis in pancreatic cancer cells in vitro and in vivo. BMC Cancer 2012; 12: 473

[28] Ahn HJ, Kim KI, Kim G, Moon E, Yang SS, et al. Atmospheric-pressure plasma jet induces apoptosis involving mitochondria via generation of free radicals. PLoS One 2011; 6: e28154

[29] Kalghatgi S, Kelly CM, Cerchar E, Torabi B, Alekseev O, et al. Effects of non-thermal plasma on mammalian cells. PLoS One 2011; 6: e16270

[30] Yan X, Xiong ZL, Zou F, Zhao SS, Lu XP, et al. Plasma-Induced Death of HepG2 Cancer Cells: Intracellular Effects of Reactive Species. Plasma Process Polymers 2012; 9: 59-66

[31] Panngom K, Baik KY, Nam MK, Han JH, Rhim H, et al. Preferential killing of human lung cancer cell lines with mitochondrial dysfunction by nonthermal dielectric barrier discharge plasma. Cell Death Dis 2013; 4: e642

[32] Schmidt A, Wende K, Bekeschus S, Bundscherer L, Barton A, et al. Non-thermal plasma treatment is associated with changes in transcriptome of human epithelial skin cells. Free Radic Res. 2013; 47: 577-592

[33] Zirnheld J, Zucker SN, DiSanto TM, Berezney R, Etemadi K. Nonthermal Plasma Needle: Development and Targeting of Melanoma Cells. IEEE Trans Plasma Sci 2010; 38: 948-952

[34] Georgescu N, Lupu AR. Tumoral and Normal Cells Treatment With High-Voltage Pulsed Cold Atmospheric Plasma Jets. IEEE Trans Plasma Sci 2010; 38: 1949-1956

[35] Iseki S, Nakamura K, Hayashi M, Tanaka H, Kondo H, et al. Selective killing of ovarian cancer cells through induction of apoptosis by nonequilibrium atmospheric pressure plasma. Appl Phys Lett 2012; 100: 13702

[36] Zucker SN, Zirnheld J, Bagati A, DiSanto TM, Des Soye B, et al. Preferential induction of apoptotic cell death in melanoma cells as compared with normal keratinocytes using a non-thermal plasma torch. Cancer Biol Ther 2012; 13: 1299-1306

[37] Kaushik NK, Attri P, Kaushik N, Choi EH A Preliminary Study of the Effect of DBD Plasma and Osmolytes on T98G Brain Cancer and HEK Non-Malignant Cells. Molecules 2013; 18: 4917-4928

[38] Arndt S, Wacker E, Li YF, Shimizu T, Thomas HM, et al. Cold atmospheric plasma, a new strategy to induce senescence in melanoma cells. Exp Dermatol 2013; 22: 284-289

[39] Koritzer J, Boxhammer V, Schafer A, Shimizu T, Klampfl TG, et al. Restoration of sensitivity in chemo - resistant glioma cells by cold atmospheric plasma. PLoS One 2013; 8: e64498

[40] Volotskova O, Hawley TS, Stepp MA, Keidar M Targeting the cancer cell cycle by cold atmospheric plasma. Sci Rep 2012; 2: 636-645

[41] Arjunan KP, Friedman G, Fridman A, Clyne AM Non-thermal dielectric barrier discharge plasma induces angiogenesis through reactive oxygen species. J R Soc Interface 2012; 9: 147-157

[42] Isbary G, Shimizu T, Li YF, Stolz W, Thomas HM, et al. Cold atmospheric plasma devices for medical issues. Expert Rev Med Devices 2013; 10: 367-377

[43] Kim JY, Ballato J, Foy P, Hawkins T, Wei Y, et al. Apoptosis of lung carcinoma cells induced by a flexible optical fiber-based cold microplasma. Biosens Bioelectron 2011; 28: 333-338

[44] Lee HJ, Shon CH, Kim YS, Kim S, Kim GC, et al. Degradation of adhesion molecules of G361 melanoma cells by a non-thermal atmospheric pressure microplasma. New Journal of Physics 2009; 11: 15026-15026

[45] Thiyagarajan M, Waldbeser L, Whitmill A THP-1 leukemia cancer treatment using a portable plasma device. Stud Health Technol Inform 2012; 173: 515-517

Table 1 List of papers presenting plasma treatment results for cancer. Abbreviations: DBD - Dielectric Barrier Discharge, SMD - Surface Micro Discharge

Authors Year Cell

Type/Tumour Cell Lines Method Plasma

Device Result Cit.

Kieft et al. 2004 chinese hamster ovary fibroblasts

CHO-K1 in vitro plasma needle (He)

technical issues, cell detachment, cell death

[1]

Kieft et al. 2005 rat vascular smooth muscle cells, bovine vascular endothelial cells

A7r5, BAEC

in vitro plasma needle (He)

technical issues, time-dependent cell detachment, dose-dependent cell death

[2]

Stoffels et al.

2006 chinese hamster and mouse fibroblasts, rat vascular smooth muscle cells, bovine vascular endothelial cells, human non-

CHO-K1, 3T3, A7r5, BAEC MR65

in vitro plasma needle (He)

technical issues, cell detachment, apoptosis

[3]

small cell lung cancer (NSCLC)

Yonson et al.

2006 human hepatocellular carcinoma, human vascular endothelial cells

HepG2, HAAE-1

in vitro plasma jet (He)

technical issues, cell detachment and reattachment

[5]

Fridman et al.

2007 human melanoma cells

A2058 in vitro FE-DBD technical issues, apoptosis

[9]

Shashurin et al.

2008 mouse fibroblasts

primary cells in vitro plasma jet (He)

technical issues, inhibition migration, detachment, cell death

[8]

Stoffels et al.

2008 rat vascular smooth muscle cells, bovine vascular endothelial cells

A7r5, BAEC in vitro plasma jet (He)

delayed cell death (low dose: apoptosis, high dose: necrosis), no detachment

[4]

Zhang et al. 2008 human hepatocellular carcinoma

BEL-7402 in vitro plasma jet (Ar)

technical issues, cell loss

[10]

Kim et al. 2009 human melanoma cells

G361 in vitro plasma jet (He)

technical issues, apoptosis

[6]

Leduc et al. 2009 human cervical cancer

HeLa in vitro plasma jet (He)

cell membrane permeabilisation

[15]

Lee et al. 2009 human melanoma cells

G361 in vitro plasma jet (He)

cell detachment, cell death, down-regulation of integrins and focal adhesion kinase

[44]

Lupu et al. 2009 human colon carcinoma cells mouse melanoma cells

COLO320DM, B16-F10

in vitro plasma jet (He)

oxygen necessary for plasma effects, increased apoptosis rate in MDR-blocked cells

[16]

Georgescu and Lupu

2010 human colon carcinoma cells mouse melanoma cells, normal macrophages

COLO320DM, B16-F10, RAW 264.7

in vitro plasma jet (He)

selctive cell death of tumour cells

[34]

Kim et al. 2010 human colon carcinoma cells

HCT-116, SW480, LoVo

in vitro plasma jet (He)

proliferation, migration, invasion

[18]

Kim et al. 2010 human breast cancer cells

MCF-7 in vitro plasma jet (He)

apoptosis [11]

Lupu et al. 2010 chinese hamster lung fibroblasts

V79-4 in vitro plasma jet (He)

apoptosis [17]

Vandamme et al.

2010 human glioblastoma cells

U87 in vivo FE-DBD technical issues, animal model, animal tolerance, tumor size

[21]

Zirnheld et al.

2010 human melanoma cells, human primary keratinocytes

1205Lu, HEK

in vitro plasma jet (He)

selective cell death of melanoma cells

[33]

Ahn et al. 2011 human cervical cancer

HeLa in vitro plasma jet apoptosis, mitochondrial dysfunction

[28]

Kalghatgi et al.

2011 human non-neoplastic

MCF10A in vitro DBD intracellular ROS, DNA damage, dose-

[29]

breast epithelial cells

dependent apoptosis

Keidar et al. 2011 mouse melanoma cells, human lung cancer cells (NSCLC), human bladder cancer cells

B16-F10, SW900, SCaBER

in vitro, in vivo

plasma jet (He)

apoptosis, decreased tumour volume, overall survival, gene expression

[24]

Kim et al. 2011 mouse lung carcinoma, mouse fibroblasts

TC-1, CL7 in vitro plasma jet (He) + endoscopic device

apoptosis [43]

Vandamme et al.

2011 human glioblastoma cells

U87 in vivo FE-DBD tumor size, survival [22]

Vandamme et al.

2012 human glioblastoma cells, human colon cancer cells

U87, HCT116 in vitro, in vivo

FE-DBD cell cycle arrest, apoptosis, tumor size, overall survival

[23]

Arjunan et al.

2012 porcine vascular endothelial cells

primary cells in vitro DBD enhanced migration of vascular cells, inhibited by ROS scavangers and anti-FGF antibodies

[41]

Barezki and Laroussi

2012 human acute lympoblastic leukemia cells

CCRF-CEM in vitro plasma jet (He)

dose dependent cell death

[13]

Brulle et al. 2012 human pancreatic cancer

MIA PaCa2-luc

in vitro in vivo

plasma jet (He)

orthotopic xenograft, significant reduction of tumour size by combination of CAP + Gemcitabine in vivo

[26]

Haertel et al. 2012 human non-neoplastic keratinocytes

HaCaT in vitro DBD down regulation of EGFR and E-Cadherin, intracellular ROS induction

[7]

Iseki et al. 2012 human ovarian cancer human non-neoplastic fibroblasts

SKOV-3, HRA, WI-38, MRC-5

in vitro plasma jet (Ar)

anti-proliferative, apoptosis, selectivity

[35]

Kaushik et al.

2012 human glioblastoma

T98G in vitro DBD time dependent micronucleus formation and reduced clonogenic growth

[12]

Partecke et al.

2012 human pancreatic adenocarcinoma murine pancreatic cancer

Colo-357, PaTu8988T, 6606PDA

in vitro in vivo

plasma jet (Ar)

dose dependent cell death (<20s apoptosis), penetration depth

[27]

Thiyagarajan et al.

2012 human acute monocytic leukemia cells

THP-1 in vitro plasma jet cell death: low doses-> apoptosis,

[45]

Conflict of interest statement

The authors disclose any financial and personal relationships with other people or

organisations that could inappropriately influence their work.

high doses -> necrosis

Volotskova et al.

2012 mouse skin cells: non-neoplastic keratinocytes, papilloma cells, skin cancer cells

WTK, 308, PAM 212

in vitro plasma jet (He)

cell cycle (G2/M) arrest

[40]

Yan et al. 2012 human hepatocellular cancer

HepG2 in vitro plasma jet (He)

intracellular ROS, apoptosis

[30]

Zucker et al. 2012 human melanoma cells, human primary keratinocytes

1205Lu, HEK

in vitro plasma jet (He)

selective cell death of melanoma cells

[36]

Arndt et al. 2013 human melanoma cells

Mel Juso, Mel Ei, Mel Ho, Mel Im, Mel Ju, HTZ19

in vitro in vivo

SMD dose dependent senescence/apoptosis

[38]

Kaushik et al.

2013 human glioblastoma cells, human embryonic kidney cells

T98G, HEK in vitro DBD CAP + low dose osmolytes selectively kills tumour cells

[37]

Köritzer et al.

2013 human glioblastoma cells

LN18, LN229, U87

In vitro SMD restoration of chemosensitivity, cell cycle (G2/M arrest)

[39]

Panngom et al.

2013 human lung cancer cells (large cell, squamous) human non-neoplastic fibroblasts

H460, HCC1588, MRC5, L132

in vitro DBD higher apoptotic rates in tumour cells

[31]

Schmidt et al.

2013 human non-neoplastic keratinocytes

HaCaT in vitro plasma jet (He)

indirect treatment, gene expression analysis: stress sensing and proliferative genes

[32]

Walk et al. 2013 mouse neuroblastoma

Neuro2a in vitro in vivo

plasma jet (He)

Decreased metabolic activity, apoptosis, reduced viability, decreased tumour volume, survival time

[19]