The Emerging Role of Gas Plasma in Oncotherapy

TIBTEC 1671 No. of Pages 16

Review

The Emerging Role of Gas Plasma in Oncotherapy Xiaofeng Dai,1,2,* Kateryna Bazaka,3,4 Derek J. Richard,4,5,6,7 Erik (Rik) W. Thompson,4,5,6,7,* and Kostya (Ken) Ostrikov3,4 Atmospheric pressure gas plasmas are emerging as a promising treatment in cancer that can supplement the existing set of treatment modalities and, when combined with other therapies, enhance their selectivity and efficacy against resistant cancers. With further optimisation in production and administration of plasma treatment, plasma-enabled therapy has a strong potential to mature as a tool for selectively curing highly resistant solid tumours. Although intense preclinical studies have been conducted to exploit the unique traits of plasma as an oncotherapy, few clinical studies are underway. This review identifies types of cancers and patient groups that most likely benefit from plasma oncotherapy, to introduce clinical practitioners to plasma therapy and accelerate the speed of translating plasma for cancer control in clinics.

Highlights CAP is a mild multimodal treatment approach that has been widely applied in the medical sector for wound healing, blood coagulation, and ulcer prevention and decontamination. The activity of CAP can be transferred to a liquid medium, which can easily be stored, transported, and administered in the form of a subcutaneous injection. CAP has demonstrated selectivity for cancer cells in many preclinical studies using various malignancy models. There is an increasing trend in investigating CAP-induced immunogenic cell death, with many positive results recently reported.

Emerging Opportunities for Gas Plasmas in Cancer Therapy Significant advances have recently been seen in the development of tailored cancer therapies; many of which are effective yet expensive and only accessible in developed countries. The need for affordable yet effective modalities for cancer treatment persists however, particularly in lowand middle-income countries, where a significant proportion of the global cancer-related deaths occur [1]. Therapy based on cold atmospheric-pressure plasma (CAP, see Glossary) (Box 1), for simplicity referred to as plasma, is one such modality that has recently been identified as an affordable yet safe and effective tool to ablate a wide range of cancers [2]. Originally developed for low-temperature decontamination and wound healing, plasma was discovered to interact with organic materials without causing thermal/electric damage to the cell surface. This eventually led to the development of a wide range of reliable and user-friendly plasma sources that can deliver mild, yet effective doses of reactive species, and electromagnetic radiation to selectively shrink tumours, restore chemo- and radiosensitivity in resistant cells, stimulate immune functions, halt metastasis, and push cancer stem cells into an apoptotic state. Figure 1 presents several examples of currently available plasma therapy devices [3–6]. Intense preclinical studies followed, demonstrating unique traits of plasma oncotherapy, such as its multimodal activity [7], synergistic interactions with conventional chemotherapy agents [8], ability to cause genetic [9] and epigenetic [10] changes that alter processes fundamental to cancer progression, and capacity to inducing immunogenic cell death (ICD) [11,12]. Several clinical studies highlight the breadth of the applications in which plasma tools can deliver clinical benefits. By saving the life of a 75-year-old incurable pancreatic cancer patient using CAP (http://www.fox32chicago.com/news/211496088-video), Keith Millikan was the first surgeon to use the chemoradiation properties of plasmas for cancer treatment and

Trends in Biotechnology, Month Year, Vol. xx, No. yy

CAP has shown synergy with many chemicals and is a promising agent for combination cancer treatment. CAP has succeeded in curing a 75year-old late-stage pancreatic cancer patient in a compassionate case. Numerous devices have been developed to enable preclinical applications of CAP.

1

Wuxi School of Medicine, Jiangnan University, Wuxi, China 2 School of Biotechnology, Jiangnan University, Wuxi, China 3 School of Chemistry, Physics and Mechanical Engineering, Queensland University of Technology, Brisbane, Queensland 4059, Australia 4 Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland 4059, Australia 5 Translational Research Institute,

https://doi.org/10.1016/j.tibtech.2018.06.010 © 2018 Elsevier Ltd. All rights reserved.

1

TIBTEC 1671 No. of Pages 16

Box 1. Plasmas and CAP Fundamentally, plasma is an ionised gas enriched with biologically and chemically reactive species, including charged electrons and ions, as well as radicals, atoms, and molecules in neutral (e.g., excited) or charged forms, where the electric charge can be positive or negative. In addition to chemical species, plasmas produce electromagnetic radiation, propagating disturbances such as shock waves and heating, among other effects. Medically relevant plasmas (termed CAP) benefit from low intensities of these individual effects, making them a gentle tool that can induce desired biological effects in a controlled manner with a good degree of spatiotemporal resolution [74]. At the same time, such a unique combination of physical and chemical effects gives rise to the unique multimodal activity of plasmas. In the laboratory, plasmas can be easily generated by applying an electric field to the process gas, typically pure helium or argon, or their mixtures with oxygen. This electric field accelerates electrons and initiates a cascade of chemical reactions that give rise to a diverse range of chemical species. The amount of applied energy, and the type and pressure of the processing gas determine both the speed (and thus the temperature) and the chemistry of this cocktail of species, and as such are commonly used to tune the properties of plasmas for a given application. In medicine, low temperature plasmas that can be generated at atmospheric pressure are sought after due to the simplicity, versatility, and affordability of such plasma devices. Clinically, the nature of the direct plasma treatment renders it highly suitable to the treatment of primary tumours that arise from skin or mucosal surfaces. This technology may compliment surgery as an adjuvant therapy. Of particular clinical interest is the ability of CAP to penetrate into tissues and effectively target cancer cells that have infiltrated healthy tissue adjacent to the tumour mass. These infiltrating cells are difficult to isolate and remove during the removal of the primary tumour and as a result surgeons often opt for the resection of large margins around the tumour. With CAP, these margins could be selectively cleared of cancer cells without the need to remove large areas of normal tissue [13] (http://www.fox32chicago.com/news/211496088-video). In addition to selective targeting of cancer cells, plasmas can be used in wound healing, where it decontaminates and stimulates tissue regeneration. When used on biomaterials, plasma treatment can remove biological and chemical contaminants, functionalise, structure, and activate the surface to control cell–surface interactions, for example, to prevent biofouling or stimulate osseointegration, and deposit functional thin films, for example, antimicrobial or drug release coatings [75].

pioneered its application as an oncotherapy. Metelmann and colleagues conducted a clinical study on 12 patients afflicted with advanced head and neck carcinomas, and the results show that CAP could partially remit superficial tumours and effectively reduce cancer ulceration contamination [13]. Canady et al. used plasma as a tool for surgery to enable complete removal of gastrointestinal tumours, and minimise the incidence of recurrence (https://iwpct2017. sciencesconf.org/resource/page/id/13). The response rates of some of these studies, although not perfect in every case, are promising and compare well with the early response rates seen with chemotherapy or radiotherapy [13]. With considerable effort currently dedicated to further optimising plasma instrumentation and treatment delivery, plasma appears set to become a valuable clinical tool. Thus, the aim of this paper is to introduce the clinical community to the key features and benefits of plasmas as a stand-alone therapeutic tool or in combination with established cancer treatment therapies, and stimulate discussions regarding the potential applications of this family of technologies. This review first briefly explains the nature of multimodal effects of plasmas, followed by demonstration of how these can be used to deliver specific clinical benefits in oncology. Patient cohorts most suited to plasma therapy are identified, with the aim of engaging the interest of clinical researchers and enabling wider adoption of plasma therapy in oncology. Lastly, we highlight clinical challenges and future trends of this field.

2

Trends in Biotechnology, Month Year, Vol. xx, No. yy

Woolloongabba 4102, Queensland, Australia 6 University of Melbourne Department of Surgery, St Vincent’s Hospital, Melbourne, Victoria 3065, VIC, Australia 7 School of Biomedical Sciences, Queensland University of Technology, Brisbane, Queensland 4059, Australia

*Correspondence: [email protected] (X. Dai) and [email protected] (E.R.W. Thompson).

TIBTEC 1671 No. of Pages 16

(A)

(C)

Glossary

(D)

(B)

(E)

Gas delivery generator

(F)

High frequency generator (electrosurgical unit)

Argon gas High frequency current

Electrode

Canady hybrid plasma™ scalpel

Tissue Neutral plate

Figure 1. Selected Examples from a Wide Range of CAP Devices Available for Clinical Use. (A) Conformité Européene-certified Ar plasma jet, kINPen, developed by the INP Greifswald and Neoplas Gmbh Greifswald, Germany. (B) Plaz4 plasma jet, constructed by Plasmology4, Inc., Scottsdale, AZ. (C) MicroPlaSter (beta), developed by ADTEC Plasma Technology Co. Ltd., Fukuyama, Japan. (D) PlasmaDerm1 VU-2010 device, constructed by CINOGY Gmbh, Duderstadt, Germany. (E) Canady Vieira Cold PlasmaTM Scalpel with connected electrosurgical system SS-200E/Argon 2 (US Medical Innovations, LLC). (F) Schematic presentation of Hybird Plasma Scalpel and its principle of operation. (A,C,D) Reproduced, with permission, from [3], (B) reproduced, with permission, from [4], (E) reproduced under a Creative Commons CC-BY license from [5], and (F) reproduced, with permission, from [6]. Abbreviation: CAP, cold atmospheric-pressure plasma.

Medical Features of Plasma Oncotherapy Early reports on the dose-dependent killing effects of CAP on mammalian cells [14] led to extensive in vitro and in vivo exploration of this technology as a promising oncotherapeutic agent. CAP has the capacity to selectively push cancer cells into an apoptotic state [15–17], enhance chemosensitivity [8,18,19], stimulate immune functions [11,12,20], halt metastasis

Aldehyde dehydrogenase 1 (ALDH1): catalyses oxidation of aldehydes and is considered a marker of cancer stemness. Cold atmospheric-pressure plasma (CAP): partially ionized gas comprising high-energy electrons, low-temperature ions, and uncharged particles, such as atoms, molecules and radicals, and UV radiation. Chemotherapy: a category of cancer treatment that uses one or more anticancer drugs as part of a standardized chemotherapy regimen. Cancer stem cell (CSC): cancer cell possessing characteristics associated with normal stem cells. CSCs are tumourigenic, capable of generating tumours through the stem cell processes of self-renewal and differentiation into multiple cell types, and are hypothesised to persist in tumours as a distinct population and cause relapse and metastasis by giving rise to new tumours. Death receptor 5 (DR5): cell surface receptor of the tumour necrosis factor receptor superfamily that binds TRAIL and mediates apoptosis. Epithelial–mesenchymal transition (EMT): process by which epithelial cells loose cell polarity and cell–cell adhesion, and gain migratory and invasive properties. EMT has been shown to occur in the initiation of metastasis in cancer progression. Helical tomotherapy: a form of computed-tomography-guided intensity-modulated radiation therapy, where the radiation is delivered slice by slice. Helical tomotherapy is better used to target treatment sites throughout the body without a pause for the patient to be moved and set up differently. Immunogenic cell death (ICD): a form of cell death caused by some cytostatic agents such as anthracyclines, oxaliplatin, and bortezomib, or radiotherapy and photodynamic therapy. ICD occurring in cancer cells can induce an effective antitumour immune response through activation of dendritic cells and consequent activation of specific T cell response. Both endoplasmic reticulum stress and ROS production are key players of intracellular signalling pathways

Trends in Biotechnology, Month Year, Vol. xx, No. yy

3

TIBTEC 1671 No. of Pages 16

[21], and eradicate cancer stem cells [22,23] (Table 1). More recently, the unique chemistry of CAP was demonstrated to transfer and be retained in plasma-treated solutions (also known as plasma-activated media, PAM), significantly expanding the scope of potential applications of CAP technologies to those cases where tumours are hard to reach, or of the size that would necessitate higher doses of plasmas. The chemical reactivity of CAP and PAM has also been explored as the means to enhance the efficacy of traditional chemotherapy agents, with promising results [18]. Selectivity for Cancer Cells Reactive oxygen species (ROS), reactive nitrogen species (RNS), charged and neutral particles, and radiation produced by plasmas deliver a variety of outcomes with direct relevance to oncology, including sterilisation of affected tissues and selective killing of a wide range of cancer cells [2,24] (Figure 2, Key Figure). For example, subsequent to tumour excision, CAP treatment can be used to treat the remaining healthy tissues to decontaminate the site, removing pathogenic microorganisms, promoting blood coagulation, and stimulating regeneration and healing of healthy tissue. Importantly, CAP treatment can directly kill any cancer cells that may have not been removed during excision, thus effectively reducing the likelihood of metastasis or tumour regrowth. ROS are considered a major player in inducing cell response to CAP treatment in vitro and in vivo [25–27]. The exact mechanism for the selectivity of CAP for cancer cells is yet to be fully understood, but may be linked to cancer cells with higher baseline ROS levels than normal cells, due to the elevated rate of ROS creation and associated genomic instability [28]; the latter being a universal hallmark of all cancers [29]. An increase in cellular ROS due to CAP treatment may be sufficient to overcome the threshold of inducing apoptosis in malignant cells, whereas normal cells, with their inherently lower baseline ROS and effective antioxidant machinery, can effectively manage this additional oxidative stress. The effect of CAP on cancer cell growth has been demonstrated to be dose dependent. Dose can be quantified by fitting treatment time, liquid surface area, thickness of medium, and cell amount to a linear model and optimised using orthogonal design in vitro [30], and constitutes a predefined combination of reactive species delivered at a specific temperature and level of UV emissions (if CAP treatment is applied directly to tissues). ROS, such as hydroxyl radical (OH
), hydrogen peroxide (H2O2), ozone (O3), and superoxide (O2), and RNS, such as nitric oxide (NO
) and anionic (OONO) and protonated (ONOOH) forms of peroxynitrite, are considered the primary biologically reactive species activated by plasma. These can be quantified following standard protocols, and their concentrations tuned by controlling key processing parameters, such as the configuration of the plasma system, for example, electrode configuration and discharge gap, chemistry of processing gases, for example, noble argon or helium as opposed to oxygen or air, duration of the treatment, proximity to and the area of interface between the plasma and the treated tissue or media, and others. When CAP treatment is applied to activate media, the chemistry of the medium will affect the reactive chemistry and thus therapeutic efficacy of PAM. The choice of the processing gas determines the type of active species produced by not only serving as a source of oxygen and nitrogen compounds, but also affecting the ionisation process due to differences in the amount of input energy required to remove electrons from different elements. During device development and optimisation, the effect of these processing parameters on temperature, UV emission and chemical composition of the plasma are carefully quantified and correlated with specific in vitro and in vivo outcomes to provide dose-type guidelines to facilitate clinical translation. Low to medium doses of CAP halt cell division leading to apoptosis, whereas 4

Trends in Biotechnology, Month Year, Vol. xx, No. yy

governing ICD. ICD is characterised by secretion of damage-associated molecular patterns (DAMPs). There are three important DAMPs: calreticulin, heat-shock proteins HSP70 and HSP90, and secreted HMGB1 and ATP. O-6-Methylguanine-DNAmethyltransferase (MGMT): crucial for genome stability, and depleting it increases carcinogenic risk. Plasma-activated medium (PAM): a liquid in which the chemical reactivity of the CAP is transferred by impregnating it with reactive species. It provides alternative routes of CAP administration, for example, injection. Penetration depth: in this context, a measure of how deep CAP can penetrate into tissue, and defined as the depth at which the intensity of CAP inside the tissue falls to 1/e (about 37%) of its original value at the surface. Plasma oncotherapy: a therapeutic modality utilising the unique combination of physical and chemical effects of cold atmospheric pressure plasma for the control or killing of malignant cells. Radioresistance: the level of ionising radiation that organisms are able to withstand. Radiotherapy: a therapy that is normally delivered by a linear accelerator and using ionizing radiation to control or kill malignant cells. Reactive species: chemical species with sufficient biological and chemical reactivity to induce genotypic and epigenetic changes, create metabolic stress, and alter microenvironment. Reactive nitrogen species (RNS): reactive chemical species containing nitrogen. Reactive oxygen species (ROS): reactive chemical species containing oxygen.

Authors

Year

Cancer type

Study material

Level

Source

Plasma feature

Refs

Schuster et al.

2016

Human head and neck cancer

Head and neck cancer patient

Clinical

Plasma jet

Selectivity for cancer cells

[44]

Thiyagarajan et al.

2012

Human acute monocytic leukaemia cells

THP1

In vitro

Plasma jet

Selectivity for cancer cells

[17]

Mirpour et al.

2014

Human breast cancer cells

MCF7

In vitro

Plasma jet

Selectivity for cancer cells

[38]

Ahn et al.

2011

Human cervical cancer cells

HeLa

In vitro

Plasma jet

Selectivity for cancer cells

[25]

Georgescu et al.

2010

Human colon cancer cells, Mouse melanoma cells

COLO320DM, B16 (murine)

In vitro

Plasma jet

Selectivity for cancer cells

[16]

Kaushik et al.

2012

Human glioblastoma cells

T98G

In vitro

DBD

Selectivity for cancer cells

[42]

Chen et al.

2017

Human glioblastoma cells

U87; U87 injected mouse

In vitro, in vivo

Plasma jet

Selectivity for cancer cells

[41]

Vandamme et al.

2012

Human glioblastoma cells, human colon cancer cells

U87, HCT116; U87 injected mouse

In vitro, in vivo

DBD

Selectivity for cancer cells

[27]

Zhang et al.

2008

Human hepatocellular cancer cells

BEL7402

In vitro

Plasma jet

Selectivity for cancer cells

[37]

Yan et al.

2012

Human hepatocellular cancer cells

HepG2

In vitro

Plasma jet

Selectivity for cancer cells

[26]

Keidar et al.

2011

Human lung cancer cells, mouse melanoma cells; human bladder cancer cells

B16, SW900; B16 & SCaBER injected mouse

In vitro, in vivo

Plasma jet

Selectivity for cancer cells

[2]

Panngom et al.

2013

Human lung cancer cells

H460, HCC1588

In vitro

DBD

Selectivity for cancer cells

[36]

Fridman et al.

2007

Human melanoma cells

A2058

In vitro

DBD

Selectivity for cancer cells

[35]

Zirnheld et al.

2010

Human melanoma cells

1205Lu

In vitro

Plasma jet

Selectivity for cancer cells

[34]

Trends in Biotechnology, Month Year, Vol. xx, No. yy

Iseki et al.

2012

Human ovarian cancer cells

SKOV3, HRA

In vitro

Plasma jet

Selectivity for cancer cells

[39]

Utsumi et al.

2014

Human ovarian cancer cells

TOV21G, ES2, SKOV3, NOS2

In vitro

PAM

Selectivity for cancer cells

[40]

Partecke et al.

2012

Human pancreatic cancer cells

Colo357, PaTu8988T, 6606PDA (murine); Colo357 injected mouse

In vitro, in vivo

Plasma jet

Selectivity for cancer cells

[43]

Ishaq et al.

2015

Human colon cancer cells

HT29 (TRAIL resistant), HCT116

In vitro

Plasma jet

Enhancing cancer chemosensitivity

[18]

Vandamme et al.

2011

Human glioblastoma cells

U87 (chemoresistance) injected mouse

In vivo

DBD

Enhancing cancer chemosensitivity

[72]

Köritzer et al.

2013

Human glioblastoma cells

LN18 (TMZ resistant), LN229, U87

In vitro

DBD

Enhancing cancer chemosensitivity, Dosedependent cell death

[19]

Xu et al.

2016

Human myeloma cells

RPMI8226, LP1 MM

In vitro

Plasma jet

Enhancing cancer chemosensitivity

[45]

TIBTEC 1671 No. of Pages 16

Table 1. List of Some Preclinical and Clinical Studies on the Features of CAP Relevant to Oncologic Therapy

5

6 Trends in Biotechnology, Month Year, Vol. xx, No. yy

Authors

Year

Cancer type

Study material

Level

Source

Plasma feature

Refs

Utsumi et al.

2013

Human ovarian cancer cells

NOS2, NOS3, NOS2TR & NOS3TR (paclitaxel resistant), NOS2CR & NOS3CR (cisplatin resistant); NOS2 & NOS2TR injected mouse

In vitro, in vivo

PAM

Enhancing cancer chemosensitivity

[70]

Kajiyama et al.

2015

Human ovarian cancer cells

K2, K2R100 (paclitaxel resistant)

In vitro

PAM

Enhancing cancer chemosensitivity

[73]

Yan et al.

2017

Human pancreatic cancer cells, human glioblastoma cells

PA-TU-8988T, U87

In vitro

PAM

Selectivity for cancer cells

[15]

Brulle et al.

2012

Human pancreatic cancer cells

MIA PaCa2-luc; MIA PaCa2-luc injected mouse

In vitro, in vivo

Plasma jet

Enhancing cancer chemosensitivity

[8]

Barezki et al.

2012

Human acute lymphoblastic leukaemia cells

CCL119

In vitro

Plasma jet

Dose-dependent cell death

[32]

Arndt et al.

2013

Human melanoma cells

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

In vitro

DBD

Dose-dependent cell death

[31]

Kaushik et al.

2016

Human glioblastoma cells, Human lung cancer cells

T98G, A549

In vitro

DBD

Stimulation of the immune system

[20]

Lin et al.

2015

Human nasopharyngeal cancer cells, human acute monocytic leukaemia cells

CNE1 (radioresistant), THP1

In vitro

DBD

Stimulation of the immune system

[11]

Mirpour et al.

2016

Mouse metastatic breast cancer cells

4T1; 4T1 injected mouse

In vitro, in vivo

Plasma jet

Halt on cancer metastasis

[51]

Zhu et al.

2016

Breast cancer cells

MDAMB231

In vitro

Plasma jet

Halt on cancer metastasis

[49]

Kim et al.

2010

Human colon cancer cells

HCT116, SW480, LoVo

In vitro

Plasma jet

Halt on cancer metastasis

[21]

Lee et al.

2009

Human melanoma cells

G361

In vitro

Plasma jet

Halt on cancer metastasis

[50]

Schmidt et al.

2015

Human melanoma cells

SKMel147

In vitro

Plasma jet

Halt on cancer metastasis

[48]

Ikeda et al.

2015

Human endometrioid cancer cells

HEC1, HEC108; HEC1 & HEC108 injected mouse

In vitro, in vivo

Plasma jet

Elimination of cancer stem cells

[22]

Ikeda

2014

Human uterine endometrioid cancer cells, human gastric cancer cells

HEC1, GCIY

In vitro

Plasma jet

Elimination of cancer stem cells

[23]

TIBTEC 1671 No. of Pages 16

Table 1. (continued)

TIBTEC 1671 No. of Pages 16

Key Figure

Clinically Relevant Features of Multimodal CAP and Cancer Patient Cohorts Most Likely to Benefit from Them 1

Selec vity for cancer cells

2

Enhancing cancer chemo-sensi vity

Electron

3

S mula on of the immune system

Excited Light molecule

4

Halt on cancer metastasis

MulƟmodal CAP

5

Elimina on of cancer stem cells

6

Wound healing Blood coagula on Ulcera on preven on Decontamina on

7

Limited penetra on depth

8

Mild doses

Radical

Clinician

Electron

Cancers lacking an effec ve targeted therapy Cancers with low penetra on depth

1

Radical Radical

7 Electron

Cancers with aesthe cs requirement

8

Radiotherapist Cancers that resist radio-therapy

2

Cancers with physical isola on

3

Pa ents with relapse or metastasis

3

Light

Oncological traits

5

Heat Heat

Surgeon Post opera ve pa ents

Light

4

Excited molecule Ion Heat

Ion

5

6

Excited molecule

Ion

Biomedical traits

Basic traits

Figure 2. Tissues treated with plasmas are exposed to chemical treatment (in a form of radicals, electrons, ions, and excited molecules), light therapy (as UV photons) and mild heat treatment, where these treatment modes act individually and synergistically to deliver an effective and selective treatment. Plasma has oncological traits including selectivity for cancer cells, enhancing cancer chemosensitivity, stimulation of the immune system, halt on cancer metastasis, and elimination of cancer stem cells; various biomedical traits, including wound healing, blood coagulation, ulceration prevention and decontamination; and basic physiochemical traits, including limited penetration depth and mild doses. These clinically relevant features are beneficial to clinicians, radiotherapists, and surgeons, as well as to cancer patients who may experience improved therapeutic outcomes and reduced side effects. Abbreviation: CAP, cold atmospheric-pressure plasma.

high doses of CAP result in cell death via necrosis. Studies using various cancer cells have shown cellular senescence or cell cycle arrest under low dose CAP treatment [19,31,32], with restoration of activity once CAP is removed. It is therefore important that the volume of tumour cells exposed to CAP is adequate to mitigate cell resistance. Intensive research is underway to fully uncover the mechanisms underlying cancer cell selectivity of CAP. Due to the apparent large therapeutic window, CAP has been proposed as an agent that could help transform the current paradigm of cancer treatment [2]. Growing preclinical evidence suggests wide applicability of CAP in the treatment of such cancers as melanoma [2,16,33–35], colon cancer [16,27], lung cancer [2,36], hepatocellular carcinoma [26,37], breast cancer [38], Trends in Biotechnology, Month Year, Vol. xx, No. yy

7

TIBTEC 1671 No. of Pages 16

ovarian cancer [39,40], cervical cancer [25], bladder cancer [2], glioblastoma [27,41,42], pancreatic cancer [43] and acute monocytic leukaemia [17] (Table 1). Clinically, plasma treatment of advanced head and neck cancer has shown great promise [44] (Figure 3). Enhancing Cancer Chemosensitivity Multiagent or combination cancer therapies can provide significantly better therapeutic outcomes for patients [19]. CAP can be viewed as a multiagent therapy as it combines electromagnetic, chemical and thermal effects at mild doses. It also combines well with other modalities to produce beneficial synergistic effects (Figure 2). For example, in a study testing the efficacy of gemcitabine–CAP cotherapy in preclinical models of pancreatic cancer, gemcitabine was found to induce cell cycle arrest by stopping cells from replicating their DNA via cytidine nucleotide depletion, making these cells more sensitive to stress from ROS and RNS generated by CAP.

(A)

(B)

Before Before

AŌer

(D)

AŌer

(C)

Before (E)

AŌer

Before

AŌer

(F)

Before

AŌer

Figure 3. Clinical Evidence of the Effects of CAP on Head and Neck Tumours. (A–D) Different appearance of tumour surfaces before and after 2 weeks of CAP treatment, where the treatment region is marked by a circle. As compared with areas of tumour progress (*) in untreated surrounding areas, treated region shows removal of microbial lawn in (A–D); a flat surface aspect with stimulated vessels in (A) and (C); tiny spots of haemorrhage in (C); a contraction of the margins of the ulceration forming a recess covered by scabs in (B); and a contraction of the margins of the ulceration forming a groove with stimulated vessels in (D). (E) CAP applied by jet stream to a spot of the tumour surface. (F) Representative microscopic photograph of squamous cell carcinoma tissue of head and neck tumour before and after CAP treatment. Tissue was exposed in vivo prior to resection and apoptotic cells were detected 24 h later. Green fluorescence results from TUNEL staining and represents apoptotic cell death, blue fluorescence results from 40 ,6diamidino-2-phenylindolestaining and represents all live and dead cells. Scale bar 100 mm. Reproduced, with permission, from [44]. Abbreviations: CAP, cold atmospheric-pressure plasma; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling.

8

Trends in Biotechnology, Month Year, Vol. xx, No. yy

TIBTEC 1671 No. of Pages 16

Another example uses a combination of CAP with temozolomide (TMZ). TMZ improves survival of patients with malignant glioma, especially those patients lacking O-6-methylguanine-DNAmethyltransferase (MGMT) expression through promoter methylation [19]. However, its efficacy is limited in patients who have intrinsic or acquired resistance, usually as a result of enhanced expression of the MGMT gene [19]. CAP can restore sensitivity of resistant cells to TMZ, with combined therapy resulting in arrested DNA replication (S phase) [19]. This is likely due to ROS-induced DNA damage from the CAP in combination with TMZ-induced DNA damage [19]. Thus, combined use of CAP and TMZ is a promising therapeutic strategy for challenging gliomas in patients with unfavourable genotypes. The synergistic effect of CAP with chemotherapy agents is likely to involve a number of mechanisms. CAP is known to cause DNA damage or genomic instability in cancer cells, perhaps through the elevated levels of free radicals. Furthermore, CAP treatment also appears to rewire tumour cells toward a more chemosensitive state by altering the expression of certain pivotal signalling factors, likely through epigenetically induced changes to the DNA. For example, tumour necrosis factor-related apoptosis inducing ligand (TRAIL) can selectively activate cancer cell apoptosis. In colon cancer, TRAIL resistance is common for cells protected from apoptosis. CAP treatment has been shown to reactivate the chemoresponsive state of these cells by enhancing the expression of death receptor 5 (DR5) [18]. Similarly, bortezomib is a first-line chemotherapy drug for myeloma patients. Some myeloma patients develop bortezomib resistance due to enhanced expression of cytochrome P450 (CYP)1A1; a protein that functions to metabolise bortezomib. It is hypothesised that the observed sensitivity increase of myeloma cells to bortezomib after CAP treatment is mediated through the suppression of CYP1A1 expression [45]. Stimulation of the Immune System One of the primary routes for tumour clearance is through the immune system. Cancers must acquire immune privilege to prevent them from being destroyed by the immune system. CAP treatment has been shown to stimulate macrophages to display enhanced migratory and cancer killing activity in vitro following low dose CAP treatment [11,12]. Moreover, CAP has been shown to induce immunogenic cell death of tumour cells by increasing their visibility to immune cells [11]. In this form of cell death, cells activate immune cells via antigen presenting cells (APCs), which stimulate T cells to circulate and destroy tumour cells with the same antigen. Importantly, by acquiring such an adaptive immune response, a small cohort of memory T cells is fostered, ready to trigger a fight response should there be a future relapse [46]. Effects of CAP on ICD induction have been demonstrated in cancers such as glioblastoma [20], lung adenocarcinoma [20], radioresistant nasopharyngeal carcinoma [11], and monocytic leukaemia [11] (Table 1). These effects may not be easily attainable through conventional treatment modalities. Halting Cancer Metastasis Metastasis of tumours to distant sites is strongly associated with poor patient outcomes. Epithelial–mesenchymal transition (EMT) is the process whereby primary carcinoma (epithelial tumour) cells lose their normal polarity and adhesive properties and develop a stem-like, therapy-resistant and migratory phenotype. Agents that may function to inhibit this process by inhibiting EMT are currently being developed [47]. CAP may affect EMT by altering the expression of migration-associated kinases, as shown in the SK-Mel-147 human melanoma cell line, where CAP treatment changed the level of these kinases by over twofold [48]. The metastasis suppressor gene 1 (MTSS1), which halts tumour progression likely through interactions with the actin cytoskeleton, was found to increase by threefold after CAP exposure Trends in Biotechnology, Month Year, Vol. xx, No. yy

9

TIBTEC 1671 No. of Pages 16

[48]. In an in vitro study of human breast cancer cells, CAP was found to downregulate the expression of several metastasis-related genes [49]. CAP-induced inhibition of cell migration has been observed in cells of several tumour types, including human melanoma [48,50], human colorectal cancer [21], and breast cancer [49,51] (Table 1). Elimination of Cancer Stem Cells Cells with tumour-initiating potential are limited to a small population of cells, namely cancer stem cells (CSCs). CSCs are believed to be responsible for cancer recurrence, and exhibit cell resistance to chemo- and radiotherapy by a variety of mechanisms, including reduced cell cycling and effluxing of antitumour chemicals [22]. CSCs have a characteristically high activity of aldehyde dehydrogenase (ALDH1), a cytosolic enzyme that plays a role in the degradation of toxins and anticancer agents [52]. Despite limited information on the effect of CAP on CSCs, early reports have shown that CAP effectively kills both CSCs and non-CSCs in both human uterine endometrioid adenocarcinoma cells and poorly differentiated human gastric carcinoma cells in vitro and in vivo, using ALDH1 as the CSC marker [22,23]. The proportion of ALDH-high cohort was reduced more than that of ALDH-low population after CAP treatment in the Aldefluor assay, while the two reduction ratios were similar in a cell viability assay [22,23]. These results suggest that CAP may interfere with the ALDH1 activity and ultimately suppress the ability of CSCs to self-detoxify and renew.

Feasible Cohorts for Clinical Trials Some patient cohorts in particular have been identified as key target groups for which plasma treatment can provide notable benefits, where the suitability increases with the number of CAP features each population utilizes (Figure 2). Importantly, CAP may provide cure for patients with cancers lacking effective treatment modalities with sufficient sensitivity and/or acceptable side effects. Cancers Lacking an Effective Targeted Therapy Cancers lacking an effective targeted therapy may benefit from the multimodal nature of CAP. Although precision therapies have been developed for some cancers, many patients do not qualify for effective targeted treatment given the diversity of tumours and their high heterogeneities. For instance, non-small-cell lung cancer patients with KRAS mutation currently do not respond to any precision treatment modalities [53]. Unlike HER2-positive and ER-positive breast cancers, which have commercially available effective drugs, such as herceptin [54] and tamoxifen [55], respectively, therapies targeting triple-negative breast cancers often fail due to either unacceptable toxicity (such as with antiangiogenic drugs [56]) or narrow patient coverage (e.g., PARP inhibitors [57]). In tumours for which targeted therapy is available, the target may mutate to evade the interactions with the active agent, or the tumours may evolve new pathways to bypass the therapeutic interference [58]. Combined approaches are thus often required. Combined inhibition of BRAF and MEK against BRAF V600E mutations in melanoma showed improved efficacy compared to individual treatments [59]. However, simultaneous alterations of more than one pathway may lead to an increase in the incidence and magnitude of potential side effects. It is possible, however, that CAP may enhance the efficacy of the chemotherapy, which would allow for reduced dosing, and subsequent reduction in treatment toxicity. Studies using CAP as a sole agent have not reported any noticeable side effects making combination treatments attractive.

10

Trends in Biotechnology, Month Year, Vol. xx, No. yy

TIBTEC 1671 No. of Pages 16

Cancers that Resist Radiotherapy The multimodal nature of CAP makes it difficult for target cells to evolve resistance, such that CAP may reduce tumours that have intrinsic or extrinsic resistance to radiotherapy. As CAP has been shown to modulate the expression of genes responsible for radioresistance, it can provide significant benefits against radioresistant cancers when used as a monotherapy or in combination with systemic therapy. Integrins are reported to be essential in mediating radioand chemoresistance of cancer cells [60]. Thus, patients with cancers that developed therapeutic resistance to radiotherapy may benefit from CAP-induced suppression of integrin signalling (Figure 2). Cancers with Physical Isolation Owing to the immune-stimulating property of CAP, which in principle can reach any spatially isolated tumour cell, cancers with physical boundaries that pose great challenges to traditional therapeutic approaches may potentially be resolved by CAP monotherapy or combination treatments. Treatment of intracranial tumours such as gliomas, meningiomas, pituitary adenomas, and nerve sheath tumours is clinically challenging due to their anatomical location. Radiotherapy is most commonly used for treating these patients, yet it can result in brain damage and neurocognitive dysfunction. Radiotherapy also damages healthy progenitor cells [61] and harms the immune system [62]. This may create severe consequences for paediatric patients. For these patients, development of treatments that completely avoid [63] or reduce the total radiation dose and brain volume exposure [64] may deliver significantly better clinical outcomes. Modern radiotherapies, for example, volumetric modulated arc therapy and helical tomotherapy, can effectively limit radiation exposure to selected parts of the brain, but it may be possible to further advance these techniques by adding CAP to existing radioprotectors such as memantine, given its demonstrated ability to provide mild yet effective stimulation of immune function [11,12,20]. It may also be possible to achieve sufficient treatment efficacy of CAP monotherapy in brain cancer patients, primary or metastatic, as it may breach the physical barrier and reach the difficultto-access tumours through ICD induction. Patients with Relapse or Metastasis Patients already with metastases may respond to CAP, as it can effectively kill CSCs and programme memory T cells against future recurrence and metastasis. Radiotherapy has been shown to increase the flux of tumour cells into the circulation by affecting tumour blood vessels [65], with more viable circulating tumour cells observed in non-small cell lung cancer [66] and bladder cancer [67] after radiotherapy. Given the accumulated in vivo and in vitro evidence on the ability of CAP to halt cancer cell migration in melanoma [48,50], colorectal cancer [21], and breast cancer [51], CAP mono- or combination therapy may deliver similar effects to radiotherapy at notably lower doses (Figure 2). Here, dose is defined as reactive species or any other therapeutic agents introduced into the patient by the treatment, which can be reflected by treatment time (min), exposed area (mm2) and penetration in-depth (mm) in terms of CAP [30] and be measured as radioactivity (Bq), energy of radiation (MV), exposure dose (R), absorbed dose (Gy), or dose-rate (Gy/min) in radiotherapy. Postoperative Patients For many types of tumours, a treatment protocol that involves surgical removal of solid tumours followed by radio- or chemotherapy is used to eradicate cancer cells and prevent relapse. However, the side effects of these adjuvant approaches are considerable due to their weak selectivity for cancer cells. The treatment is typically split into multiple cycles and fractions with Trends in Biotechnology, Month Year, Vol. xx, No. yy

11

TIBTEC 1671 No. of Pages 16

the aim to deliver the minimum effective dose, and provide a period of rest to allow for the recovery of healthy cells. Accurate prognosis and appropriate dosing are crucial to achieve desirable treatment outcome. However, personalised dosing poses great challenges to clinicians as precise simulations of the electron path in tissues, and penetration depth and direction, are difficult. CAP, with its sensitising role, may be combined with chemo- and/or radiotherapy to achieve favourable clinical results at lower, or even trace chemo- and radiotherapy doses. Alternatively, CAP may be sufficiently effective as a monotherapy in eradicating the residual cancer cells after surgery, which may become a novel postsurgical approach with reduced or minimal adverse effects to achieve equivalent or more favourable outcomes (Figure 2). Cancers That Require Low Penetration Depth Superficial cancers, such as melanoma, Kaposi’s sarcoma, and squamous cell carcinoma are suitable to be treated using most common, simple plasma devices due to low penetration depth required for the treatment (Figure 2). Since these tumours are typically solid cancer masses that are visible, the treatment efficacy can be readily observed by clinicians. These features make clinical trials involving CAP or PAM treatments easy to design, implement, and evaluate. Cancers with Aesthetic Requirements Cancers located in the head and neck may be associated with poor cosmetic outcomes. Traditional surgery and chemo- and radiotherapy may be associated with psychological effects associated with negative changes in appearance as well as physiological implications of functional defects of affected facial organs [68]. Such patients may benefit from CAP treatment given its mild yet effective selectivity on cancer cells (Figure 2).

Clinical Challenges for Clinical Trials The key to achieving clinical success using CAP is closely related to the type of tumour for which treatment is sought. For superficial cancers, plasma-generated effects triggering, for example, cancer cell apoptosis and immune response involving ICD, could be directly delivered to the tumour in a highly controlled manner as long as the plasma is stable and has been optimised for this purpose. For internal tumours, however, the increased penetration depth may necessitate further development of the device and more sophisticated modelling of the interactions between plasma-generated effects and various types of tissues. CAP surface treatment can penetrate approximately 50 mm into the tissue [43], which is not sufficient to reach internal cancers. Classical immunology suggests that exposing a small portion of tumour mass to CAP treatment could lead to systemic immune responses necessary for tumour cell elimination [69]. It thus may be possible to trigger the avalanche of immune cell-mediated tumour eradication events by exposing only a fraction of the deep tumour to CAP. PAM may also be useful in reaching internal tumours as, being in the liquid form, it can be easily delivered to the tumour site by injection. Significant tumour size reduction was observed after PAM treatment on ovarian cancer cells both in vitro and in vivo [70]. The clinical uses of plasma in oncotherapy, either as direct CAP or indirect PAM, all demand treatment delivery systems that are safe, effective, and convenient for clinicians to use, which pose challenges for their design and fabrication (Box 2). The development and clinical use of the Canady Helios Cold Plasma Scalpel in cancer patients has demonstrated that it is feasible to design and use such a device in a real-life clinical setting. However, to deliver more advanced plasma features for effective cancer control, sources with, for example, higher penetration depth and more evenly spread long-lasting ROS induction, are urgently called for and are subject of active research efforts. For instance, Mirpour and colleagues designed a CAP device 12

Trends in Biotechnology, Month Year, Vol. xx, No. yy

TIBTEC 1671 No. of Pages 16

Box 2. Sources of CAP Therapy CAP can be generated using a range of device configurations [76], each with their own merits in the clinical setting. Typical configurations include dielectric barrier discharge (DBD), plasma jet, and plasma torch [3]. In the DBD configuration, high sinusoidal voltages in the kHz frequency range are applied to plate electrodes that are covered by quartz, glass, or another insulator. Plasma is produced and geometrically confined within a gap separating the electrodes (or the electrode and the surface of the tissue) typically using air or helium and oxygen mixture as a processing gas. In a jet configuration, the plasma is once again generated between electrodes by applying pulsed DC, radiofrequency, and microwave energy, however a controlled flow of gas pushes a plasma out from the confinement of electrodes into the ambient in the form of a plume that propagates in the direction of the flow. Under certain conditions, while appearing uniform, the plume may be composed of a series of tiny of donut-like plasma bullets that travel along the interface between the processing gas and ambient air at tens of kilometres per second, greatly exceeding the velocity of gas. Using external electric fields, it is possible to tune the initiation time, velocity, and distance the plasma bullets travel, and even deflect the charged species. By controlling the formation and propagation of bullets, it may be possible to efficiently generate and deliver highly-specific doses of plasma-generated reactive species and photons with micrometre and nanosecond precision while maintaining temperature of the treated area very low. Several CAP sources have been certified as medical devices based on physical, biological, preclinical, and clinical characterisation of their safety and efficacy. One of the best-described medically relevant plasma jets, the Ar-driven CAP jet kINPen MED (neoplas GmbH, Greifswald, Germany) is shown in Figure 1A in the main text [77]. Its medical safety has been demonstrated by several clinical studies, where direct CAP treatment of the fingertips of four healthy male volunteers to mimic a plasma disinfection protocol was shown not to cause skin damage or dryness [78]. In a wound healing study involving five individuals and 20 laser lesions, no precancerous skin feature was observed up to 1 year after kINPen CAP treatment [79]. In a series of five clinical case reports involving four men and one woman, CAP showed promise for effective wound healing with high aesthetic scoring and, importantly, no adverse effects [80]. A pilot study focusing on the antiseptic effect of CAP involving treatment of chronic leg ulcers in six men and ten women also reported no cytotoxicity of CAP, highlighting its potential for effective antimicrobial applications [81]. In a clinical follow-up of 12 advanced head and neck patients, CAP was demonstrated to be a suitable tool for reducing cancer ulceration contamination without severe side effects [13]. Another promising plasma jet, the Plaz4 electrosurgical generator (see Figure 1B in main text), produces CAP at 26 C and was effective in pathogenic bacteria control and coagulation reduction during surgery, without any safety concerns [4]. Yet another clinically tested plasma source is the microwave-driven Ar-plasma torch MicroPlaSter (ADTEC Plasma Technology Co. Ltd., Fukuyama, Japan; see Figure 1C in main text) [82]. After a Phase I study on the safety parameters and optimum doses of MicroPlaSter for effective antibacterial performance, a prospective randomized Phase II clinical trial was conducted on decreasing bacterial load of 38 chronic infected wounds in 36 patients. Analysis of 291 treatments in this study revealed significant microorganism reduction with no side effects among all patients [82]. In a similar study involving 70 treatments in 24 patients, both MicroPlaSter (alpha) and its updated version MicroPlaSter (beta) proved effective in chronic wound control with no adverse effects [83]. A randomized placebo-controlled clinical study involving 40 patients with skin graft donor sites on the upper leg showed positive results for DBD-produced CAP on wound healing without any side effect [84]. Tested over the long term by physicians and clinicians, MicroPlaSter, now offered as SteriPlas (ADTEC, Hunslow, UK), has proven to have no mutagenic potential [85] and be well-tolerated by virtually all patients [3]. The PlasmaDerm1 VU-2010 device (CINOGY GmbH Duderstadt, Germany) is based on DBD and uses atmospheric air for plasma generation (see Figure 1D in main text). PlasmaDerm achieved European certification as a medical device in 2013, after its success in a 1-year clinical trial on 14 patients for chronic wound treatment [86]. The Canady Helios Cold PlasmaTM Scalpel (US Medical Innovations, LLC) is an example of a plasma electrosurgical systems (see Figure 1E in main text) [87], which is another tool with significant clinical potential. It has been shown to clinically decrease tissue damage, blood loss, surgical time and transfusion rate, and to promote the survival of late stage cancer patients without side effects (http://www.fox32chicago.com/news/211496088-video).

capable of producing a plasma jet of 250 mm in diameter [51]. This device has a greater penetration depth of 5 cm and causes significant tumour growth recession in vivo [51]. Ongoing efforts with multichannel in situ plasma jet design may offer an acupuncture-like delivery system that is minimally invasive. An adaptive CAP platform has been established to improve treatment outcomes in vitro [71]. In contrast, micro-sized CAP devices have been developed to solve other issues raised by plasma irradiation for internal cancer treatment. For instance, Chen and

Trends in Biotechnology, Month Year, Vol. xx, No. yy

13

TIBTEC 1671 No. of Pages 16

coworkers established the mCAP device and showed its efficacy in treating glioblastoma both in vitro and in vivo, which can circumvent problems such as high voltage, discharge formation in the organ, gas delivery, and plasma probe volume given, its reduced gas flow rate and size [41].

Concluding Remarks The discovery of the anticancer features of CAP and its demonstrated preclinical and clinical efficacies has the potential to underpin the development of a diverse repertoire of synergistic and personalised plasma-enabled therapeutics. These therapies have the potential to not only provide a tool for mild yet effective cancer monotherapy with a wide therapeutic window and high selectivity, but also advance existing therapies toward safer, more effective treatment modalities. While the mechanistic bases of plasma multimodal activity are being actively investigated, clinical trials that demonstrate the utility of CAP in oncotherapy are required (see Outstanding Questions). Furthermore, although there has been a large number of cancers investigated for their susceptibility to plasma-enabled therapies, their selection has not necessarily been driven by our understanding of how plasma-generated effects interact with different types of cells. Indeed, linking specific genetic and epigenetic markers of cancers with the potential sensitivity of the latter to CAP treatment modalities would facilitate screening of cancers for their suitability for CAP treatment. CAP ability to sensitize resistant cancer cells to conventional therapies also warrants significant attention, and therefore more intense efforts should be made to understand potential synergistic and antagonistic events that may arise when CAP is used as part of combination therapy or as an adjuvant therapy. Another important area that deserves attention and would be critical for development and translation of CAP therapy into clinical setting concerns the development of a regulatory framework of industry standards and benchmarks for CAP therapy. Such a framework would enable comparison of CAP efficacy between different plasma device designs and with conventional cancer therapies to provide developers, doctors, and patients with an effective means for selection of the most appropriate device, treatment approach, and therapeutic dose for a given cancer with consideration of patient health status. Acknowledgements

Outstanding Questions Although numerous studies have demonstrated the efficacy of CAP as an oncotherapy, its clinical use has been restricted by the limited penetration depth. So far, only superficial tumours such as melanoma or areas after surgery can be directly treated by CAP. Is there a way to eject plasma inside tissues? How to solve problems imposed by in situ plasma administration such as discharge formation in the organ, gas delivery, and emission to benefit patients carrying nonsuperficial solid tumours from CAP technologies? Many nonsuperficial solid tumours develop in internal organs. How to guide the plasma ejector bypass healthy organs to reach targeted internal tumours is an important question to address before widely taking CAP to clinics for solid tumour treatment and making plasma oncotherapy minimally invasive. Can we take advantage of some monitoring systems in this process? As CAP can be administered to patients in the liquid form, its clinical applications can be largely expanded. For example, it can be easily mixed with drugs to create synergy as a combinatorial chemotherapy with improved efficacy and reduced adverse effects. How to maximize the clinical efficacy of plasma for the benefits of patients give such properties?

We thank Professor Bryan Burmeister, a consulting Radiation Oncologist and the Chair of the Australian and New Zealand Melanoma Trials Group, for his extensive and valuable contributions to the clinical take we have in this paper. This study was funded by the National Natural Science Foundation of China (Grant No. 31471251), Natural Science Foundation of Jiangsu Province (Grant No. BK20161130), the Six Talent Peaks Project in Jiangsu Province (Grant No. SWYY-128), National Science and Technology Major project (Grant No. 2018ZX10302205-004-002), Research Funds for the Medical School of Jiangnan University ESI special cultivation project (Grant No. 1286010241170320), Postgraduate Education Reform Project of Jiangsu Province, and the Australian Research Council Discovery Early Career Research Award (Grant No. DE130101550). These funding sources have no role in the writing of the manuscript or the

Although much preclinical evidence has suggested the oncotherapeutic efficacy of CAP, clinical studies are still lacking. What features of plasma will be most likely taken advantages of in clinical trials? Is it possible to create synergies with canonical treatment modalities to achieve enhanced therapeutic efficacy for cancer patients?

decision to submit it for publication. The Translational Research Institute is supported by a grant from the Australian Government.

Disclaimer Statement The authors declare no conflicts of interest.

Supplemental Information Supplemental information associated with this article can be found, in the online version, at https://doi.org/10.1016/j. tibtech.2018.06.010.

14

Trends in Biotechnology, Month Year, Vol. xx, No. yy

Which cancers or cancer subtypes may benefit most from CAP treatment but have not been tested in vitro? What are the clinically relevant features of CAP that enable such applications? In which, if any, aspects can current conventional cancer treatments outperform plasma, and how might CAP

TIBTEC 1671 No. of Pages 16

References 1. Gelband, H. et al. (2016) Costs, affordability, and feasibility of an essential package of cancer control interventions in low-income and middle-income countries: key messages from Disease Control Priorities, 3rd edition. Lancet 387, 2133–2144 2. Keidar, M. et al. (2011) Cold plasma selectivity and the possibility of a paradigm shift in cancer therapy. Br. J. Cancer 105, 1295– 1301 3. Isbary, G. et al. (2013) Non-thermal plasma – more than five years of clinical experience. Clin. Plasma Med. 1, 19–23 4. Kulaga, E.M. et al. (2016) The use of an atmospheric pressure plasma jet to inhibit common wound-related pathogenic strains of bacteria. Plasma Med. 6, 1–12 5. Shashurin, A. et al. (2015) Electric discharge during electrosurgery. Sci. Rep. 5, 9946 6. Guild, G.N., III et al. (2017) Efficacy of hybrid plasma scalpel in reducing blood loss and transfusions in direct anterior total hip arthroplasty. J. Arthroplasty 32, 458–462 7. Yan, D. et al. (2017) Cold atmospheric plasma, a novel promising anti-cancer treatment modality. Oncotarget 8, 15977–15995 8. Brulle, L. et al. (2012) Effects of a non thermal plasma treatment alone or in combination with gemcitabine in a MIA PaCa2-luc orthotopic pancreatic carcinoma model. PLoS One 7, e52653 9. Hou, J. et al. (2015) Non-thermal plasma treatment altered gene expression profiling in non-small-cell lung cancer A549 cells. BMC Genomics 16, 435 10. Park, S.B. et al. (2015) Differential epigenetic effects of atmospheric cold plasma on MCF-7 and MDA-MB-231 breast cancer cells. PLoS One 10, e0129931 11. Lin, A. et al. (2015) Uniform nanosecond pulsed dielectric barrier discharge plasma enhances anti-tumor effects by induction of immunogenic cell death in tumors and stimulation of macrophages. Plasma Process. Polym. 12, 1392–1399 12. Miller, V. et al. (2016) Why target immune cells for plasma treatment of cancer. Plasma Chem. Plasma Process. 36, 259–268 13. Metelmann, H.R. et al. (2015) Head and neck cancer treatment and physical plasma. Clin. Plasma Med. 3, 17–23 14. Kieft, I.E. et al. (2004) Electric discharge plasmas influence attachment of cultured CHO K1 cells. Bioelectromagnetics 25, 362–368 15. Yan, D. et al. (2017) The specific vulnerabilities of cancer cells to the cold atmospheric plasma-stimulated solutions. Sci. Rep. 7, 4479

24. Kong, M.G. et al. (2009) Plasma medicine: an introductory review. New J. Phys. 11 25. Ahn, H.J. et al. (2011) Atmospheric-pressure plasma jet induces apoptosis involving mitochondria via generation of free radicals. PLoS One 6, e28154

Are there any regulatory or licensing concerns the field might face?

28. Keidar, M. (2015) Plasma for cancer treatment. Plasma Sources Sci. Technol. 24, 033001 29. Hanahan, D. and Weinberg, R.A. (2011) Hallmarks of cancer: the next generation. Cell 144, 646–674 30. Xu, X.Y. et al. (2018) Quantitative assessment of cold atmospheric plasma anti-cancer efficacy in triple-negative breast cancers. Plasma Process. Polym. http://dx.doi.org/10.1002/ ppap. 201800052 31. Arndt, S. et al. (2013) Cold atmospheric plasma, a new strategy to induce senescence in melanoma cells. Exp. Dermatol. 22, 284–289 32. Barekzi, N. and Laroussi, M. (2012) Dose-dependent killing of leukemia cells by low-temperature plasma. J. Phys. D Appl. Phys. 45, 422002 33. Chernets, N. et al. (2015) Reaction chemistry generated by nanosecond pulsed dielectric barrier discharge treatment is responsible for the tumor eradication in the B16 melanoma mouse model. Plasma Process. Polym. 12, 1400–1409 34. Zirnheld, J.L. et al. (2010) Nonthermal plasma needle: development and targeting of melanoma cells. IEEE Trans. Plasma Sci. 38, 948–952 35. Fridman, G. et al. (2007) Floating electrode dielectric barrier discharge plasma in air promoting apoptotic behavior in melanoma skin cancer cell lines. Plasma Chem. Plasma Process. 27, 163–176 36. Panngom, K. et al. (2013) Preferential killing of human lung cancer cell lines with mitochondrial dysfunction by nonthermal dielectric barrier discharge plasma. Cell Death Dis. 4, e642 37. Zhang, X.H. et al. (2008) Ablation of liver cancer cells in vitro by a plasma needle. Appl. Phys. Lett. 93

17. Thiyagarajan, M. et al. (2012) THP-1 leukemia cancer treatment using a portable plasma device. Stud. Health Technol. Inform. 173, 515–517

39. Iseki, S. et al. (2012) Selective killing of ovarian cancer cells through induction of apoptosis by nonequilibrium atmospheric pressure plasma. Appl. Phys. Lett. 100, 113702

18. Ishaq, M. et al. (2015) Atmospheric-pressure plasma- and TRAILinduced apoptosis in TRAIL-resistant colorectal cancer cells. Plasma Process. Polym. 12, 574–582

40. Utsumi, F. et al. (2014) Selective cytotoxicity of indirect nonequilibrium atmospheric pressure plasma against ovarian clear-cell carcinoma. Springerplus 3, 398

19. Köritzer, J. et al. (2013) Restoration of sensitivity in chemoresistant glioma cells by cold atmospheric plasma. PLoS One 8, e64498

41. Ribassin-Majed, L. et al. (2017) What is the best treatment of locally advanced nasopharyngeal carcinoma? An individual patient data network meta-analysis. J. Clin. Oncol. 35, 498–505

20. Kaushik, N.K. et al. (2016) Cytotoxic macrophage released tumor necrosis factor-alpha (TNF-a) as a killing mechanism for cancer cell death after cold plasma activation. J. Phys. D Appl. Phys. 49, 084001

42. Kaushik, N.K. et al. (2012) Micronucleus formation induced by dielectric barrier discharge plasma exposure in brain cancer cells. Appl. Phys. Lett. 100

23. Ikeda, J. (2014) Effect of nonequilibrium atmospheric pressure plasma on cancer-initiating cells. Plasma Med. 4, 49–56

those

27. Vandamme, M. et al. (2012) ROS implication in a new antitumor strategy based on non-thermal plasma. Int. J. Cancer 130, 2185– 2194

38. Mirpour, S. et al. (2014) The selective characterization of nonthermal atmospheric pressure plasma jet on treatment of human breast cancer and normal cells. IEEE Trans. Plasma Sci. 42, 315–322

22. Ikeda, J. et al. (2015) Anti-cancer effects of nonequilibrium atmospheric pressure plasma on cancer-initiating cells in human endometrioid adenocarcinoma cells. Plasma Process. Polym. 12, 1370–1376

overcome

26. Yan, X. et al. (2012) Plasma-induced death of HepG2 cancer cells: intracellular effects of reactive species. Plasma Process. Polym. 9, 59–66

16. Georgescu, N. and Lupu, A.R. (2010) Tumoral and normal cells treatment with high-voltage pulsed cold atmospheric plasma jets. IEEE Trans. Plasma Sci. 38, 1949–1955

21. Kim, C.H. et al. (2010) Induction of cell growth arrest by atmospheric non-thermal plasma in colorectal cancer cells. J. Biotechnol. 150, 530–538

treatment challenges?

43. Partecke, L.I. et al. (2012) Tissue tolerable plasma (TTP) induces apoptosis in pancreatic cancer cells in vitro and in vivo. BMC Cancer 12, 473 44. Schuster, M. et al. (2016) Visible tumor surface response to physical plasma and apoptotic cell kill in head and neck cancer. J. Craniomaxillofac. Surg. 44, 1445–1452 45. Xu, D. et al. (2016) The effects of cold atmospheric plasma on cell adhesion, differentiation, migration, apoptosis and drug sensitivity of multiple myeloma. Biochem. Biophys. Res. Commun. 473, 1125–1132

Trends in Biotechnology, Month Year, Vol. xx, No. yy

15

TIBTEC 1671 No. of Pages 16

46. Klebanoff, C.A. et al. (2005) Central memory self/tumor-reactive CD8+ T cells confer superior antitumor immunity compared with effector memory T cells. Proc. Natl. Acad. Sci. U. S. A. 102, 9571–9576 47. Chaffer, C.L. et al. (2016) EMT, cell plasticity and metastasis. Cancer Metastasis Rev. 35, 645–654 48. Schmidt, A. et al. (2015) Cell migration and adhesion of a human melanoma cell line is decreased by cold plasma treatment. Clin. Plasma Med. 3, 24–31

postoperative radiation therapy: a case study. Cancer. Biol. Ther. 15, 683–687 68. Sterba, K.R. et al. (2016) Quality of life in head and neck cancer patient-caregiver dyads: a systematic review. Cancer Nurs. 39, 238–250 69. Knutson, K.L. and Disis, M.L. (2005) Tumor antigen-specific T helper cells in cancer immunity and immunotherapy. Cancer Immunol. Immunother. 54, 721–728

49. Zhu, W. et al. (2016) Synergistic effect of cold atmospheric plasma and drug loaded core-shell nanoparticles on inhibiting breast cancer cell growth. Sci. Rep. 6, 21974

70. Utsumi, F. et al. (2013) Effect of indirect nonequilibrium atmospheric pressure plasma on anti-proliferative activity against chronic chemo-resistant ovarian cancer cells in vitro and in vivo. PLoS One 8, e81576

50. Lee, H.J. et al. (2009) Degradation of adhesion molecules of G361 melanoma cells by a non-thermal atmospheric pressure microplasma. New J. Phys. 11, 13

71. Gjika, E. et al. (2018) Adaptation of operational parameters of cold atmospheric plasma for in vitro treatment of cancer cells. ACS Appl. Mater. Interfaces 10, 9269–9279

51. Mirpour, S. et al. (2016) Utilizing the micron sized non-thermal atmospheric pressure plasma inside the animal body for the tumor treatment application. Sci. Rep. 6, 29048

72. Vandamme, M. et al. (2011) Response of human glioma U87 xenografted on mice to non thermal plasma treatment. Plasma Med. 1, 27–43

52. Vasiliou, V. et al. (2000) Role of aldehyde dehydrogenases in endogenous and xenobiotic metabolism. Chem. Biol. Interact. 129, 1–19

73. Kajiyama, H. et al. (2015) Possible therapeutic option of aqueous plasma for refractory ovarian cancer. Clin. Plasma Med. 4, 14–18

53. Ludovini, V. et al. (2011) Phosphoinositide-3-kinase catalytic alpha and KRAS mutations are important predictors of resistance to therapy with epidermal growth factor receptor tyrosine kinase inhibitors in patients with advanced non-small cell lung cancer. J. Thorac. Oncol. 6, 707–715

74. Hoffmann, C. et al. (2013) Cold atmospheric plasma: methods of production and application in dentistry and oncology. Med. Gas Res. 3, 21

54. Murray, S. (2006) Trastuzumab (Herceptin) and HER2-positive breast cancer. CMAJ 174, 36–37 55. Yu, F.L. and Bender, W. (2002) A proposed mechanism of tamoxifen in breast cancer prevention. Cancer Detect. Prev. 26, 370–375 56. Mailliez, A. et al. (2010) Nasal septum perforation: a side effect of bevacizumab chemotherapy in breast cancer patients. Br. J. Cancer 103, 772–775 57. Liedtke, C. and Kiesel, L. (2011) Current issues of targeted therapy in metastatic triple-negative breast cancer. Breast Care (Basel) 6, 234–239 58. Glickman, M.S. and Sawyers, C.L. (2012) Converting cancer therapies into cures: lessons from infectious diseases. Cell 148, 1089–1098 59. Flaherty, K.T. et al. (2012) Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations. N. Engl. J. Med. 367, 1694–1703 60. Eke, I. and Cordes, N. (2015) Focal adhesion signaling and therapy resistance in cancer. Semin. Cancer Biol. 31, 65–75 61. Attia, A. et al. (2014) Treatment of radiation-induced cognitive decline. Curr. Treat. Options Oncol. 15, 539–550 62. Talebian Yazdi, M. et al. (2016) Standard radiotherapy but not chemotherapy impairs systemic immunity in non-small cell lung cancer. Oncoimmunology 5, e1255393 63. Rutkowski, S. et al. (2005) Treatment of early childhood medulloblastoma by postoperative chemotherapy alone. N. Engl. J. Med. 352, 978–986 64. Merchant, T.E. et al. (2014) Critical combinations of radiation dose and volume predict intelligence quotient and academic achievement scores after craniospinal irradiation in children with medulloblastoma. Int. J. Radiat. Oncol. Biol. Phys. 90, 554–561 65. Martin, O.A. et al. (2014) Mobilization of viable tumor cells into the circulation during radiation therapy. Int. J. Radiat. Oncol. Biol. Phys. 88, 395–403 66. Dorsey, J.F. et al. (2015) Tracking viable circulating tumor cells (CTCs) in the peripheral blood of non-small cell lung cancer (NSCLC) patients undergoing definitive radiation therapy: pilot study results. Cancer 121, 139–149 67. Ju, M. et al. (2014) Application of a telomerase-based circulating tumor cell (CTC) assay in bladder cancer patients receiving

16

Trends in Biotechnology, Month Year, Vol. xx, No. yy

75. Rizwan, M. et al. (2018) Surface modification of valve metals using plasma electrolytic oxidation for antibacterial applications: A review. J. Biomed. Mater. Res. A 106, 590–605 76. Lu, X. et al. (2016) Reactive species in non-equilibrium atmospheric-pressure plasmas: Generation, transport, and biological effects. Phys. Rep. 630, 1–84 77. Bekeschus, S. et al. (2016) The plasma jet kINPen – a powerful tool for wound healing. Clin. Plasma Med. 4, 19–28 78. Daeschlein, G. et al. (2012) Cold plasma is well-tolerated and does not disturb skin barrier or reduce skin moisture. J. Dtsch. Dermatol. Ges. 10, 509–515 79. Metelmann, H.R. et al. (2013) Scar formation of laser skin lesions after cold atmospheric pressure plasma (CAP) treatment: A clinical long term observation. Clin. Plasma Med. 1, 30–35 80. Metelmann, H.R. et al. (2012) Experimental recovery of CO2-laser skin lesions by plasma stimulation. Am. J. Cosmet. Surg. 29, 52–56 81. Ulrich, C. et al. (2015) Clinical use of cold atmospheric pressure argon plasma in chronic leg ulcers: a pilot study. J. Wound Care 24, 202–203 82. Isbary, G. et al. (2010) A first prospective randomized controlled trial to decrease bacterial load using cold atmospheric argon plasma on chronic wounds in patients. Br. J. Dermatol. 163, 78–82 83. Isbary, G. et al. (2012) Successful and safe use of 2 min cold atmospheric argon plasma in chronic wounds: results of a randomized controlled trial. Br. J. Dermatol. 167, 404–410 84. Heinlin, J. et al. (2013) Randomized placebo-controlled human pilot study of cold atmospheric argon plasma on skin graft donor sites. Wound Repair Regen. 21, 800–807 85. Maisch, T. et al. (2017) Investigation of toxicity and mutagenicity of cold atmospheric argon plasma. Environ. Mol. Mutagen. 58, 172–177 86. Brehmer, F. et al. (2015) Alleviation of chronic venous leg ulcers with a hand-held dielectric barrier discharge plasma generator (PlasmaDerm1 VU-2010): results of a monocentric, twoarmed, open, prospective, randomized and controlled trial (NCT01415622). J. Eur. Acad. Dermatol. Venereol. 29, 148– 155 87. Gjika, E. et al. (2017) The cutting mechanism of the elctrosurgical scalpel. J. Phys. D Appl. Phys. 50