Vandana Miller

Why Target Immune Cells for Plasma Treatment of Cancer

This paper addresses the challenge of using non-equilibrium plasma as a therapeutic approach for diseases of body systems not readily accessible to plasma-generated factors. The role of plasma stimulation of the immune system is discussed as a conceivable mechanism to deliver effects. This is especially important for treatment of cancers since the pathogenesis and progression of cancers are directly influenced by immune function. By optimizing plasma parameters to induce immunogenic cell death in tumors locally, it is possible to trigger specific, protective immune responses systemically. The observations from in vitro and in vivo investigations on this subject are reviewed here. An in depth understanding of the interaction between plasma components and the cells of the immune system may provide necessary information for use of plasmas in treatment of many systemic diseases. The clinical implications of treating cancers with non-equilibrium plasma are considered. The paper also identifies some hurdles that must be overcome before plasma immunotherapy becomes a clinical reality.

Cytotoxic macrophage-released tumour necrosis factor-alpha (TNF-α) as a killing mechanism for cancer cell death after cold plasma activation

The present study aims at studying the anticancer role of cold plasma-activated immune cells. The direct anti-cancer activity of plasma-activated immune cells against human solid cancers has not been described so far. Hence, we assessed the effect of plasma-treated RAW264.7 macrophages on cancer cell growth after co-culture. In particular, flow cytometer analysis revealed that plasma did not induce any cell death in RAW264.7 macrophages. Interestingly, immunofluorescence and western blot analysis confirmed that TNF-α released from plasma-activated macrophages acts as a tumour cell death inducer. In support of these findings, activated macrophages down-regulated the cell growth in solid cancer cell lines and induced cell death in vitro. Together our findings suggest plasma-induced reactive species recruit cytotoxic macrophages to release TNF-α, which blocks cancer cell growth and can have the potential to contribute to reducing tumour growth in vivo in the near future.

Nanosecond-Pulsed DBD Plasma-Generated Reactive Oxygen Species Trigger Immunogenic Cell Death in A549 Lung Carcinoma Cells through Intracellular Oxidative Stress

A novel application for non-thermal plasma is the induction of immunogenic cancer cell death for cancer immunotherapy. Cells undergoing immunogenic death emit danger signals which facilitate anti-tumor immune responses. Although pathways leading to immunogenic cell death are not fully understood; oxidative stress is considered to be part of the underlying mechanism. Here; we studied the interaction between dielectric barrier discharge plasma and cancer cells for oxidative stress-mediated immunogenic cell death. We assessed changes to the intracellular oxidative environment after plasma treatment and correlated it to emission of two danger signals: surface-exposed calreticulin and secreted adenosine triphosphate. Plasma-generated reactive oxygen and charged species were recognized as the major effectors of immunogenic cell death. Chemical attenuators of intracellular reactive oxygen species successfully abrogated oxidative stress following plasma treatment and modulated the emission of surface-exposed calreticulin. Secreted danger signals from cells undergoing immunogenic death enhanced the anti-tumor activity of macrophages. This study demonstrated that plasma triggers immunogenic cell death through oxidative stress pathways and highlights its potential development for cancer immunotherapy.

Successful treatment of actinic keratoses using nonthermal atmospheric pressure plasma: A case series

Latitude Highest* 7.2 6.4 Lowesty 14.0 15.2 Age, y \50 3.4 6.0

Microsecond-pulsed dielectric barrier discharge plasma stimulation of tissue macrophages for treatment of peripheral vascular disease

80 8.8 11.8 Sex Female 3.0 6.8 Male 7.4 11.0 Sun sensitivity #25th percentile 5.0 10.2

A Comparison of Floating-Electrode DBD and kINPen Jet: Plasma Parameters to Achieve Similar Growth Reduction in Colon Cancer Cells Under Standardized Conditions

75th percentile 9.8 13.7 History of 5-FU use No 6.1 9.9 Yes 12.3 15.4 No. of prior invasive SCCs Lowest quintile 5.3 11.3 Highest quintile 13.6 16.0 No. of prior in situ SCCs Lowest quintile 6.5 10.5 Highest quintile 12.4 12.8 Photodamage Absent/mild 6.5 n/a Severe 11.4 n/a Focal dermatitis None 5.6 n/a Moderate/severe 14.3 n/a Rosacea with rhinophyma None 6.3 n/a Moderate/severe 12.0 n/a

Use of cold atmospheric pressure plasma to treat warts: a potential therapeutic option

Angiogenesis is the formation of new blood vessels from pre-existing vessels and normally occurs during the process of inflammatory reactions, wound healing, tissue repair, and restoration of blood flow after injury or insult. Stimulation of angiogenesis is a promising and an important step in the treatment of peripheral artery disease. Reactive oxygen species have been shown to be involved in stimulation of this process. For this reason, we have developed and validated a non-equilibrium atmospheric temperature and pressure short-pulsed dielectric barrier discharge plasma system, which can non-destructively generate reactive oxygen species and other active species at the surface of the tissue being treated. We show that this plasma treatment stimulates the production of vascular endothelial growth factor, matrix metalloproteinase-9, and CXCL 1 that in turn induces angiogenesis in mouse aortic rings in vitro. This effect may be mediated by the direct effect of plasma generated reactive oxygen species on tissue.

Abstract 18: Therapeutic potential of cold physical plasma in palliative cancer care: Introduction and perspectives

A comparative study of two plasma sources (floating-electrode dielectric barrier discharge, DBD, Drexel University; atmospheric pressure argon plasma jet, kINPen, INP Greifswald) on cancer cell toxicity was performed. Cell culture protocols, cytotoxicity assays, and procedures for assessment of hydrogen peroxide (H2O2) were standardized between both labs. The inhibitory concentration 50 (IC50) and its corresponding H2O2 deposition was determined for both devices. For the DBD, IC50 and H2O2 generation were largely dependent on the total energy input but not pulsing frequency, treatment time, or total number of cells. DBD cytotoxicity could not be replicated by addition of H2O2 alone and was inhibited by larger amounts of liquid present during the treatment. Jet plasma toxicity depended on peroxide generation as well as total cell number and amount of liquid. Thus, the amount of liquid present during plasma treatment in vitro is key in attenuating short-lived species or other physical effects from plasmas. These in vitro results suggest a role of liquids in or on tissues during plasma treatment in a clinical setting. Additionally, we provide a platform for correlation between different plasma sources for a predefined cellular response.

Contribution of electric fields and active species in nanosecond pulsed DBD plasma treatment for stimulation of murine mesenchymal C3H10T1/2 cells

Treating warts can be frustrating for patients and dermatologists alike, as they can recur frequently and often need multiple treatments, which carry a risk of adverse effects. Cold atmospheric-pressure plasma (CAP) is an ionized gas that has been extensively studied for medical applications, including clinical trials for skin conditions. Safety of CAP use on skin has been well demonstrated in the past. Patient 1 was a 33-year-old man, who presented with four warts on his fingers. The warts failed to improve with over-the-counter products and liquid nitrogen treatments performed over 6 months. Patient 2 was a 28-year-old man, who presented with a wart on his wrist. He refused cryotherapy because of possible hypopigmentation and he could not afford topical medications. Both patients were enrolled in a study approved by the Western Institutional Review Board (protocol number: 20130084 and registered on ClinicalTrials.gov (registration number: NCT02759900). Our CAP source was a pulse generator (FPG10– 01NM10; FID GmbH, Burbach, Germany) supplying 20 kV pulse of 20 ns pulse width at 400 Hz to a quartzcovered copper electrode (10 cm in length, 1 mm thick quartz cover, 5 mm total diameter (Fig. 1). Duration of treatment was 2 min/lesion. The thickest (right thumb) lesion of Patient 1 cleared after two treatment, and the other lesions after three treatments (Fig. 2), and remained clear 7 and 6 months later, respectively. Four months after the start of treatment, a new wart appeared on the left thumb, which cleared with three treatments. Patient 2 was treated twice and, according to him but not confirmed by us, the wart improved but never resolved. He never returned for subsequent treatments. By the time of his 5-month evaluation, the wart had recurred (Fig. 2). We treated two patients with a total of six warts. Our first patient had resolution of all lesions; he later developed a new wart during the observation period, which also cleared after three treatments. This suggests that our results were due to a local CAP effect and not a generalized immune response. The second patient reported improvement of his single lesion, but it recurred after treatment was stopped, indicating that CAP did not have a lasting inhibitory effect on the partially cleared wart. It is unknown if continuing treatments would have altered the outcome. We are not aware of previous in vivo or in vitro models to explain the effect of CAP on warts, but CAP has been shown to selectively induce apoptosis in malignant cells in vitro. In vivo efficacy of CAP in treating actinic keratosis has also been reported. It can be hypothesized that CAP may induce apoptosis in warts. It has been reported that CAP inhibits adenoviruses, possibly by altering the virus capsid. It is feasible that the capsid of human papilloma virus, a similarly nonenveloped DNA virus, is also susceptible to CAP-induced damage. Although the mechanism of action is unclear, and the number of treated lesions was very small, meaning that larger follow-up studies are needed, the results of this proof-of-concept study are promising, with clinical improvement of warts achieved by CAP treatment and lack of recurrence of fully cleared lesions after several months. Encouragingly, the cost of a market-ready CAP device could be on par with current electrosurgery tools. Figure 1 Device used for wart treatment. Custom-built cold atmospheric pressure plasma device with handheld electrode.

Uniform Nanosecond Pulsed Dielectric Barrier Discharge Plasma Enhances Anti‐Tumor Effects by Induction of Immunogenic Cell Death in Tumors and Stimulation of Macrophages

Background: Patients suffering from advanced head and neck tumors frequently suffer from superinfected chronic wounds caused by necrotic tissue due to progressive tumor growth, weak systemic and local immunologic response and various accompanying illnesses. Because of wound vulnerability, local antiseptic wound care of microbial-contaminated tumor areas is frequently complicated by bleeding, pain and patient dissatisfaction. Plasma medicine in cancer is of palliative benefit in these cases and among the fastest growing of the current applications of cold atmospheric pressure physical plasma (CAP). Since several plasma medical devices have received approval for treatment of infected skin and ulcerations by the relevant agencies, limited clinical tests have begun on humans and palliative treatment of cancer patients with contaminated ulcers. This report as part of a large-scale study program illustrates one recent impressive application of CAP to a patient suffering from advanced head and neck cancer. Materials and Methods: After curably intended surgical cancer treatment of a well-differentiated squamous cell carcinoma of the left cheek January 2015, the 51-year-old patient noticed a rapidly progressive swelling on the left neck in June. CT scan indicated a large contrast enhancing mass, which was suspected to be tumor recurrence. Operative findings revealed inoperability due to infiltrating the vascular wall of the external carotid. After a palliative intended combined radio-chemotherapy, the tumor was characterized by progressive growth with exulceration. Due to the vulnerability of the extended bacterially contaminated wound and the underlying carotid artery, wound care was difficult. Since October 2015, a supportive palliative cancer treatment using CAP has been started with the patient9s written consent. The exulcerative tumor growth region received treatment with the kINPenMED (Neoplas GmbH, Greifswald, Germany) by scanning the surface for 5 minutes in a meandering manner. Plasma treatment continued to be performed every 3 days. Wound care was implemented in conjunction with an antiseptic wound dressing. Results: The superinfected necrotic tumor areas appeared to be clean of cell detritus and bacteria. Microbiological examination revealed a reduction of bacterial colonization which led to decrease of wound odor, too. Due to the decrease of inflammation, vulnerability and pain have been reduced significantly. Upon CAP therapy a partial tumor response with tumor mass reduction was observed. The ulcerated tumor area has been reduced to one-quarter of its original size. The underlying carotid artery is still intact and ultrasound investigation revealed a regular blood flow. Histologic examinations revealed an increased amount of apoptic tumor cells and a local increase of immune defense. Furthermore, a desmoplastic reaction of the conjunctive tissue represented by a higher proliferation rate of fibroblasts could be depicted. No plasma-relevant systemic side effects have occurred. Conclusion: By sufficient reduction of bacterial colonization, decrease of inflammation, wound vulnerability and pain, CAP constitutes an innovative and valuable treatment option in palliative cancer care. Local tumor mass reduction is an unexpected and promising response during CAP treatment and has to be further examined. Plasma-unique synergies between reactive species, charges and electric fields must be more fully explored and understood. The impressive demonstration of medical efficacy of CAP in cancer ulcerations supports optimism for trials of plasma medical devices with the intention of an adaptive cancer treatment protocol. Citation Format: Christian Seebauer, Thomas von Woedtke, Klaus-Dieter Weltmann, Vandana Miller, Masaru Hori, Hans-Robert Metelmann. Therapeutic potential of cold physical plasma in palliative cancer care: Introduction and perspectives [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 18.