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International audience; Non-thermal plasmas, produced from dielectric barrier discharges in air or from rare gas plasma jet devices, generate at room temperature, long and short lifetime reactive species (RS) in gaseous phase or in the liquid solution on which they impinge, ionic species, UV photons and intense transient electric fields (EF). In the last decade, the main focus was drawn on the RS to understand and optimize their action in the frame of the broad topic of “Plasma medicine”. More recently, the characterization and implication of EF in biomedical applications is studied, considering the generation of ns to μs duration, kV/cm amplitude features of plasma EF. Plasma have been shown to be a potential alternative to more “conventional technologies” for gene transfer, cell permeabilization, drug delivery in various applications including for cancer therapy. Quite recently, the concepts of reversible electroporation, especially the models of so called pore formation in cell membranes were broaden and detailed involving not only the key role of EF but also the oxidation of cell membrane associated with RS. Thus it appears we attend to a unique opportunity to reach more insights and achieve cell manipulation process optimization with considering the potential complementary or synergistic delivery of pulsed electric fields and plasma for in vitro and in vivo protocols.Besides a quick overview of some significant works focused on the use of non-thermal plasmas in interaction with cell membranes, the lecture will document the results obtained so far with the Plasma Gun developed at GREMI for first, cancer cell permeabilization with various plasma delivery protocols, and second on the first demonstrations of the positive combined action of pulsed electric fields (PEF) and plasma for antitumor action. Cell permeabilization has been performed in vitro for different adherent cell lines, exposed to plasma jet delivered at various pulse repetition rates for short duration and followed with cell incubation in plasma treated solution. The combination of plasma and PEF is studied in vitro and with murine models considering the delivery of pulsed voltage and current pulses (electroporation) together with plasma treated solutions in contact with biological targets (suspended cancer cells in electroporation cuvettes or tumors) at different delays with respect to the electroporation step.AcknowledgmentsWork supported by PLASCANCER project INCa-PlanCancer-n°17CP087-00 and GDR CNRS 2025 HAPPYBIO

International audience; The last decade has seen an impressive increase of the research dedicated to the biomedical applications of low temperature plasmas, especially with plasma sources working at atmospheric pressure. In this new trend, beside decontamination/sterilization and surface treatment that have already a quite long story through low-pressure plasma research and developments, medical applications are tacking an increasing place underlined by the actual numerous clinical trials. Medical applications of low temperature plasmas now concern a very wide range of domains, including primary haemostasis and blood coagulation, dental care, skin decontamination and hygiene, wound and ulcer treatment, dermatology, cancer treatment. Biological applications are also now extended to agriculture and, more recently, to cosmetic. Despite the huge number of in vitro and in vivo experiments there are still numerous challenges to overcome linked to the nature of the encountered target (biological tissues and materials, organs and their direct environment, liquids) that have a direct effect on the produced plasma itself and on the generated species. It is clear that the extremely strong coupling between the characteristics of the plasma and those of the target, as already shown, will play a very important role in the results observed during the treatments. A variation in the chemical or physical characteristics of the target will involve significant differences in the gas flow, the local temperature, or the induced electric field, resulting de facto in variations in the production of the reactive species. It also concerns the transposition of the results between the in vitro and the in vivo experiments that are carried out under extremely different conditions, especially concerning the equivalent electric circuit of the reactor / plasma / biological target assembly. These problems directly affect the identification of the processes involved and currently limit the possibility of a definition of a "dose" in plasma treatments. Recently, study reported led to reflections on non-sustainable tumor response. The loss of effectiveness under long-term plasma treatment of cancer tissue opens questions about plasma application and protocol. It must be considered that the treated area is morphologically and chemically changing over the time, from activated surrounding to more normal tissues that are less humid and bacteriologically cleaner. This aspect is particularly important for the development of efficient systems and protocols in plasma cancer treatment but also for any other plasma therapeutically approaches. It induce a in real-time in situ control of plasma production and at longer term a protocol adaptation taking into account the biological target evolution. Some progress are already done in that domain

International audience; Compelling evidence suggests that Cold Atmospheric Pressure Plasma (CAPPs) has potential as a new cancer therapy. However, knowledge about cellular signaling events and toxicity subsequent to plasma treatment of is still poorly documented. The aim of this study was to focus on the interaction between 3 different types of plasma (He, He-O2, He-N2) and human epithelial cell lines to gain better insight into plasma-cell interaction. We choose epithelial cell lines (human keratinocytes, human fibroblasts, human colorectal carcinoma and skin melanoma) to gain insight into plasma-cell interaction and to determine which cellular pathways are induced by CAPP treatment in normal and malignant cells. We provide evidence that the proteasome, a major intracellular proteolytic system which is important for tumor cell growth and survival, is a target for (He or He-N2) CAPP. This is accompanied by apoptosis, DNA damage, mitochondrial dysfunction and mild oxidative protein damage. By differential label-free quantitative proteomic analysis we found that CAPP triggers antioxidant and cellular defense but also seen cancerous processes in keratinocytes. Moreover we found that malignant cells are more resistant to CAPP treatment than normal cells. However, reactiveoxygen and nitrogen species (RONS) are not the only actors involved in cell death; electric field and charged particles could play a significant role especially for He-O2 CAPP. Particular attention was paid to the contribution of the electric field (EF) and RONS in cellular response to plasma treatment. Taken together, our findings provide insight into potential mechanisms of CAPP-induced cell death.

International audience; The antitumor action of Low Temperature Non Thermal Plasma (LTNTP) has been now widely demonstrated both in vitro and in vivo (on animal models and more recently on human [1]) on a large variety of cancer cell lines and tumor types. Nevertheless, due to the great variety of plasma components able to play a role, the precise understanding of the process chain leading to the observed effects is far to be reached. It is not easy to isolate or to measure the plasma component individual action or, in contrast, to evaluate the potential benefit or synergy of combined ones. Up to now, many experiments in vitro have been realized with that intention, but the translation to in vivo results is rather complicated, even, in many cases, impossible due to the complex environment of the cells in vivo, including vascularization (then blood flow and oxygenation/physioxia) and the body response (signaling, recruitment, immune system). Generally, the first step of dedicated studies concerns in vitro cell viability or toxicity in culture media with the analysis of both the induced changes in the cells and in the medium. It must be stressed that the used experimental conditions together with the fact to treat cells in multiwell plates outside their incubation chamber may generate cell stress that can affect their behavior and then leading to biased results. This presentation will show some of the issues and challenges associated with in vitro cancerous cells treatments by a plasma jet (here a Plasma Gun), also in relation with in vivo conditions associated with the hypoxic/physioxic environment.We will present results on direct effect of plasma treatment on both the culture medium and cells, including liquid evaporation, oxygen deprivation, extra and intracellular RONS generation, membrane poration, cell depolarization, with the potential role of electric field in the treated and adjacent wells [2]. We will particularly emphasize the importance of cell oxygenation level which is a key factor in the proliferation/regression of tumors in hypoxic environment. It will be shown that an uncontrolled plasma jet in vitro treatments can lead to drastic changes in the oxygen pressure (and temperature) in cell culture during the experiment with sequences of maxima (leading to normoxia) and minima (potentially leading to strong hypoxia). The latter contrasts with the plasma oxygen pressure and blood flow increases measured in the in vivo conditions [3]. This also means that a great attention must paid in the modeling of interactions of plasma jets with physiological liquid media since the initial concentration of oxygen can be greatly affected. AcknowledgmentsThis work is founded by ARD 2020 Cosmetoscience PLASMACOSM project. X.D. is supported by INEL/Region Centre-Val de Loire fellowship.References[1] Metelmann et al, Clinical Plasma Medicine, 3 17-23 (2015).[2] E. Robert, T. Darny, S. Dozias, S. Iseni, and J. M. Pouvesle, Phys. Plasmas 22, 122007 (2015).[3] G. Collet, E. Robert, A. Lenoir, M. Vandamme, T. Darny, S. Dozias, C. Kieda, and J. M. Pouvesle, Plasma Sources Sci. Technol.23, 012005 (2014).

International audience; The antitumor effect of Non Thermal Plasmas (NTP) has been clearly shown in vivo on murine models with various cancer types (bladder, colon, glioblastoma, melanoma, ovary, pancreas). Although the mechanism is far from being fully understood, the therapeutic effect is now totally admitted. To date, the literature reports mainly induction of apoptosis, effect which is not restricted to cancer cells and probably mask other aspects of the NTP for cancer therapy. In case of plasma jet experiments, the observed effect are most of the time attributed to the very rich chemistry generated by the interaction of the rare gas plasma plume with the surrounding environment constituted either from the ambient air, or this last one in complex interaction with liquids at the interface with the targeted organ. Our recent experiments performed on tissue oxygenation1 or breast cancer treatments on immunocompetent mice2 lead to the conclusion that probably the involved chemistry couldn’t, alone, completely allow to describe the observed phenomena. This, especially under very soft treatment conditions, is suggesting possible triggering of some immune system chain processes, but also possible modifications in the microenvironment of tissue and tumors. In this context, there is still an unknown role of the electric field associated with the ionization front or generated in the environment of the plasma plume tip. Taking into consideration the recent vessel normalization based-cancer treatment, the NTP effect should be further investigated in view of blood vessels structure and function (blood flow) as well as tumor hypoxia compensation to confirm a possible NTP-based adjuvant approach for cancer treatments. These results suggest new ways, especially combined therapy, to consider the plasma and its therapeutic delivery in NTP-based tumor therapy. All these aspects will be discussed during the presentation, including recent results on electric field measurements in plasma biological application conditions. This work is supported by the APR Region Centre PLASMEDNORM.References[1] G. Collet, E. Robert, A. Lenoir, M. Vandamme, T. Darny, S. Dozias, C. Kieda, , J.M. Pouvesle, “Plasma jet-induced tissue oxygenation: potentialities for new therapeutic strategies” Plasma Sources Sci. Technol. 23 (2014) 012005[2] G. Collet, L. Ridou, E. Robert, M. Vandamme, T. Darny, S. Dozias, C. Kieda, , J.M. Pouvesle “NTP antitumor soft treatment: evidence of a triggering effect? ”, ICPM5, May 18-23, 2014, Nara (Japan)