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We developed a volatile organic compound (VOC)-based probe cocktail for monitoring the dysregulation of tumour-associated enzymes via the detection of volatile tracers in animal breath, thereby allowing the discovery of a new tumour targeted therapy.

The discovery of tumour-associated markers is of major interest for the development of selective cancer chemotherapy. Within this framework, we introduced the concept of induced-volatolomics enabling to monitor simultaneously the dysregulation of several tumour-associated enzymes in living mice or biopsies. This approach relies on the use of a cocktail of volatile organic compounds (VOCs)-based probes that are activated enzymatically for releasing the corresponding VOCs. Exogenous VOCs can then be detected in the breath of mice or in the headspace above solid biopsies as specific tracers of enzyme activities. Our induced-volatolomics modality highlighted that the up-regulation of N-acetylglucosaminidase was a hallmark of several solid tumours. Having identified this glycosidase as a potential target for cancer therapy, we designed an enzyme-responsive albumin-binding prodrug of the potent monomethyl auristatin E programmed for the selective release of the drug in the tumour microenvironment. This tumour activated therapy produced a remarkable therapeutic efficacy on orthotopic triple-negative mammary xenografts in mice, leading to the disappearance of tumours in 66 % of treated animals. Thus, this study shows the potential of induced-volatolomics for the exploration of biological processes as well as the discovery of novel therapeutic strategies.

International audience; Porous materials, such as zeolites, have great potential for biomedical applications, thanks to their ability to accommodate positively charged metal-ions and their facile surface functionalization. Although the latter aspect is important to endow the nanoparticles with chemical/colloidal stability and desired biological properties, the possibility for simple ion-exchange enables easy switching between imaging modalities and/or combination with therapy, depending on the envisioned application. In this study, the nanozeolite Linde type L (LTL) with already confirmed magnetic resonance imaging properties, generated by the paramagnetic gadolinium (GdIII) in the inner cavities, was successfully radiolabeled with a positron emission tomography (PET)-tracer zirconium-89 (89Zr). Thereby, exploiting 89Zr-chloride resulted in a slightly higher radiolabeling in the inner cavities compared to the commonly used 89Zr-oxalate, which apparently remained on the surface of LTL. Intravenous injection of PEGylated 89Zr/GdIII-LTL in healthy mice allowed for PET-computed tomography evaluation, revealing initial lung uptake followed by gradual migration of LTL to the liver and spleen. Ex vivo biodistribution confirmed the in vivo stability and integrity of the proposed multimodal probe by demonstrating the original metal/Si ratio being preserved in the organs. These findings reveal beneficial biological behavior of the nanozeolite LTL and hence open the door for follow-up theranostic studies by exploiting the immense variety of metal-based radioisotopes.

Background/aim To develop and validate a nebulizer device for anti-cancer research on pressurized intraperitoneal aerosol supply in a preclinical peritoneal metastases (PM) rat model. Material and methods For aerosol generation, an ultrasonic nebulizer (USN) was modified. Aerosol analyses were performed ex-vivo by laser diffraction spectrometry (LDS). Intraperitoneal (IP) 99mtechnetium sodium pertechnetate (99mTc) aerosol distribution and deposition were quantified by in-vivo single photon emission computed tomography (SPECT/CT) and compared to liquid IP instillation of equivalent volume/doses of 99mTc with and without capnoperitoneum. PM was induced by IP injection of HCT116-Luc2 human colon cancer cells in immunosuppressed RNU rats. Tumor growth was monitored by bioluminescence imaging (BLI), 18F-FDG positron emission tomography (PET) and tissues examination at necropsy. Results The USN was able to establish a stable and reproducible capnoperitoneum at a pressure of 8 to 10 mmHg. LDS showed that the USN provides a polydisperse and monomodal aerosol with a volume-weighted diameter of 2.6 μm. At a CO2 flow rate of 2 L/min with an IP residence time of 3.9 s, the highest drug deposition efficiency was found to be 15 wt.-%. In comparison to liquid instillation, nebulization showed the most homogeneous IP spatial drug deposition. Compared to BLI, 18F-FDG-PET was more sensitive to detect smaller PM nodules measuring only 1–2 mm in diameter. BLI, 18F-FDG PET and necropsy analyses showed relevant PM in all animals. Conclusions The USN together with the PM rat model are suitable for robust and species-specific preclinical pharmacological studies regarding intraperitoneal delivery of pressurized aerosolized drugs and cancer research.

Pressurized Intraperitoneal Aerosol Chemotherapy (PIPAC) is a promising approach with a high optimization potential for the treatment of peritoneal carcinomatosis. To study the efficacy of PIPAC and drugs, first rodent cancer models were developed. But inefficient drug aerosol supply and knowledge gaps concerning spatial drug distribution can limit the results based on such models. To study drug aerosol supply/deposition, computed tomography scans of a rat capnoperitoneum were used to deduce a virtual and a physical phantom of the rat capnoperitoneum (RCP). RCP qualification was performed for a specific PIPAC method, where the capnoperitoneum is continuously purged by the drug aerosol. In this context, also in-silico analyses by computational fluid dynamic modelling were conducted on the virtual RCP. The physical RCP was used for ex-vivo granulometric analyses concerning drug deposition. Results of RCP qualification show that aerosol deposition in a continuous purged rat capnoperitoneum depends strongly on the position of the inlet and outlet port. Moreover, it could be shown that the droplet size and charge condition of the drug aerosol define the deposition efficiency. In summary, the developed virtual and physical RCP enables detailed in-silico and ex-vivo analyses on drug supply/deposition in rodents.

Simple Summary Intraperitoneal chemotherapy (IPC) is a locoregional treatment option in patients with peritoneal metastases (PM). Small animal models are valuable research tools allowing for rapid, reproducible, and inexpensive study and optimization of different forms of IPC. Here, we present a mouse model of ovarian cancer-derived PM and demonstrate its suitability for various modes of IPC, including pressurized intraperitoneal chemotherapy (PIPAC) using a micro-nozzle for aerosolized drug delivery. Abstract Intraperitoneal chemotherapy (IPC) is a locoregional treatment option in patients with peritoneal metastases (PM). Here, we present an ovarian cancer (OC)-derived PM mouse model for the study of different forms of IPC. Xenograft cell proliferation (luciferase-transfected OVCAR3 and SKOV3 clones) and growth kinetics were assessed using PET scan, bioluminescence imaging (BLI), and histological tumor analysis. Liquid IPC was achieved by intraperitoneal injection with/without capnoperitoneum (6–7 mmHg). Pressurized intraperitoneal aerosol chemotherapy (PIPAC) was mimicked using an intratracheal drug aerosol administration system (micro-nozzle), which, as demonstrated by ex vivo granulometric analysis using laser diffraction spectrometry, produced a polydisperse, bimodal aerosol with a volume-weighted median diameter of (26.49 ± 2.76) µm. Distribution of Tc-99m-labeled doxorubicin in mice was characterized using SPECT and was dependent on the delivery mode and most homogeneous when the micro-nozzle was used. A total of 2 mg doxorubicin per kg body weight was determined to be the optimally effective and tolerable dose to achieve at least 50% tumor reduction. Repeated PIPAC (four times at seven-day-intervals) with doxorubicin in SKOV3-luc tumor-bearing mice resulted in halted tumor proliferation and tumor load reduced after the second round of PIPAC versus controls and the number of tumor nodules was significantly reduced (27.7 ± 9.5 vs. 57.3 ± 9.5; p = 0.0003). Thus, we established the first mouse model of OC PM for the study of IPC using a human xenograft with SKOV3 cells and an experimental IPC setup with a miniaturized nozzle. Repeated IPC was feasible and demonstrated time-dependent anti-tumor activity.

Intraperitoneal chemotherapy (IPC) is a locoregional treatment option in patients with peritoneal metastases (PM). Here, we present an ovarian cancer (OC)-derived PM mouse model for the study of different forms of IPC. Xenograft cell proliferation (luciferase-transfected OVCAR3 and SKOV3 clones) and growth kinetics were assessed using PET scan, bioluminescence imaging (BLI), and histological tumor analysis. Liquid IPC was achieved by intraperitoneal injection with/without capnoperitoneum (6–7 mmHg). Pressurized intraperitoneal aerosol chemotherapy (PIPAC) was mimicked using an intratracheal drug aerosol administration system (micro-nozzle), which, as demonstrated by ex vivo granulometric analysis using laser diffraction spectrometry, produced a polydisperse, bimodal aerosol with a volume-weighted median diameter of (26.49 ± 2.76) µm. Distribution of Tc-99m-labeled doxorubicin in mice was characterized using SPECT and was dependent on the delivery mode and most homogeneous when the micro-nozzle was used. A total of 2 mg doxorubicin per kg body weight was determined to be the optimally effective and tolerable dose to achieve at least 50% tumor reduction. Repeated PIPAC (four times at seven-day-intervals) with doxorubicin in SKOV3-luc tumor-bearing mice resulted in halted tumor proliferation and tumor load reduced after the second round of PIPAC versus controls and the number of tumor nodules was significantly reduced (27.7 ± 9.5 vs. 57.3 ± 9.5; p = 0.0003). Thus, we established the first mouse model of OC PM for the study of IPC using a human xenograft with SKOV3 cells and an experimental IPC setup with a miniaturized nozzle. Repeated IPC was feasible and demonstrated time-dependent anti-tumor activity.

Cytosolic Ca2+ oscillations provide signaling input to several effector systems of the cell. These include neuronal development, migration and networking. Although similar signaling events are hijacked by highly aggressive cancer cells, the complexity of the ‘neuron-like’ remodeling in metastasis remains to be explored. Here, using a variety of in vitro and in vivo techniques we show that strongly metastatic prostate cancer cells acquire specific Na+/Ca2+ signature required for persistent invasion. We identify the ‘neuronal’ Na+ leak channel, NALCN, at the hot spots of the Ca2+ wave initiation and invadopodia formation. Mechanistically, NALCN associates functionally with plasmalemmal and mitochondrial Na+/Ca2+ exchangers, reactive oxygen species and store-operated channels to generate intracellular Ca2+ oscillations. In turn, this stimulates the activity of protooncogene Src kinase co-localized with NALCN, actin remodeling and secretion of proteolytic enzymes, thus increasing an invasive potential of the cancer cells and metastatic lesions in vivo (accessed in pre-clinical models). Overall, our findings provide new insight into the signaling pathway specific for metastatic cells where NALCN plays the role of the persistent invasion “launcher and controller”.

In this study we implemented an axillary SLN invasion model to develop highly sensitive imaging strategies enabling detection of a very small amount of tumor cells. A highly diffusible molecular probe targeting αvβ3 and αvβ5 integrins was investigated either via IV or locoregional injections. We additionally documented the potential interferences of this Near Infrared Fluorescence Probe with Blue Patente V and ICG dyes routinely used to facilitate lymph node detection during surgery. The human mammary adenocarcinoma MDA-MB-231-luc model was injected into the forepaw of nude female rats to obtain a controlled invasion of the axillary LN. Thanks to its high sensitivity, BLI was selected to achieve in vivo quantitation of tumor cells in SLNs and determine eligible animals for the study. NIRF of integrins was performed at 680 nm both in vivo and ex vivo using spectral unmixing to suppress auto-fluorescence signal and preserve sensitivity. In vivo BLI was quite reliable in estimating discrete invasion by cancer cells in the LN with thresholds of detection and quantitation of about 500 and 1500 cells respectively. For fluorescence at 680 nm, in vivo imaging is not suitable to detect micro-invasion, but ex vivo fluorescence with spectral unmixing of SLNs confirmed the presence of a tumor burden as low as 1500 cells expressing αvβ3/αvβ5 integrins. Targeting few tumor cells inside a micro-invaded sentinel lymph node by molecular probes is not sensitive enough to provide direct in vivo or peroperative imaging. At the time NIRF is performed on the excised specimen, high sensitivity imaging associated with spectral unmixing allowed such detection within less than 1 minute of examination.

International audience; Multimodal probes capable of combining imaging modalities within a single molecule are in high demand today as they can provide information at both molecular and anatomical levels. Herein, a study was conducted on a series of gallium(III)/lanthanide(III) bis(12-MC-4) metallacrowns (MCs) with the general composition {Ln[12-MCGa III N(shi) -4]}2 (iph)4 (Ln-Ix , x=0, 4, 8, 12), where shi and iph are salicylhydroximate and isophthalate ligands, respectively, or their iodinated derivatives. For Yb-Ix , the attenuation in X-ray computed tomography (XCT) imaging and near-infrared (NIR) luminescence properties can be finely tuned by controlled structural modifications based on iodo groups. Solutions of Yb-Ix appear to be 22-40 times more efficient as XCT agents in comparison to the commercially available iobitridol, while providing an intense emission signal in the NIR range with total quantum yields up to 8.6 %, which are among the highest values reported so far. Therefore, these molecules are promising potential bimodal agents for combined NIR luminescence and XCT imaging.

We propose quantitative assessment of zinc by combining nuclear and MR imaging. We use a cocktail of a Gd3+-complex providing a Zn2+-dependent MRI response and its 165Er3+ analogue allowing for concentration assessment. 165Er is readily obtained in a cyclotron and purified, which is indispensable for successful quantification of metal ions.

We propose quantitative assessment of zinc by combining nuclear and MR imaging. We use a cocktail of a Gd3+-complex providing a Zn2+-dependent MRI response and its 165Er3+ analogue allowing for concentration assessment. 165Er is readily obtained in a cyclotron and purified, which is indispensable for successful quantification of metal ions.

Human small cell lung carcinoma (SCLC) is the most aggressive type of lung cancer but no clinically relevant animal model has been developed to date. Such a model would be valuable to study the molecular aspects of tumour progression and to test the effectiveness of new treatment agents. We generated a reproducible and reliable nude mouse orthotopic model of human SCLC with NCI-H209 tumour cells genetically modified to express firefly luciferase. Cells were analysed for long-term stability of bioluminescence and a clone was passaged twice subcutaneously to enhance tumorigenicity. Cells resuspended in Matrigel and/or EDTA RPMI medium containing a (99m)Tc-labelled tin colloid used as tracer were implanted intrabronchially with a catheter inserted into the trachea and positioned in the main bronchus using X-ray-guided imaging. Deposition of cells into the lung was then assessed by scintigraphy. The growth of the primary tumour was sensitively and non-invasively followed by bioluminescence imaging that allowed real-time monitoring of tumour progression in the same animals over a 2-12-week period. Additional 3D bioluminescence imaging and computed tomography scanning were used to document tumour location and measurements that were confirmed by histological analyses. In conclusion, this original nude mouse orthotopic model resembles various stages of human small cell lung cancer, and therefore could be used to evaluate new treatment strategies.

This work was conducted to evaluate the potential antitumor effect of an in vivo plasma treatment on a U87-luc glioma tumor. A pulsed DBD with µs pulses at moderate power was used for treatment. Electrical analyses and spectroscopic measurements were realized to characterize plasma properties. We showed that the plasma treatment was safe for mice. However, the reiterated long time plasma treatment (20 min, 3 consecutive days) produces a superficial burn. During plasma treatment, we observed an increase of subcutaneous temperature and a cutaneous skin pH reduction. After 5 d of plasma treatment, we observed a dramatical U87 bioluminescence decrease associated with a reduction of tumor volume in U87 glioma-bearing mice

Galactomannans act as storage reserves for the seeds in some plants, such as guar (Cyamopsis tetragonoloba) and coffee (Coffea arabica and Coffea canephora). In coffee, the galactomannans can represent up to 25 % of the mass of the mature green coffee grain, and they exert a significant influence on the production of different types of coffee products. The objective of the current work was to isolate and characterize cDNA encoding proteins responsible for galactomannan synthesis in coffee and to study the expression of the corresponding transcripts in the developing coffee grain from C. arabica and C. canephora, which potentially exhibit slight galactomannan variations. Comparative gene expression analysis was also carried out for several other tissues of C. arabica and C. canephora.cDNA banks, RACE-PCR and genome walking were used to generate full-length cDNA for two putative coffee mannan synthases (ManS) and two galactomannan galactosyl transferases (GMGT). Gene-specific probe-primer sets were then generated and used to carry out comparative expression analysis of the corresponding genes in different coffee tissues using quantitative RT-PCR.Two of the putative galactomannan biosynthetic genes, ManS1 and GMGT1, were demonstrated to have very high expression in the developing coffee grain of both Coffea species during endosperm development, consistent with our proposal that these two genes are responsible for the production of the majority of the galactomannans found in the grain. In contrast, the expression data presented indicates that the ManS2 gene product is probably involved in the synthesis of the galactomannans found in green tissue.The identification of genes implicated in galactomannan synthesis in coffee are presented. The data obtained will enable more detailed studies on the biosynthesis of this important component of coffee grain and contribute to a better understanding of some functional differences between grain from C. arabica and C. canephora.

Objectives We have developed a relevant preclinical model associated with a specific imaging protocol dedicated to onco-pharmacology studies in mice. Materials and Methods We optimized both the animal model and an ultrasound imaging procedure to follow up longitudinally the lung tumor growth in mice. Moreover we proposed to measure by photoacoustic imaging the intratumoral hypoxia, which is a crucial parameter responsible for resistance to therapies. Finally, we compared ultrasound data to x-ray micro computed tomography and volumetric measurements to validate the relevance of this approach on the NCI-H460 human orthotopic lung tumor. Results This study demonstrates the ability of ultrasound imaging to detect and monitor the in vivo orthotopic lung tumor growth by high resolution ultrasound imaging. This approach enabled us to characterize key biological parameters such as oxygenation, perfusion status and vascularization of tumors. Conclusion Such an experimental approach has never been reported previously and it would provide a nonradiative tool for assessment of anticancer therapeutic efficacy in mice. Considering the absence of ultrasound propagation through the lung parenchyma, this strategy requires the implantation of tumors strictly located in the superficial posterior part of the lung.

Olfactory receptors (ORs) are expressed in the olfactory epithelium, where they detect odorants, but also in other tissues with additional functions. Some ORs are even overexpressed in tumor cells. In this study, we identified ORs expressed in enterochromaffin tumor cells by RT-PCR, showing that single cells can co-express several ORs. Some of the receptors identified were already reported in other tumors, but they are orphan (without known ligand), as it is the case for most of the hundreds of human ORs. Thus, genes coding for human ORs with known ligands were transfected into these cells, expressing functional heterologous ORs. The in vitro stimulation of these cells by the corresponding OR odorant agonists promoted cell invasion of collagen gels. Using LNCaP prostate cancer cells, the stimulation of the PSGR (Prostate Specific G protein-coupled Receptor), an endogenously overexpressed OR, by beta-ionone, its odorant agonist, resulted in the same phenotypic change. We also showed the involvement of a PI3 kinase gamma dependent signaling pathway in this promotion of tumor cell invasiveness triggered by OR stimulation. Finally, after subcutaneous inoculation of LNCaP cells into NSG immunodeficient mice, the in vivo stimulation of these cells by the PSGR agonist beta-ionone significantly enhanced metastasis emergence and spreading.

International audience; Monoclonal antibodies (mAbs) are usually delivered systemically, but only a small proportion of the drug reaches the lung after intravenous injection. The inhalation route is an attractive alternative for the local delivery of mAbs to treat lung diseases, potentially improving tissue concentration and exposure to the drug while limiting passage into the bloodstream and adverse effects. Several studies have shown that the delivery of mAbs or mAb-derived biopharmaceuticals via the airways is feasible and efficient, but little is known about the fate of inhaled mAbs after the deposition of aerosolized particles in the respiratory system. We used cetuximab, an anti-EGFR antibody, as our study model and showed that, after its delivery via the airways, this mAb accumulated rapidly in normal and cancerous tissues in the lung, at concentrations twice those achieved after intravenous delivery, for early time points. The spatial distribution of cetuximab within the tumor was heterogeneous, as reported after i.v. injection. Pharmacokinetic (PK) analyses were carried out in both mice and macaques and showed aerosolized cetuximab bioavailability to be lower and elimination times shorter in macaques than in mice. Using transgenic mice, we showed that FcRn, a key receptor involved in mAb distribution and PK, was likely to make a greater contribution to cetuximab recycling than to the transcytosis of this mAb in the airways. Our results indicate that the inhalation route is potentially useful for the treatment of both acute and chronic lung diseases, to boost and ensure the sustained accumulation of mAbs within the lungs, while limiting their passage into the bloodstream.

Tissue factor pathway inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin, a protease which is involved in tumour progression by activating (MMPs). This therefore makes TFPI-2 a potential inhibitor of invasiveness and the development of metastases. In this study, low levels of TFPI-2 expression were found in 65% of patients with small cell lung cancer (SCLC), the most aggressive type of lung cancer. To study the impact of TFPI-2 in tumour progression, TFPI-2 was overexpressed in NCI-H209 SCLC cells which were orthotopically implanted in nude mice. Investigations showed that TFPI-2 inhibited lung tumour growth. Such inhibition could be explained in vitro by a decrease in tumour cell viability, blockade of G1/S phase cell cycle transition and an increase in apoptosis shown in NCI-H209 cells expressing TFPI-2. We also demonstrated that TFPI-2 upregulation in NCI-H209 cells decreased MMP expression, particularly by downregulating MMP-1 and MMP-3. Moreover, TFPI-2 inhibited phosphorylation of the MAPK signalling pathway proteins involved in the induction of MMP transcripts, among which MMP-1 was predominant in SCLC tissues and was inversely expressed with TFPI-2 in 35% of cases. These results suggest that downregulation of TFPI-2 expression could favour the development of SCLC., Highlights • The Tissue Factor Pathway Inhibitor-2 inhibits small cell lung cancer growth • Monitoring of small cell lung cancer growth in a mouse orthotopic model by imaging • Increasing information on the role of TFPI-2 in human lung tumour cells • Increasing information on TFPI-2 and protease expression in human tissue samples

Introduction The aim of this study is to develop new strategies for the exploration of relevant preclinical lung tumor models dedicated to onco-pharmacology studies in mice. Here we refine a translational approach while overcoming physical ultrasound (US) imaging limitations allowing lung tumors exploration. Our data were compared to Computed Tomography (CT) and Bioluminescence (BLI) aiming to validate this promising approach. Methods Human lung cancer cells NCI-H460-luc2 were orthotopically xenografted in Balb/c nude mice. BLI was performed 7 days after implantation then once a week until the end of the study (Day 28) using an IVIS-Lumina II (Perkin Elmer). Tumors were imaged with the Vevo®LAZR System (FUJIFILM VisualSonics Inc.), 3D scans of US, Contrast Enhanced Ultrasound (CEUS) (Vevo MicroMarkerTM) and photoacoustic (PA) image being recorded digitally. A comparison between in vivo US and CT was achieved with additional ex vivo weight and volumetric measurements. Results The control of the tumor cell implantation was determined by BLI allowing the early quantification of tumor burden. 3D US measurements made possible longitudinal tumor volumes assessment. Compared to the other in vivo measurements, results clearly indicated the gross correlation of volumes processed with US and CT (R2 = 0.75). Such a correlation is also observed with ex vivo US weight and volumetric measurements (R2 = 0.72, R2 = 0.75 and R2 = 0.65 respectively). PA imaging evidenced the hemoglobin saturation with oxygen inside tumors, and targeted CEUS imaging allowed assessing the relative VEGFR2 expression in lung tumors. Conclusions This study proved the ability of US imaging to detect and control the in vivo orthotopic lung tumor growth. This real-time approach is fast while being completely inert regarding the in situ proliferation, as compared to dosimetry related to repeated CT examinations. Furthermore it allows overcoming the limitation of BLI at the time tumors become hypoxic since the luciferase/luciferin reaction is strictly dependent upon O2 and ATP. We described here a PA and US imaging strategy to follow the orthotopic lung tumors growth and consider biomarkers such as VEGFR2 expression or hypoxia in vivo for the first time. This procedure would be of great interest to longitudinally investigate tumor proliferation in animals when evaluating new anticancer therapies efficacy, avoiding any perturbation of the tumor progression compared to other available imaging modalities. Citation Format: Florian Raes, Julien Sobilo, Sharuja Natkunarajah, Philippe Trochet, Dieter Fuchs, Stephanie Lerondel, Alain Le Pape. Novel imaging strategy to assess the antitumor efficacy of treatments in an orthotopic mouse lung cancer model using ultrasound and photoacoustic imaging. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4200.

International audience; Non-thermal plasma (NTP) is generated by ionizing neutral gas molecules/atoms leading to a highly reactive gas at ambient temperature containing excited molecules, reactive species and generating transient electric fields. Given its potential to interact with tissue or cells without a significant temperature increase, NTP appears as a promising approach for the treatment of various diseases including cancer. The aim of our study was to evaluate the interest of NTP both in vitro and in vivo. To this end, we evaluated the antitumor activity of NTP in vitro on two human cancer cell lines (glioblastoma U87MG and colorectal carcinoma HCT-116). Our data showed that NTP generated a large amount of reactive oxygen species (ROS), leading to the formation of DNA damages. This resulted in a multiphase cell cycle arrest and a subsequent apoptosis induction. In addition, in vivo experiments on U87MG bearing mice showed that NTP induced a reduction of bioluminescence and tumor volume as compared to nontreated mice. An induction of apoptosis was also observed together with an accumulation of cells in S phase of the cell cycle suggesting an arrest of tumor proliferation. In conclusion, we demonstrated here that the potential of NTP to generate ROS renders this strategy particularly promising in the context of tumor treatment.

International audience; This abstract summarizes the experimental results obtained and plasma delivery strategy developed in GREMI for the evaluation of antitumor action of dielectric barrier discharge and plasma gun for cancer treatment. Detailed analysis of biological effects following cold plasma application for both in vitro and in vivo experiments reveals the role of ROS, DNA damage, cell cycle modification and apoptosis induction. Recent characterization of plasma splitting and mixing in capillary geometry, using the plasma gun, together with preliminary tolerance study dealing with lung and colon treatment indicate that endoscopic plasma delivery may be a new and valuable therapy in cancerology.This work deals with the development of two non thermal plasma sources and their application in cancerology. A floating electrode dielectric barrier discharge device (FE-DBD) and a plasma jet generator, labelled plasma gun [1], are used for both in vitro and in vivo assessment of cold atmospheric pressure plasma as a new therapy for cancer treatment. The first experiments were performed using the FE-DBD as an external plasma applicator on mice subcutaneously grafted with a tumor. The operation of the FE-DBD at low electrical power, a few watt, and correspondingly moderate plasma flux on the mouse skin surface allow repetitive plasma delivering for a few minutes during a few consecutive days. Following this plasma application protocol neither systemic behaviour, cardiac and pulmonary rhythm alterations nor severe skin burns were measured during tolerance studies. A five day DBD plasma treatment was shown to induce a significant delay and slowing down of the growth of U87 glioma cancer, in comparison with non treated control group [2]. This first demonstration of in vivo plasma therapeutic effect on a resistant cancer target was at the origin of three main experimental investigations: the study of the plasma delivery protocol, in vitro studies on different cell lines and the development of a new plasma source likely to allow endoscopic treatments. In vitro studies concern U87 (brain tumor) and HCT116 (colon tumor) cell lines for which both FE-DBD and plasma gun were used. The two plasma sources were shown to be efficient to kill the cell after plasma application over periods of a few tens of seconds. A more detailed analysis of FE-DBD action mechanism in in vitro cancerous cell treatment has been performed. The crucial role of ROS was proven by the comparable effect of a direct DBD plasma exposure and an indirect treatment protocol for which the culture medium was exposed to plasma and then transferred in culture wells containing cancerous cells. The use of ROS scavenger confirms that these species are, in vitro, the main agent in the cell destruction. DNA damage characterization, apoptosis quantification, and cell cycle analysis have then been performed. Briefly, formation of DNA double strand break damages and an accumulation of cells in the S phase were measured. Significant enhancement of the portion of cells in an apoptotic stage was also measured following in vitro plasma treatment. Dealing with the in vitro effect of plasma, the analogy of our results with plasma exposure with conventional cancer treatment strategies such as radio and chemo therapies, leads to suggest the following scenario to explain the cancerous cell destruction under plasma action. The key element is associated with in situ ROS generation leading successively to DNA damage, cell cycle arrest, and apoptosis induction. A very promising but questioning results concern the effect of plasma treatment during in vivo application. Apoptosis increasing and cell cycle alteration leading to an accumulation of cells in the S phase, were also measured by analyzing in vivo plasma treated tumors with the same detection techniques. The figure 1 presents the cell cycle measurements for in vitro and in vivo studies. Comparable G0/G1 fraction decrease, S phase accumulation and G2/M level are detected for in vivo and in vitro treated cells in comparison with the control group distribution. Figure 1 Cell cycle distribution in control and plasma treated groups for in vitro (left) and in vivo (right) U87 tumor.The role of ROS, reported to be of greater influence for in vitro plasma action, during in vivo treatment remains unclear, the main issue lying in the potential diffusion of these active species through the mouse skin to the tumor volume. In vivo plasma effects may also be correlated with plasma triggered in situ, i.e. in tumor environment, ROS release through local surface modification of charge density, temperature, or pH value. Such local pH modification was for instance observed on the skin surface where acidification occurs while subcutaneously a slight increase of the pH value was measured. Such chemical modification may induce in situ ROS release which may then interact with the tumor cells eventually through similar pathways as those encountered during in vitro trials. If this assumption sounds, the tumor treatment will probably be greatly optimized by delivering chemically active plasma in the close vicinity of the targeted cells. To check the efficiency of this mode of plasma application, there exists a need for a plasma source likely to allow endoscopic treatment. The main targets of our study concern colorectal and lung cancers for which access requires a specific care. The plasma gun set up is based on a pulsed nanosecond DBD reactor coupled with high aspect ratio flexible capillaries through which ionization wave sustained plasma propagation occurs. The possibility to use the plasma gun in multi branched volume, including branch splitting and connection at different locations, was recently experienced with success. Thus the plasma propagation was shown to be possible at large distances from the primary DBD plasma up to a few tens of centimetres through capillary structure exhibiting both splitting and connection of different branches. This unique specificity of the plasma gun among all other various plasma jet sources appears especially relevant for in vivo plasma application. The possibility to perform plasma delivery through micrometric catheter suitable for mice treatment by coloscopy or tracheotomy and flushed with low rare gas flow was recently achieved together with preliminary evidence for a reasonable tolerance of both colorectal and lung tissues under plasma exposure of a few minutes. The feasibility of such plasma application in combination with a proper matching of the plasma gun characteristics allows in situ in vivo study and optimization of antitumor action of cold plasma. Recent studies on the plasma jet action [3] on colorectal cell lines confirms cell growth arrest [4] and report a selective apoptosis induction for different tumoral and normal cells [5]. References[1] E. Robert, E. Barbosa, S. Dozias, M. Vandamme, C. Cachoncinlle, R. Viladrosa and J.M. Pouvesle, Plasma Process Polym. 6, 795-802 (2009).[2] M. Vandamme, E. Robert, S. Dozias, J. Sobilo, S. Lerondel, A. Le Pape, and J.M. Pouvesle, Plasma Medicine, 1 (1), 27-43 (2011).[3] X. 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International audience; Non thermal atmospheric plasma is a new promising candidate in anticancer therapy. We have already reported the absence of skin damage induced by our plasma treatment. Preliminary results suggested that this treatment could also induce an antitumor effect on U87 malignant glioma xenografts, and we conducted this work to evaluate the antitumor efficacy of plasma in this model. Antitumor effects were assessed by tumor volume measurement and bioluminescence imaging (BLI). Plasma treatment was applied during five consecutive days in open air with a μs-duration pulsed floating electrode dielectric barrier discharge (FE-DBD), which delivered about 0.75 W at 200 Hz on the mouse skin. Our results showed a significant tumor volume decrease of 56% for treated mice at the end of the treatment, with a concomitant decrease of BLI intensity. Moreover, this tumor volume reduction translated into an increase of mouse life span of 60%, median survival being 9.5 and 15.0 days for control and plasma-treated mice, respectively. In conclusion, our study demonstrates a marked antitumor effect of plasma treatment in U87 glioma xenografts. These results, obtained in both a radio and chemoresistant model, are very promising and highlight the potential of plasma treatment as an anticancer treatment with little or no toxic side effects.

Background Over the past few years, nonthermal atmospheric pressure plasma has emerged as a novel promising tool in medicine. Plasma is a partially ionized gas sustained by a supply of energy in various forms (electric, thermal, radiative . . .). Plasma contains free charges (electrons, ions), free radicals, excited molecules, energetic photons (UV) and generates transient electric fields. Studies have demonstrated an antitumor effect of plasma treatment on melanoma cells lines and our group has recently demonstrated an antitumor effect of plasma treatment on U87 glioma bearing mice. The aim of this work was to better understand the mechanisms involved in the antitumor effect of plasma treatment observed on U87 glioma cells in vitro and in vivo. Methods The experiments were performed using a floating electrode DBD plasma reactor. For in vitro experiments, 24-well plate were seeded with U87 cells previously transfected by the luciferase gene (U87-Luc). Cells were treated by a single fraction of plasma. Effects of treatment on proliferation and viability were assessed by bioluminescence imaging (BLI), MTT assay and by trypan blue assay. For in vivo experiments, malignant glioma U87 model was heterotopically implanted in nude mice. When tumors reached 150 mm3, mice were randomly assigned into two groups: CTRL and Plasma. Plasma was delivered as a daily fraction of 6 min at 200Hz for five consecutive days and tumors were imaged by bioluminescence before treatment and 24h after the first and the last fraction. Treatment effects on tumor cells proliferation and apoptosis were assessed 24h after the last fraction by determination of cell cycle distribution and detection of annexin V positive cells, respectively. Results and Discussion Plasma treatment showed a significant antitumoral effect on U87 glioma in vitro and in vivo. In vitro plasma induced a decrease of BLI resulting from a massive cell death. Moreover, in vivo experiments, plasma treated tumors presented an important BLI decrease of ∼70% associated with a ∼60% reduction of tumor volume as compared to CTRL. Such results are particularly encouraging in this high chemo and radioresistant model. Cell cycle distribution and annexin V analysis showed a marked apoptosis induction in the treated group. To our knowledge, only in vitro experiments have reported an apoptosis induction of plasma treatment on melanoma cell lines. DNA double strand break and ROS formation have been reported in vitro during plasma treatment and these mechanisms may participate in the induction of apoptosis observed in vivo. Our data highlight a major antitumor effect of plasma treatment both in vitro and in vivo and this effect may be linked to an apoptosis induction. Further studies are ongoing to elucidate mechanism of action of plasma in apoptosis induction and to understand the implication of the different plasma components in the cytotoxic effect. These results will allow us to develop more efficient plasma types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 505.