Développement de stratégies d'imagerie multimodalités pour la pharmacologie des agents anticancéreux

Preclinical imaging in oncology is booming. It allows, using representative animal models of human cancers, to understand the mechanisms of development of pathologies and to assess the therapeutic efficiency of a new treatment. The main objective of this work was to develop two orthotopic models of cancer (pancreas and colon) and to assess on them the reference treatments as well as a new therapeutic strategy by non thermal plasma so called Plasma Gun. The two cancer models developed showed good representation in relation to human cancers, with the appearance of distant metastases and hypoxia. 5-fluorouracil for the HCT116-luc orthotopic model of colorectal carcinoma and gemcitabine for the MIA PaCa2-luc pancreatic adenocarcinoma model, have induced discrete effects at low dose wich can be detected thanks imaging modalities. After validation of our experimental steps, a new therapeutic strategy, Plasma Gun was evaluated and showed significant effects on tumor growth inhibition. The second objective was to carry out tools for the induction and the characterization of bone metastases and for high resolution imaging of the vasculature. On the one hand, bone metastases obtained by injection of PC3M-luc cells intracardially, was evaluated and quantified with different imaging modalities (bioluminescence, scintigraphy and Computed Tomography). And the other hand, the achievement of a high resolution imaging of vascularization, was possible by the casting method that restores the 3D structure of the vascular architecture following injection of a resin in the circulation.L’imagerie préclinique dans le domaine de la cancérologie est en plein essor. Elle permet grâce à des modèles animaux représentatifs de cancers humains de comprendre les mécanismes de développement des pathologies et d’évaluer l’efficacité thérapeutique d’un nouveau traitement. Le principal objectif de ce travail a été de développer deux modèles orthotopiques de cancer (pancréas et colon) et d’évaluer des traitements de références ainsi qu’une nouvelle stratégie thérapeutique par plasma froid fibré appelée Plasma Gun. Les 2 modèles de cancers développés ont montré une bonne représentativité vis-à-vis des cancers humains, avec l’apparition de métastases à distance et la présence de zones hypoxiques. Le 5-fluorouracile pour le modèle orthotopique de carcinome colorectal HCT116-luc et la gemcitabine pour le modèle d’adénocarcinome pancréatique MIA PaCa2-luc ont induit à faible dose des effets discrets pouvant être mis en évidence grâce aux modalités d’imageries mises en oeuvre. Après validation de nos démarches expérimentales une nouvelle stratégie thérapeutique, le Plasma Gun, a été évaluée et a montré des effets significatifs sur l’inhibition de la croissance tumorale. Le second objectif de ma thèse a été de mettre en oeuvre des outils pour l’induction et la caractérisation des métastases osseuses ainsi que pour l’imagerie haute résolution de la vascularisation. D’une part, les métastases osseuses obtenues par injection de cellules PC3M-luc en intracardiaque ont été évaluées et quantifiées grâce à différentes modalités d’imagerie (bioluminescence, scintigraphie et scanner X). D’autre part, la réalisation d’une imagerie haute résolution de la vascularisation a été possible grâce à la technique de casting qui permet de recréer la structure 3D de l’architecture vasculaire suite à l’injection d’une résine dans la circulation. Les développements réalisés lors de cette thèse ont ainsi permis d’apporter des outils pour l’évaluation préclinique de nouvelles thérapies anticancéreuses

Development of multimodal imaging strategies for the pharmacology of anticancer agents

L’imagerie préclinique dans le domaine de la cancérologie est en plein essor. Elle permet grâce à des modèles animaux représentatifs de cancers humains de comprendre les mécanismes de développement des pathologies et d’évaluer l’efficacité thérapeutique d’un nouveau traitement. Le principal objectif de ce travail a été de développer deux modèles orthotopiques de cancer (pancréas et colon) et d’évaluer des traitements de références ainsi qu’une nouvelle stratégie thérapeutique par plasma froid fibré appelée Plasma Gun. Les 2 modèles de cancers développés ont montré une bonne représentativité vis-à-vis des cancers humains, avec l’apparition de métastases à distance et la présence de zones hypoxiques. Le 5-fluorouracile pour le modèle orthotopique de carcinome colorectal HCT116-luc et la gemcitabine pour le modèle d’adénocarcinome pancréatique MIA PaCa2-luc ont induit à faible dose des effets discrets pouvant être mis en évidence grâce aux modalités d’imageries mises en oeuvre. Après validation de nos démarches expérimentales une nouvelle stratégie thérapeutique, le Plasma Gun, a été évaluée et a montré des effets significatifs sur l’inhibition de la croissance tumorale. Le second objectif de ma thèse a été de mettre en oeuvre des outils pour l’induction et la caractérisation des métastases osseuses ainsi que pour l’imagerie haute résolution de la vascularisation. D’une part, les métastases osseuses obtenues par injection de cellules PC3M-luc en intracardiaque ont été évaluées et quantifiées grâce à différentes modalités d’imagerie (bioluminescence, scintigraphie et scanner X). D’autre part, la réalisation d’une imagerie haute résolution de la vascularisation a été possible grâce à la technique de casting qui permet de recréer la structure 3D de l’architecture vasculaire suite à l’injection d’une résine dans la circulation. Les développements réalisés lors de cette thèse ont ainsi permis d’apporter des outils pour l’évaluation préclinique de nouvelles thérapies anticancéreuses.Preclinical imaging in oncology is booming. It allows, using representative animal models of human cancers, to understand the mechanisms of development of pathologies and to assess the therapeutic efficiency of a new treatment. The main objective of this work was to develop two orthotopic models of cancer (pancreas and colon) and to assess on them the reference treatments as well as a new therapeutic strategy by non thermal plasma so called Plasma Gun. The two cancer models developed showed good representation in relation to human cancers, with the appearance of distant metastases and hypoxia. 5-fluorouracil for the HCT116-luc orthotopic model of colorectal carcinoma and gemcitabine for the MIA PaCa2-luc pancreatic adenocarcinoma model, have induced discrete effects at low dose wich can be detected thanks imaging modalities. After validation of our experimental steps, a new therapeutic strategy, Plasma Gun was evaluated and showed significant effects on tumor growth inhibition. The second objective was to carry out tools for the induction and the characterization of bone metastases and for high resolution imaging of the vasculature. On the one hand, bone metastases obtained by injection of PC3M-luc cells intracardially, was evaluated and quantified with different imaging modalities (bioluminescence, scintigraphy and Computed Tomography). And the other hand, the achievement of a high resolution imaging of vascularization, was possible by the casting method that restores the 3D structure of the vascular architecture following injection of a resin in the circulation

Plasma antitumor action and the potential role of plasma-induced electric fields

International audienceAfter a brief review of the main progress in the plasma cancer domain, experiments dealing with cancer treatment ran by the Orleans group with DBD and, mainly, with the Plasma Gun in different setups will be presented. From the first in vivo demonstration of air DBD plasma antitumor effect, antitumor action with helium or neon fed plasma jets delivered in ambient air on different tumor bearing organs in mice have been achieved. The positive combination of plasma jet treatment with chemotherapeutic agents was also demonstrated on pancreatic tumors [1]. The reasons for this positive combination are under study to suggest new combined protocols likely to promote the antitumor action of plasma jets and afford a new adjuvant approach for cancer treatment. Both loco regional plasma-induced tumor environment oxygenation and blood flow modulation, as recently demonstrated for healthy mice [2], and the potential role of intense transient electric fields associated with plasma delivery over targets, are under study. The synchronized in situ delivery of so called ROS/RNS and transient electric fields may be a unique feature of plasma treatments likely to involve either electroporation or chemical agents but also their combined action. First measurements, including the temporal profile and intensity, of the electric field associated with plasma jets delivery over different targets will be presented.Brullé et al, Plos one, 2012, 7 (12) e52653.Collet et al, Plasma Sources Sci. Technol., 2014, 23 012005Keywords: plasma medicine, electric fields, antitumor action, plasma jet

Physics and applications of the Plasma Gun: a user friendly tool for atmospheric pressure room temperature plasma delivery including endoscopic potentialities

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Targeted Shiga toxin-drug conjugates prepared via Cu-free click chemistry.

International audienceThe main drawback of the anticancer chemotherapy consists in the lack of drug selectivity causing severe side effects. The targeted drug delivery appears to be a very promising strategy for controlling the biodistribution of the cytotoxic agent only on malignant tissues by linking it to tumor-targeting moiety. Here we exploit the natural characteristics of Shiga toxin B sub-unit (STxB) as targeting carrier on Gb3-positive cancer cells. Two cytotoxic conjugates STxB-doxorubicin (STxB-Doxo) and STxB-monomethyl auristatin F (STxB-MMAF) were synthesised using copper-free 'click' chemistry. Both conjugates were obtained in very high yield and demonstrated strong tumor inhibition activity in a nanomolar range on Gb3-positive cells

Antitumoral effects of combined non thermal plasma and gemcitabine treatments on Mia Paca-luc orthotopic pancreatic carcinoma model

International audienceCancer of the exocrine pancreas and most particularly ductal adenocarcinoma, the most common form of pancreatic cancer1, is rarely curable and has an overall survival rate of less than 4%. Moreover, chemotherapy and radiotherapy of pancreatic cancer are only, up to now, palliative treatments. New therapeutic approaches are then necessary. Recent results we obtained on the treatment of glioblastoma and colon carcinoma2,3 led us to evaluate the antitumoral effect of non-thermal plasma (NTP) alone or in combination with Gemcitabine, reference chemotherapeutic agent, in the case of pancreas cancer. Experiments were carried out using the Plasma Gun developed in GREMI both in vitro on MIA PaCa2-luc cell lines (ductal adenocarcinoma cells) and in vivo on a MIA PaCa Luc orthotopic Pancreatic carcinoma murin model (Female Swiss nude mice from Charles River Laboratoires France). As we verified with Gemcitabine, Plasma Gun NTP had an in vitro significant antitumor activity with an IC50 of 13s exposure duration. In vivo experiments were carried out using four mouse groups: one control group, one group treated only with Gemcitabine, one group treated only using the Plasma Gun, and one group treated using a combination of Gemcitabine and Plasma Gun treatment. The striking results of this series of experiments is that, not only, the plasma treatment appeared more efficient than Gemcitabine alone, but that the combination of both led to the most effective tumor growth arrest, indicating a possible synergetic effect between NTP and chemotherapeutical agent. 1. M.P. Wescott and A.K. Rustgi Cancer Prevention Research 2008;1:503-6.2. M Vandamme, E Robert, S Dozias et al, Plasma Medicine 2011;1:27-43.3. M Vandamme, E Robert, S Lerondel et al, Int J Cancer 2011________________________________* Work supported by APR Région Centre PLASMED project and CG 45

A new delivery system for auristatin in STxB-drug conjugate therapy

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Effects of combined plasma jet and gemcitabine treatments on tumor proliferation of a murine orthotopic pancreatic carcinoma model

International audienceCancer of the exocrine pancreas is rarely curable and has an overall survival rate of less than 4%. While it is relatively rare for the time being, pancreatic cancer is also one of the most formidable and its incidence appears to increase significantly with number of cases of diabetes. Chemotherapy and radiotherapy treatments showed limited efficacy, development of new therapeutic strategies is then necessary.Recent results were obtained on the treatment of glioblastoma [1] and colon carcinoma [2] using non thermal plasma (NTP). They led us to assess the antitumoral effect of NTP alone or in combination with gemcitabine a reference chemotherapeutic agent with radiosensitizing properties, on pancreatic cancer.Experiments were carried out using the Plasma Gun developed in GREMI both in vitro on MIA PaCa2-luc cell lines (pancreatic cancer cells) then in vivo on orthotopically grafted tumor cells to induce a pancreatic carcinoma model in immunodeficient mice.Plasma Gun showed an in vitro significant antitumor activity with an IC50 corresponding to 13s exposure duration. In vivo experiments were carried out using four mouse groups: one control group, one group treated only with gemcitabine (200 mg/kg), one group treated only using the Plasma Gun, and one group treated using a combination of gemcitabine (200 mg/kg) and Plasma Gun.Our data showed a significant inhibition of tumor growth in NTP and/or gemcitabine treated mice, this from 20th day post treatment. We demonstrated that plasma gun induced an inhibition of MIA PaCa2-luc cell proliferation in vitro and in vivo and that this effect is enhanced when combined with gemcitabine, a radiosensitive agent, this later being reported for the first time in vivo. Given these results, the possibility to use NTP in combination with a chemotherapeutical agent to increase its effects seems of very high interest for further developments in oncology involving cold plasmas, eventually delivered through an endoscopic approach. There is also a need for optimization of the sequence of chemotherapeutic agent administration and NTP exposition. This will be done in a forthcoming study.References [1]Vandamme M, Robert E, Pesnel S, Barbosa E, Dozias S, Sobilo J, Lerondel S, Le Pape A, Pouvesle JM. Antitumor Effect of Plasma Treatment on U87 Glioma Xenografts: Preliminary Results. Plasma processes and polymers 2010;7:264.[2]Vandamme M, Robert E, Lerondel S, Sarron V, Ries D, Dozias S, Sobilo J, Gosset D, Kieda C, Legrain B, Pouvesle JM, Pape AL. ROS implication in a new antitumor strategy based on non-thermal plasma. Int J Cancer 2011

Plasmas froids atmosphériques pour des applications thérapeutiques

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In vitro antitumor activity of plasma Gun

International audienceOur group has recently showed a marked antitumor effect of DBD plasma treatment in vivo on U87 glioma bearing mice. Beside these results in vivo, various studies have showed an antitumor effect of plasma treatment on various cancer cells lines in vitro by apoptosis induction using DBD or plasma jet. In our lab, development of a plasma jet so called plasma gun lead to new very interesting perspectives in a context of tumor treatment via small catheter. The main goal of this work was to investigate the in vitro cells sensitivity to plasma and major cells mechanisms induce by plasma gun treatment. Experiments were performed using the plasma gun previously described by our group. Antitumor activity was evaluated on HCT116, Mia Paca and H460 cancer cells which are representative models of colorectal carcinoma, pancreatic ductal adenocarcinoma and lung tumors respectively. These cells lines are potential future target for in situ plasma application. In vitro effects of plasma treatment on proliferation and viability were assessed by bioluminescence imaging (BLI). Figure 1: Effect of plasma gun treatment on tumor growth in vivoPlasma treatment induced a significant antitumor effect the different cells lines. The most sensitive cell line to plasma treatment was Mia Paca cells with a LD50 of 12s, then HCT-116 (LD50 18s) and H460 (LD50 28s) (figure 1). In HCT-116, plasma induces a decrease of cell proliferation and a decrease of cell mobility. This decrease of cell proliferation was the consequence of a massive cell cycle arrest in G2/M after plasma treatment. Moreover, an increase of apoptosis was observed in the treated cells. These results obtained with the plasma gun were closed to previous results obtained with our DBD and to other research group. Recent studies suggest the major importance of p53 in response to ROS generated by plasma treatment which is implicated in the cell cycle control. Our results on various cell lines including intracellular mutations like p53 or PTEN could explain the difference of cell sensitivity. Indeed, the most resistant cell line (H460) has various mutations including p53, KRAS and PIK3 while a Mia PACA cell has KRAS and p53 mutations. Implications of theses mutations in plasma cell sensitivity need to be further investigated. Non thermal plasma applied with plasma appears to be very promising and experiments are ongoing to evaluate antitumor activity of this strategy on representative cancer models in vivo