La lectine de Xerocomellus chrysenteron, un nano-objet théranostique pour l'imagerie et le traitement des cancers épithéliaux : preuve de concept appliquée aux carcinoses péritonéales d'origine ovarienne

Le développement de thérapies ciblées et des techniques d'imagerie est un défi majeur en santé, particulièrement dans les pathologies cancéreuses. Les carcinoses péritonéales sont habituellement causées par une dissémination de cellules tumorales au sein de la cavité abdominale, ce qui est le cas de 85% des patientes atteintes d'un cancer ovarien et plus de 10% des patients atteints d'un cancer colorectal. Dans les deux cas, les traitements consistent en une chirurgie (cytoréduction), aussi complète que possible, accompagnée de chimiothérapies. L'amélioration de la survie globale des patients passe par le développement de technologies parallèles comme de nouveaux outils diagnostiques pour détecter des implantations précoces dans le péritoine, le blocage de la dissémination de cellules cancéreuses pendant et après la chirurgie, ou encore la combinaison de chimiothérapies et de traitements ciblés intrapéritonéaux. Ce projet de thèse consiste à aborder ces différents aspects par l'utilisation d'un nanocontainer protéique multifonctionnel. L'objectif est d'optimiser cette protéine, appartenant à la famille des lectines, pour envisager son développement en tant qu'outil théranostique dans le cadre du diagnostic et des traitements de cancers épithéliaux. La lectine de Xerocomellus chrysenteron, à l'origine extraite d'un champignon supérieur comestible, présente une forte affinité pour un biomarqueur glycosidique des carcinomes, l'antigène de Thomsen-Friedenreich (antigène TF ou CD176). De plus, la présence d'une large cavité hydratée au centre de cet assemblage protéique (homotétramère) permet d'envisager le confinement et l'adressage spécifique de molécules thérapeutiques à des cellules épithéliales cancéreuses. Nous avons commencé par établir la preuve de concept de la délivrance ciblée de molécules thérapeutiques dans plusieurs lignées d'adénocarcinomes ovariens humains (OVCAR-3, SKOV-3, IGROV-1). Le marquage de la lectine dans le proche infrarouge a permis de confirmer le mécanisme de délivrance et prouver que la molécule thérapeutique avait bien été endocytée grâce à son confinement dans le nanocontainer. La protéine marquée a également été utilisée pour valider son utilisation comme nanosonde pour la détection de nodules tumoraux submillimétriques dans le péritoine. Cette détection est faite par imagerie de fluorescence in vivo dans des modèles précliniques de carcinose péritonéale ovarienne préalablement développés à partir de lignées cellulaires. La combinaison des deux propriétés de la protéine (sonde et container) permet d'envisager son utilisation en nanothéranostique intrapéritonéale. Afin de confirmer ce développement prometteur, il sera nécessaire d'établir la preuve de concept sur des modèles murins plus pertinents de la situation clinique développés à partir de tumeurs issues de patientes (Patient Derived Xenografts, PDX).The development of targeted therapy and imaging tools is a major challenge in human health, particularly in cancer pathologies. Peritoneal carcinomatosis is usually caused by scattering of cancer cells within the abdominal cavity, which is the case for 85% of ovarian cancer patients and more than 10% of colorectal cancer patients. In both cases treatments include a cytoreductive surgery, as complete as possible, and chemotherapies. Patients overall survival improvement can be reach with the development of parallel technologies such as new diagnostic tools to detect early implantations in the peritoneal cavity, agents to block the spreading of cancer cells detached during the surgical procedure, or combining chemotherapies and intraperitoneal targeted drug delivery. This project involves reaching all those aspects by using a unique multifunctional nanocontainer protein. The aim is to maximize this protein, which belongs to the lectin family, to consider its development as a theranostic tool as part of epithelial cancers diagnostic and treatment. Xerocomellus chrysenteron lectin, originally extracted from an edible higher mushroom, has a strong affinity for a carcinoma glycan biomarker, the Thomsen-Friedenreich antigen (TF antigen). Furthermore, a large hydrated inner cavity located in the middle of the tetrameric assembly of the protein led us to consider the containment and specific addressing of therapeutic molecules to epithelial cancerous cells expressing TF antigen. We first established the proof of concept for the targeted drug delivery of therapeutic molecules in several human ovarian adenocarcinoma cell lines (OVCAR-3, SKOV-3, IGROV-1). The labelling of the lectin in near infrared allowed us to confirm the mechanism implicated in the delivery and prove that the uptake of the molecule within the cells was due to its containment in the nanocontainer. The labelled protein was also used also to validate it as a nanoprobe for the detection of submillimeter nodules in the peritoneal cavity. This detection was made by in vivo fluorescence imaging in preclinical models of ovarian peritoneal carcinomatosis developed beforehand using established cell lines. The combination of these two properties of the protein (probe and container) permits to consider its use in intraperitoneal nanotheranostic. To confirm this promising development, it will be necessary to establish the proof of concept for theranostic aspects in mice models closer to clinic situations developed from patients' tumors (patient derived xenografts, PDX)

Xerocomellus chrysenteron lectin, a theranostic nanotool for imaging and treatment of epithelial cancers : proof of concept applied to ovarian peritoneal carcinomatosis

Le développement de thérapies ciblées et des techniques d’imagerie est un défi majeur en santé, particulièrement dans les pathologies cancéreuses. Les carcinoses péritonéales sont habituellement causées par une dissémination de cellules tumorales au sein de la cavité abdominale, ce qui est le cas de 85% des patientes atteintes d'un cancer ovarien et plus de 10% des patients atteints d’un cancer colorectal. Dans les deux cas, les traitements consistent en une chirurgie (cytoréduction), aussi complète que possible, accompagnée de chimiothérapies. L'amélioration de la survie globale des patients passe par le développement de technologies parallèles comme de nouveaux outils diagnostiques pour détecter des implantations précoces dans le péritoine, le blocage de la dissémination de cellules cancéreuses pendant et après la chirurgie, ou encore la combinaison de chimiothérapies et de traitements ciblés intrapéritonéaux.Ce projet de thèse consiste à aborder ces différents aspects par l'utilisation d'un nanocontainer protéique multifonctionnel. L’objectif est d’optimiser cette protéine, appartenant à la famille des lectines, pour envisager son développement en tant qu'outil théranostique dans le cadre du diagnostic et des traitements de cancers épithéliaux. La lectine de Xerocomellus chrysenteron, à l’origine extraite d’un champignon supérieur comestible, présente une forte affinité pour un biomarqueur glycosidique des carcinomes, l’antigène de Thomsen-Friedenreich (antigène TF ou CD176). De plus, la présence d’une large cavité hydratée au centre de cet assemblage protéique (homotétramère) permet d’envisager le confinement et l’adressage spécifique de molécules thérapeutiques à des cellules épithéliales cancéreuses.Nous avons commencé par établir la preuve de concept de la délivrance ciblée de molécules thérapeutiques dans plusieurs lignées d’adénocarcinomes ovariens humains (OVCAR-3, SKOV-3, IGROV-1). Le marquage de la lectine dans le proche infrarouge a permis de confirmer le mécanisme de délivrance et prouver que la molécule thérapeutique avait bien été endocytée grâce à son confinement dans le nanocontainer. La protéine marquée a également été utilisée pour valider son utilisation comme nanosonde pour la détection de nodules tumoraux submillimétriques dans le péritoine. Cette détection est faite par imagerie de fluorescence in vivo dans des modèles précliniques de carcinose péritonéale ovarienne préalablement développés à partir de lignées cellulaires. La combinaison des deux propriétés de la protéine (sonde et container) permet d’envisager son utilisation en nanothéranostique intrapéritonéale. Afin de confirmer ce développement prometteur, il sera nécessaire d’établir la preuve de concept sur des modèles murins plus pertinents de la situation clinique développés à partir de tumeurs issues de patientes (Patient Derived Xenografts, PDX).The development of targeted therapy and imaging tools is a major challenge in human health, particularly in cancer pathologies. Peritoneal carcinomatosis is usually caused by scattering of cancer cells within the abdominal cavity, which is the case for 85% of ovarian cancer patients and more than 10% of colorectal cancer patients. In both cases treatments include a cytoreductive surgery, as complete as possible, and chemotherapies. Patients overall survival improvement can be reach with the development of parallel technologies such as new diagnostic tools to detect early implantations in the peritoneal cavity, agents to block the spreading of cancer cells detached during the surgical procedure, or combining chemotherapies and intraperitoneal targeted drug delivery.This project involves reaching all those aspects by using a unique multifunctional nanocontainer protein. The aim is to maximize this protein, which belongs to the lectin family, to consider its development as a theranostic tool as part of epithelial cancers diagnostic and treatment. Xerocomellus chrysenteron lectin, originally extracted from an edible higher mushroom, has a strong affinity for a carcinoma glycan biomarker, the Thomsen-Friedenreich antigen (TF antigen). Furthermore, a large hydrated inner cavity located in the middle of the tetrameric assembly of the protein led us to consider the containment and specific addressing of therapeutic molecules to epithelial cancerous cells expressing TF antigen. We first established the proof of concept for the targeted drug delivery of therapeutic molecules in several human ovarian adenocarcinoma cell lines (OVCAR-3, SKOV-3, IGROV-1). The labelling of the lectin in near infrared allowed us to confirm the mechanism implicated in the delivery and prove that the uptake of the molecule within the cells was due to its containment in the nanocontainer. The labelled protein was also used also to validate it as a nanoprobe for the detection of submillimeter nodules in the peritoneal cavity. This detection was made by in vivo fluorescence imaging in preclinical models of ovarian peritoneal carcinomatosis developed beforehand using established cell lines. The combination of these two properties of the protein (probe and container) permits to consider its use in intraperitoneal nanotheranostic. To confirm this promising development, it will be necessary to establish the proof of concept for theranostic aspects in mice models closer to clinic situations developed from patients’ tumors (patient derived xenografts, PDX)

Optimization of protein electroextraction from microalgae by a flow process

International audienceClassical methods, used for large scale treatments such as mechanical or chemical extractions, affect the integrity of extracted cytosolic protein by releasing proteases contained in vacuoles. Our previous experiments on flow processes electroextraction on yeasts proved that pulsed electric field technology allows preserving the integrity of released cytosolic proteins, by not affecting vacuole membranes. Furthermore, large cell culture volumes are easily treated by the flow technology. Based on this previous knowledge, we developed a new protocol in order to electro-extract total cytoplasmic proteins from microalgae (Nannochloropsis salina, Chlorella vulgaris and Haematococcus pluvialis). Given that induction of electropermeabilization is under the control of target cell size, as the mean diameter for N. salina is only 2.5 μm, we used repetitive 2 ms long pulses of alternating polarities with stronger field strengths than previously described for yeasts. The electric treatment was followed by a 24 h incubation period in a salty buffer. The amount of total protein release was observed by a classical Bradford assay. A more accurate evaluation of protein release was obtained by SDS-PAGE. Similar results were obtained with C. vulgaris and H. pluvialis under milder electrical conditions as expected from their larger size

E. coli electroeradication on a closed loop circuit by using milli-, micro- and nanosecond pulsed electric fields: Comparison between energy costs

International audienceOne of the different ways to eradicate microorganisms, and particularly bacteria that might have an impact on health consists in the delivery of pulsed electric fields (PEFs). The technologies of millisecond (ms) or microsecond (μs) PEF are still well known and used for instance in the process of fruit juice sterilization. However, this concept is costly in terms of delivered energy which might be too expensive for some other industrial processes. Nanosecond pulsed electric fields (nsPEFs) might be an alternative at least for lower energetic cost. However, only few insights were available and stipulate a gain in cost and in efficiency as well. Using Escherichia coli, the impact of frequency and low rate on eradication and energy consumption by msPEF, μsPEF and nsPEF have been studied and compared. While a 1 log 10 was reached with an energy cost of 100 and 158 kJ/L with micro-and millisecond PEFs respectively, nsPEF reached the reduction for similar energy consumption. The best condition was obtained for a 1 log 10 deactivation in 0.5 h, for energy consumption of 143 kJ/L corresponding to 0.04 W•h when the field was around 100 kV/cm. Improvement can also be expected by producing a generator capable to increase the electric field

Development of a near infrared protein nanoprobe targeting Thomsen-Friedenreich antigen for intraoperative detection of submillimeter nodules in an ovarian peritoneal carcinomatosis mouse model

International audienceThe epithelial ovarian cancer is one of the most lethal gynecological malignancy due to its late diagnostic and many relapses observed after first line of treatment. Once diagnose, the most important prognostic factor is the completeness of cytoreductive surgery. To achieve this goal, surgeons have to pinpoint and remove nodules, especially the smallest nodules. Recent advances in fluorescence-guided surgery led us to develop a recombinant lectin as a nanoprobe for the microscopic detection of nodules in the peritoneal cavity of tumor-bearing mice. This lectin has an intrinsic specificity for a carcinoma-associated glycan biomarker, the Thomsen-Friedenreich antigen. In this study, after its labelling by a near infrared dye, we first demonstrated that this nanoprobe allowed indirect detection of nodules already implanted in the peritoneal cavity, through tumor microenvironment targeting. Secondly, in a protocol mimicking the scattering of cells during surgery, we obtained a direct and long-lasting detection of tumor cells in vivo. This lectin as already been described as a nanocontainer able to do targeted delivery of a therapeutic compound to carcinoma cells. Future developments will focus on the combination of the nanoprobe and nanocontainer aspects in an intraperitoneal nanotheranostic approach

Reduction of NADPH-oxidase activity ameliorates the cardiovascular phenotype in a mouse model of Williams-Beuren Syndrome

A hallmark feature of Williams-Beuren Syndrome (WBS) is a generalized arteriopathy due to elastin deficiency, presenting as stenoses of medium and large arteries and leading to hypertension and other cardiovascular complications. Deletion of a functional NCF1 gene copy has been shown to protect a proportion of WBS patients against hypertension, likely through reduced NADPH-oxidase (NOX)–mediated oxidative stress. DD mice, carrying a 0.67 Mb heterozygous deletion including the Eln gene, presented with a generalized arteriopathy, hypertension, and cardiac hypertrophy, associated with elevated angiotensin II (angII), oxidative stress parameters, and Ncf1 expression. Genetic (by crossing with Ncf1 mutant) and/or pharmacological (with ang II type 1 receptor blocker, losartan, or NOX inhibitor apocynin) reduction of NOX activity controlled hormonal and biochemical parameters in DD mice, resulting in normalized blood pressure and improved cardiovascular histology. We provide strong evidence for implication of the redox system in the pathophysiology of the cardiovascular disease in a mouse model of WBS. The phenotype of these mice can be ameliorated by either genetic or pharmacological intervention reducing NOX activity, likely through reduced angII–mediated oxidative stress. Therefore, anti-NOX therapy merits evaluation to prevent the potentially serious cardiovascular complications of WBS, as well as in other cardiovascular disorders mediated by similar pathogenic mechanism.This work was supported by grants from the Spanish Ministry of Science and Innovation of Health (FIS 07/0059 to VC, FIS 10/2512 to LAP-J, RD06/0020/0001 to XRB) and the VI Framework Programme of the European Union (LSHG-CT-2006-037627 to LAP-J). VC is a Miguel Servet FIS Investigator (CP04/00068). MS-P is supported by a CIBERER Fellowship. MM-M is supported by the CSIC JAE-Doc program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Hybrid Peptide-Alkoxyamine Drugs: A Strategy for the Development of a New Family of Antiplasmodial Drugs

International audienceThe emergence and spread of drug-resistant Plasmodium falciparum parasites shed a serious concern on the worldwide control of malaria, the most important tropical disease in terms of mortality and morbidity. This situation has led us to consider the use of peptide-alkoxyamine derivatives as new antiplasmodial prodrugs that could potentially be efficient in the fight against resistant malaria parasites. Indeed, the peptide tag of the prodrug has been designed to be hydrolysed by parasite digestive proteases to afford highly labile alkoxyamines drugs, which spontaneously and instantaneously homolyse into two free radicals, one of which is expected to be active against P. falciparum. Since the parasite enzymes should trigger the production of the active drug in the parasite’s food vacuoles, our approach is summarized as “to dig its grave with its fork”. However, despite promising sub-micromolar IC50 values in the classical chemosensitivity assay, more in-depth tests evidenced that the anti-parasite activity of these compounds could be due to their cytostatic activity rather than a truly anti-parasitic profile, demonstrating that the antiplasmodial activity cannot be based only on measuring antiproliferative activity. It is therefore imperative to distinguish, with appropriate tests, a genuinely parasiticidal activity from a cytostatic activity

Peptide-Alkoxyamine Drugs: An Innovative Approach to Fight Schistosomiasis: “Digging Their Graves with Their Forks”

The expansion of drug resistant parasites sheds a serious concern on several neglected parasitic diseases. Our recent results on cancer led us to envision the use of peptide-alkoxyamines as a highly selective and efficient new drug against schistosome adult worms, the etiological agents of schistosomiasis. Indeed, the peptide tag of the hybrid compounds can be hydrolyzed by worm’s digestive enzymes to afford a highly labile alkoxyamine which homolyzes spontaneously and instantaneously into radicals—which are then used as a drug against Schistosome adult parasites. This approach is nicely summarized as digging their graves with their forks. Several hybrid peptide-alkoxyamines were prepared and clearly showed an activity: two of the tested compounds kill 50% of the parasites in two hours at a concentration of 100 µg/mL. Importantly, the peptide and alkoxyamine fragments that are unable to generate alkyl radicals display no activity. This strong evidence validates the proposed mechanism: a specific activation of the prodrugs by the parasite proteases leading to parasite death through in situ alkyl radical generation