Blood contains circulating cell-free respiratory competent mitochondria.

Mitochondria are considered as the power-generating units of the cell due to their key role in energy metabolism and cell signaling. However, mitochondrial components could be found in the extracellular space, as fragments or encapsulated in vesicles. In addition, this intact organelle has been recently reported to be released by platelets exclusively in specific conditions. Here, we demonstrate for the first time, that blood preparation with resting platelets, contains whole functional mitochondria in normal physiological state. Likewise, we show, that normal and tumor cultured cells are able to secrete their mitochondria. Using serial centrifugation or filtration followed by polymerase chain reaction-based methods, and Whole Genome Sequencing, we detect extracellular full-length mitochondrial DNA in particles over 0.22 µm holding specific mitochondrial membrane proteins. We identify these particles as intact cell-free mitochondria using fluorescence-activated cell sorting analysis, fluorescence microscopy, and transmission electron microscopy. Oxygen consumption analysis revealed that these mitochondria are respiratory competent. In view of previously described mitochondrial potential in intercellular transfer, this discovery could greatly widen the scope of cell-cell communication biology. Further steps should be developed to investigate the potential role of mitochondria as a signaling organelle outside the cell and to determine whether these circulating units could be relevant for early detection and prognosis of various diseases

Evaluation of circulating DNA analysis in the context of tumorigenesis and as a diagnostic tool

L’analyse de l’ADN circulant dans le contexte de la tumorogenèse et comme outil diagnostic Le projet de thèse ici décrit est fondé sur la découverte remarquable qu'une quantité importante d’ADN circule dans le sang de patients atteints de cancer [1-5]. Le développement d'une technologie basée sur la détection de l'ADN circulant représente une avancée scientifique et médicale pour le diagnostic et le suivi dans la prise en charge thérapeutique des patients atteints de cancer. Malgré de nombreuses études menées au cours des dix dernières années [4,5] sur l'ADN circulant, les origines de la libération de l'ADN circulant dans les liquides biologiques sont hypothétiques et sa structure n'est pas élucidée. Ces données ne valident pas jusqu'à présent l'ADN circulant en tant que biomarqueur. Pour cette raison, les objectifs du groupe dirigé par Alain Thierry sont axés sur l'élucidation des formes structurelles de l'ADN circulant. Ainsi, en utilisant des souris nude xénogreffées avec des lignées cellulaires tumorales humaines de cancer colorectal ainsi que des échantillons sanguins cliniques provenant de patients atteints de cancer colorectal, l'équipe a montré que la concentration en ADN circulant était corrélée positivement avec la taille de la tumeur [6, 7] et ces résultats se sont révélés optimaux pour des tailles inférieures à 100 pb. Une discrimination significative entre les individus sains et les patients du cancer a été observée grâce à l'analyse de la fragmentation de l'ADN circulant. L'originalité de ces découvertes a donné naissance à la technologie Intplex récemment breveté par le CNRS [8]. L'objectif de la thèse est de valider la quantification et la fragmentation de l'ADN circulant comme un outil de diagnostic et de suivi de la maladie dans la prise en charge du cancer en analysant de près les facteurs qui peuvent influencer la quantification et la fragmentation de l'ADN circulant. Grâce au modèle animal développé par l’équipe et l’étroite collaboration avec les centres anti-cancéreux, différents paramètres seront analysés. Une partie de la thèse se concentrera sur la comparaison et la standardisation des résultats en fonction de nombreux facteurs spécifiques à la tumeur, comme son type, sa progression, sa différenciation et sa localisation tissulaire. Le travail de thèse portera également sur l'influence de facteurs individuels pouvant affecter la quantité et la fragmentation de l'ADN circulant: âge, sexe, antécédents médicaux, états physiologiques spécifiques, situations physiopathologiques. L'influence du traitement sera également explorée. Des études seront menées afin de standardiser l'analyse biologique: influence du rythme circadien, prise de nourriture .... Techniquement, la variation analytique et l'influence des facteurs pré-analytiques seront déterminées afin d’établir un guide de bonnes pratiques analytiques pour éliminer tout artefact susceptible d’affecter la quantité et l'intégrité de l'ADN circulant dans les échantillons. Ces deux paramètres seront testés dans une évaluation clinique prospective multicentrique sur une cohorte de 450 patients atteints de cancer colorectal. Ce travail garantit un impact considérable dans la littérature et dans la pratique clinique comme test non invasif de diagnostic et de suivi et comme un outil pour améliorer les connaissances de base sur l'ADN circulant et le cancer.Analysis of circulating DNA circulating in the context of tumorigenesis and as a diagnostic tool The thesis project described here is based on the remarkable discovery that a significant amount of DNA circulates in blood of cancer patients [1-5]. The development of a technology based on the detection of circulating DNA represents a scientific and medical breakthrough for diagnosis and follow up in therapeutic care of cancer patients. Despite numerous studies conducted over the last decade [4,5] on circulating DNA, origins of release of circulating DNA in biological fluids are hypothetical and its structure is unclear. These data do not validate so far circulating DNA as a biomarker. For this reason, objectives of the group led by Alain Thierry are focused on elucidating structural forms of circulating DNA. Thus, using nude mice xenografted with human tumor cell lines of colorectal cancer as clinical samples from colorectal cancer patients, the team showed that the concentration was positively correlated with tumor size [6 , 7] and these results were optimal for sizes below 100 bp. A significative discrimination between healthy individuals and cancer patients was found by the analysis of circulating DNA fragmentation. The originality of these discoveries gave rise to the Intplex technology recently patented by the CNRS [8]. The aim of the thesis is to validate quantification and fragmentation of circulating DNA as a diagnostic and follow-up test in the management of cancer by closely analyzing the factors that may influence quantification and fragmentation of circulating DNA. Thanks to mouse model developed by the team and close collaboration with clinical cancer centers, different parameters will be analyzed. One part of the thesis will be focused on comparison and standardization of the different results depending on many factors specific to the tumor, such as its type, its progression, its differentiation and its tissue localization. The thesis work will also focus on the influence of individual factors that may affect the quantity and the fragmentation of circulating DNA: age, sex, medical history, specific physiological states, pathophysiological situations. The influence of treatment will also be explored. Studies will be undertaken in order to standardize the biological analysis: influence of circadian rhythm, food intake.... Technically, the analytical variation and the influence of pre-analytical factors will be determined to establish a good practice guide to eliminate any artifacts altering the amount and integrity of circulating DNA in the samples. These two parameters will be tested in a prospective multicentric clinical evaluation on a cohort including 450 patients with colorectal cancer. This work warrants a significant impact in the literature and in cancer clinical practice as a non invasive diagnostic and follow-up test and as a tool to improve the basic knowledge on circulating DNA and cancer

Multi-marker analysis of circulating cell-free DNA toward personalized medicine for colorectal cancer.

International audienceDevelopment of a Q-PCR-based assay for the high-performance analysis of circulating cell-free DNA (ccfDNA) requires good knowledge of its structure and size. In this work, we present the first visual determination of ccfDNA by Atomic Force Microscopy (AFM) on plasma samples from colorectal cancer (CRC) patients and healthy donors. In addition to the examination of fragment size distribution profile as performed by Q-PCR, this analysis confirms that ccfDNA is highly fragmented and that more than 80% of ccfDNA fragments in CRC plasma are below 145 bp. We adapted an Allele-Specific Blocker (ASB) Q-PCR to small ccfDNA fragments to determine simultaneously the total ccfDNA concentration, the presence of point mutation, the proportion of mutated allele, and a ccfDNA integrity index. The data validated analytically these four parameters in 124 CRC clinical samples and 71 healthy individuals. The multi-marker method, termed Intplex, enables sensitive and specific non-invasive analysis of tumor ccfDNA, which has great potential in terms of cost, quality control, and easy implementation in every clinical center laboratory

Imidazoquinoxaline anticancer derivatives and imiquimod interact with tubulin: Characterization of molecular microtubule inhibiting mechanisms in correlation with cytotoxicity

International audienceDisplaying a strong antiproliferative activity on a wide variety of cancer cells, EAPB0203 and EAPB0503 belong to the imidazo[1,2-a]quinoxalines family of imiquimod structural analogues. EAPB0503 has been shown to inhibit tubulin polymerization. The aim of the present study is to characterize the interaction of EAPB0203 and EAPB0503 with tubulin. We combine experimental approaches at the cellular and the molecular level both in vitro and in silico in order to evaluate the interaction of EAPB0203 and EAPB0503 with tubulin. We examine the influence of EAPB0203 and EAPB0503 on the cell cycle and fate, explore the binding interaction with purified tubulin, and use a computational molecular docking model to determine the binding modes to the microtubule. We then use a drug combination study with other anti-microtubule agents to compare the binding site of EAPB0203 and EAPB0503 to known potent tubulin inhibitors. We demonstrate that EAPB0203 and EAPB0503 are capable of blocking human melanoma cells in G2 and M phases and inducing cell death and apoptosis. Second, we show that EAPB0203 and EAPB0503, but also unexpectedly imiquimod, bind directly to purified tubulin and inhibit tubulin polymerization. As suggested by molecular docking and binding competition studies, we identify the colchicine binding site on β-tubulin as the interaction pocket. Furthermore, we find that EAPB0203, EAPB0503 and imiquimod display antagonistic cytotoxic effect when combined with colchicine, and disrupt tubulin network in human melanoma cells. We conclude that EAPB0203, EAPB0503, as well as imiquimod, interact with tubulin through the colchicine binding site, and that the cytotoxic activity of EAPB0203, EAPB0503 and imiquimod is correlated to their tubulin inhibiting effect. These compounds appear as interesting anticancer drug candidates as suggested by their activity and mechanism of action, and deserve further investigation for their use in the clinic

Metabolism and Pharmacokinetics of EAPB0203 and EAPB0503, Two Imidazoquinoxaline Compounds Previously Shown to Have Antitumoral Activity on Melanoma and T-Lymphomas

International audienceFor several years, our group has been developing quinoxalinic compounds. Two of them, N-methyl-1-(2-phenethyl)imidazo[1,2-a]qui-noxalin-4-amine (EAPB0203) and 1-(3-methoxyphenyl)-N-methylimi-dazo[1,2-a]quinoxalin-4-amine (EAPB0503), have emerged as the most promising anticancer drugs. In the present work, we determined metabolism pathways using liver microsomes from four mam-malian species including human. We identified the cytochrome P450 isoform(s) involved in the metabolism and then investigated the phar-macokinetics and metabolism of EAPB0203 and EAPB0503 in rat after intravenous and intraperitoneal administration. Biotransformation of the compounds involved demethylation and hydroxylation reactions. Rat and dog metabolized the compounds at a higher rate than mouse and human. In all species, CYP1A1/2 and CYP3A isoforms were the predominant enzymes responsible for the metabolism. From human liver microsomes, unbound intrinsic clearances were approximately 56 ml/(min ⅐ g) protein. EAPB0203 and EAPB0503 were extensively bound to human plasma proteins, mainly human serum albumin (HSA) (ϳ98-99.5%). Thus, HSA could act as carrier of these compounds in human plasma. Scatchard plots showed patterns in which the plots yielded upwardly convex hyperbolic curves. On the basis of the Hill coefficients, there appears to be interaction between the binding sites of HSA, suggesting positive cooperativity. The main in vitro metabolites were identified in vivo. Total clearances of EAPB0203 and EAPB0503 [3.2 and 2.2 l/(h ⅐ kg), respectively] were notably lower than the typical cardiac plasma output in rat. The large volumes of distribution of these compounds (4.3 l/kg for EAPB0203 and 2.5 l/kg for EAPB0503) were consistent with extensive tissue binding. After intraperitoneal administration, bioavailability was 22.7% for EAPB0203 and 35% for EAPB0503 and a significant hepatic first-pass effect occurred

Circulating DNA as a Strong Multimarker Prognostic Tool for Metastatic Colorectal Cancer Patient Management Care

International audienc

Quantifying circulating cell-free DNA in humans

Abstract To our knowledge, this is the first comprehensive study on the influence of several pre-analytical and demographic parameters that could be a source of variability in the quantification of nuclear and mitochondrial circulating DNA (NcirDNA and McirDNA). We report data from a total of 222 subjects, 104 healthy individuals and 118 metastatic colorectal cancer (mCRC) patients. Approximately 50,000 and 3,000-fold more mitochondrial than nuclear genome copies were found in the plasma of healthy individuals and mCRC patients, respectively. In healthy individuals, NcirDNA concentration was statistically influenced by age (p = 0.009) and gender (p = 0.048). Multivariate analysis with logistic regression specified that age over 47 years-old was predictive to have higher NcirDNA concentration (OR = 2.41; p = 0.033). McirDNA concentration was independent of age and gender in healthy individuals. In mCRC patients, NcirDNA and McirDNA levels were independent of age, gender, delay between food intake and blood collection, and plasma aspect, either with univariate or multivariate analysis. Nonetheless, ad hoc study suggested that menopause and blood collection time might have tendency to influence cirDNA quantification. In addition, high significant statistical differences were found between mCRC patients and healthy individuals for NcirDNA (p < 0.0001), McirDNA (p < 0.0001) and McirDNA/NcirDNA ratio (p < 0.0001). NcirDNA and McirDNA levels do not vary in the same way with regards to cancer vs healthy status, pre-analytical and demographic factors