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International audience; Endometrial cancer (EC) is one of the most common gynecological cancers worldwide. Sentinel lymph node (SLN) status could be a major prognostic factor in evaluation of EC, but several prospective studies need to be performed. Here we report an in-depth proteomics analysis showing significant variations in the SLN protein landscape in EC. We show that SLNs are correlated to each tumor grade, which strengthens evidence of SLN involvement in EC. A few proteins are overexpressed specifically at each EC tumor grade and in the corresponding SLN. These proteins, which are significantly variable in both locations, should be considered potential markers of overall survival. Five major proteins for EC and SLN (PRSS3, PTX3, ASS1, ALDH2, and ANXA1) were identified in large-scale proteomics and validated by immunohistochemistry. This study improves stratification and diagnosis of individuals with EC as a result of proteomics profiling of SLNs.

International audience; Tissue diagnosis is critical in the clinical management of cancer patients. Mass Spectrometry (MS) has long been of interest for discriminating cells using tissue sections of patient biopsies. However, the main challenge arises in the context of intraoperative tissue analyses, where the MS instrument must operate within a surgical environment. To date, only the Intelligent Knife (iKnife) system allowed real-time monitoring during surgery by collecting aerosol released during tissue excision with an electric scalpel or bipolar forceps [1]. Recently we have demonstrated that in vivo real-time analyses can be less invasive using laser ablation. In our prototype, a fibered IR Optical Parametric Oscillator (OPO) is tuned at 2.94 µm to excite the most intense vibrational band (O-H stretching mode) of water molecules found abundantly in all biological tissues. Water acts as a natural endogenous Matrix-Assisted Laser Desorption-Ionization (MALDI) matrix leading to the production of ions that can be transported over a few meters to a MS instrument for analysis without requiring further post-ionization handling. The molecular patterns (metabolites, lipids and proteins) thus retrieved are specific to cell phenotypes and benign versus cancer regions can easily be differentiated [2]. We present the first assessment of our prototype in a veterinary surgery room [3]. For this purpose, a series of benchmark studies have been initiated with the aim of building up an extensive databank able to relate distinct molecular profiles to a specific type of pathology, namely the dog sarcoma. These studies are accompanied by the development of a real-time query interface. Classification models based on tumor grade (cancer/normal/necrotic) and cancer subtype developed in this work showed a minimum of ~98% correct classification when put to use. The system demonstrated clear-cut margin detection capabilities that have been validated in correlation with histology. Finally, this instrument enables real-time diagnostics by the immediate interrogation of classification models established ahead of time.

International audience; In Mammals, microglial cells are considered as the resident immune cells in central nervous system (CNS). Many studies demonstrated that, after injury, these cells are activated and recruited at the lesion site. Leech microglia present a similar pattern of microglial activation and migration upon experimental lesion of CNS. This activation is associated with the release of a large amount of extracellular vesicles (EVs). We collected EVs released by microglia primary culture and compared two different protocols of isolation: one with differential ultracentrifugation (UC) and one using an additional Optiprep™ Density Gradient (ODG) ultracentrifugation. Nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM) were used to assess vesicles size and morphology. The protein content of isolated EVs was assessed by mass spectrometry approaches. Results showed the presence of EV-specific proteins in both procedures. The extensive proteomic analysis of each single ODG fractions confirmed the efficiency of this protocol in limiting the presence of co-isolated proteins aggregates and other membranous particles during vesicles isolation. The present study permitted for the first time the characterisation of microglial EV protein content in an annelid model. Interestingly, an important amount of proteins found in leech vesicles was previously described in EV-specific databases. Finally, purified EVs were assessed for neurotrophic activity and promote neurites outgrowth on primary cultured neurons.

A protocol to follow the processing of angiotensin I into angiotensin II by rabbit angiotensin-converting enzyme (ACE) and its inhibition by a novel natural antagonist, the leech osmoregulator factor (LORF) using capillary zonal electrophoresis is described. The experiment was carried out using the Beckman PACE system and steps were taken to determine (a) the migration profiles of angiotensin and its yielded peptides, (b) the minimal amount of angiotensin II detected, (c) the use of different electrolytes and (d) the concentration of inhibitor. We demonstrated that LORF (IPEPYVWD), a neuropeptide previously found in leech brain, is able to inhibit rabbit ACE with an IC(50) of 19.8 micro m. Interestingly, its cleavage product, IPEP exhibits an IC(50) of 11.5 micro m. A competition assay using p-benzoylglycylglycylglycine and insect ACE established that LORF and IPEP fragments are natural inhibitors for invertebrate ACE. Fifty-four percent of insect ACE activity is inhibited with 50 micro m IPEP and 35% inhibition with LORF (25 mm). Extending the peptide at both N- and C-terminus (GWEIPEPYVWDES) and the cleavage of IPEP in IP abolished the inhibitory activity of both peptides. Immunocytochemical data obtained with antisera raised against LORF and leech ACE showed a colocalization between the enzyme and its inhibitor in the same neurons. These results showed that capillary zonal electrophoresis is a useful technique for following enzymatic processes with small amounts of products and constitutes the first evidence of a natural ACE inhibitor in invertebrates.

Despite important differences between infectious diseases and cancers, tumour development (neoplasia) can nonetheless be closely compared to infectious disease because of the similarity of their effects on the body. On this basis, we predict that many of the life-history (LH) responses observed in the context of host-parasite interactions should also be relevant in the context of cancer. Parasites are thought to affect LH traits of their hosts because of strong selective pressures like direct and indirect mortality effects favouring, for example, early maturation and reproduction. Cancer can similarly also affect LH traits by imposing direct costs and/or indirectly by triggering plastic adjustments and evolutionary responses. Here, we discuss how and why a LH focus is a potentially productive but under-exploited research direction for cancer research, by focusing our attention on similarities between infectious disease and cancer with respect to their effects on LH traits and their evolution. We raise the possibility that LH adjustments can occur in response to cancer via maternal/paternal effects and that these changes can be heritable to (adaptively) modify the LH traits of their offspring. We conclude that LH adjustments can potentially influence the transgenerational persistence of inherited oncogenic mutations in populations.

Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) allows to map endogenous and exogenous molecules along thin tissue sections without requiring any labelling. The aim of the Gliomic study (NCT 02473484) is to take advantage of MALDI-MSI to classify high-grade gliomas (HGG) by integration of MALDI-MSI to spatially-resolved proteomics data and correlation with clinical data for diagnosis and prognosis.

Background: Since its introduction 10 years ago by Caprioli and associates, MALDI mass spectrometry imaging has enabled spatial analysis of drugs, lipids, peptides, and polypeptides. In polypeptides, the detectable mass range is limited to small proteins with a mass less than 25 kDa. This is a limitation, as many proteins, including cytokines, growth factors, enzymes, and receptors have molecular weights, exceeding 25 kDa. In the present work, we report the development of a novel strategy to observe higher mass proteins up to 30 kDa. Material/Methods: We investigated the development of sample preparation methods based on hexafluoroisopropanol (1,1,1,3,3,3-hexaluoro-2-propanol) and 2,2,2-trifluoroethanol solvents for protein solubilization optimized for high-mass proteins. Results: We were, for the first time in mass spectrometry imaging, able to detect to proteins up to 70 kDa directly from tissue. These developments indicate future avenues by which the sensitivity of protein mass spectrometry imaging can be further improved. We applied these developments to ovarian cancer and demonstrate that protein are similar to that which can be obtained using 2D gel based analyses. Conclusions: Increasing the possibility of detecting proteins and high-mass proteins is key for developing direct tissue proteomics and especially any potential functional investigation. These data will open the door of a novel step in mass spectrometry imaging.

n° de priorité : US20060796535P 20060502 ; également publié en tant que : WO2007128751 (A3) 2008-09-18 ; EP2022076 (A2) 2009-02-11 ; US2009197295 (A1) 2009-08-06 ; JP2009535631 (A) 2009-10-01

The invention concerns improved methods for studying peptides/proteins expression in a tissue section or for determining at least one compound, in particular a protein, expression map in a tissue section, using ionic MALDI matrices.

The invention concerns a method for determining at least one target molecule map in a tissue section, using at least one (A-X)n-B conjugate, wherein A is a tag molecule of known molecular weight, X is a linker that is cleaved during sample desorption/ionization, n is an integer of at least l,and B is a binding molecule that binds specifically to said target molecule. When using MALDI mass spectrometry, said linker molecule X may be cleaved by photodissociation during sample laser irradiation if photocleavable at the wavelength of said MALDI laser. Alternatively, when using UV- MALDI, IR-MALDI, SIMS or DESI mass spectrometry, said linker molecule X may be cleaved by fragmentation during sample desorption/ionization.

This paper reports on simple, efficient and economical methods to increase MALDI image resolution. It is not currently possible to focus the diameter of a MALDI laser down to 50x50µm², i.e. 2500µm², without decreasing signal intensity. But using Silicon masks openings features, it might even be decreased to about 15x50µm², i.e. 750µm². Furthermore, the presence of a mask on the tissue section sample may result in a significant increase in the signal intensity for high m/z ratios. Finally, a huge advantage of the masks is their ease of adaptability on any MALDI mass spectrometer.

Within the growing field of proteomics, mass spectrometry is now established as Q powerful tool for peptide and protein identification and discovery from purified samples. A new era is now beginning, with the development of MALDI imaging, maintaining the sensitivity and efficacy of both discovery and identification while additionally preserving the anatomical integrity of biomolecules like peptides, proteins, oligonucleotides and lipids within tissues. Crucial developments for sample preparations have made leaps and bounds, as it is now possible to work with freezed conserved biopsies (- 80 degrees c) of more than 6 months or even conserved after paraformaldehyde fixation and paraffin embedding. The latter development has opened the door to archived tissues in hospital libraries and biomarkers hunting from tissues derived from these libraries are now a key objective. The relationship between MALDI imaging and immunocytochemistry used by the pathologist is important. The development of specific MALDI imaging using probes with a tag (peptide or organic) called << Tag-Mass >> adds a whole new perspective, It is possible henceforth to localize a protein with its specific mRNA and more specifically, with its signalling pathway on the same sections or within a pathology expression phenotype from a biopsy. Development of such a technology is similar to the one that occurred several years ago for nuclear magnetic resonance (NMR) that leads the development of imaging technologies called MRI in hospital which is intensively used for pathology diagnostics.

International audience

International audience; MALDI direct analysis and MALDI imaging of tissues have shown to be a very powerful tool by localizing molecules in tissue and by avoiding extraction, pre-purification, separation. Nevertheless, this new method requires new developments to increase sensitivity and specificity. We propose here a new concept and evolutions of each step of MALDI Imaging analysis. New matrices called Ionic matrices were synthesized to be especially well adapted for a good crystallization, sensitivity and resolution compared to the classical matrices. Moreover, for tissue kept in hospital libraries, FFPE tissues we developed a new strategy of in situ tissue enzymatic digestion was developed in combination with automatic microspotting MALDI Imaging. In order to add a dimension of specificity to imaging by MS, we have developed specific imaging using designed probes directed against specific targets with mass spectrometry detection. This strategy has been developed for different class of biomolecules with a specific highlight, to mRNA and peptides/proteins. This concept is based of indirect detection of specific targeted molecule using a photocleavable tagged probe. The introduction of the specific imaging give another dimension by targeting specific disease-marker-gene RNA transcripts, following their expression within tissues and then confirming their translation by targeting their specific proteinproducts or metabolites

International audience; La protéomique connaît un essor considérable depuis ces dernières années comme le démontre les nombreux travaux réalisés dans le domaine. Elle a permis de conduire à l'identification de nombreuses protéines. Cependant, malgré la puissance de cette stratégie, l'information concernant la localisation des composés au sein des tissus ne peut être conservée. En revanche, le développement de méthodologies nouvelles, comme l'analyse directe de tissus par spectrométrie de masse permet d'obtenir cette information. Dans le cadre de l'analyse des biomolécules sous leur forme intacte, les sources MALDI sont bien adaptées. Ainsi, il est possible de réaliser l'analyse directe de composés au sein des tissus de biomolécules en MALDI. L'automatisation de cette méthode ouvre vers l'imagerie par spectrométrie de masse MALDI au travers de cartes de répartition moléculaires dans le tissu. Néanmoins, cette méthodologie nécessite encore de nombreux développements afin d'améliorer les qualités d'analyse et la résolution des images. Nous nous sommes plus intéressés à développer différents aspects de cette technologie. Ces développements ont été appliqués à l'étude du cancer de l'ovaire et la recherche de nouveaux biomarqueurs de cette pathologie. L'analyse directe différentielle de biopsies de patientes présentant une tumeur et de patientes témoins (descente d'organe) a conduit à la mise en évidence de plusieurs biomarqueurs potentiels.