Glioblastoma characterization and monitoring of its chemotherapies by MALDI imaging coupled to top down analysis

Le glioblastome est la forme la plus agressive des tumeurs du système nerveux central. Le traitement de référence consiste en l'exérèse chirurgicale, suivie d'une radiothérapie associée à une chimiothérapie concomitante et adjuvante par le témozolomide. Son bénéfice est démontré par une médiane de survie entre 12 et 14 mois. Le glioblastome est caractérisé par une population cellulaire hétérogène hautement infiltrante, angiogénique et résistante à la chimiothérapie. Dans le but d'optimiser l'effet des molécules thérapeutiques, un suivi de leur pharmacocinétique ainsi qu'une bonne caractérisation tumorale sont nécessaires. L'imagerie par désorption laser assistée par matrice en spectrométrie de masse (IMS MALDI) a été utilisée pour l'identification de marqueurs diagnostiques, pronostiques et prédictifs de réponse aux traitements. Elle a aussi permis de suivre la pharmacocinétique in situ des chimiothérapies.L'identification de protéines directement sur tissu par fragmentation en source a permis la mise en évidence de différents isotypes de tubuline, une des cibles majeures en thérapie anticancéreuse. Le couplage de cette stratégie d'identification à l'imagerie MALDI a permis d'identifier et de localiser dans des zones tumorales, des protéines impliquées dans la tumorigenèse. La distribution intra-tissulaire du bévacizumab et du témozolomide a été étudiée pour la première fois.Des marqueurs de réponse aux traitements ont ensuite été identifiés par comparaison des profils d'expression protéique de tumeurs avec et sans traitement. Ces résultats montrent l'intérêt de l'imagerie MALDI pour l'étude des chimiothérapies et permettent d'envisager son utilisation clinique future.Glioblastoma is the most aggressive of the gliomas, a collection of tumors arising from glia or their precursors within the central nervous system. The current standard of care, comprised of surgical resection followed by radiation and the chemotherapeutic agent temozolomide, only provides patients with a 12-14 months survival period post-diagnosis. The glioblastoma is characterized by a heterogeneous population of cells that are highly infiltrative, angiogenic and resistant to chemotherapy. In order to optimize the therapy effect, a pharmacokinetic monitoring and a better understanding and characterization of tumor biology are needed. For this purpose, matrix assisted laser desorption/ionization imaging mass spectrometry imaging mass spectrometry (MALDI IMS) technology was applied to identify diagnostic, prognostic and predictive markers of therapy response; and to understand/follow the pharmacokinetic of chemotherapies. The top-down in-source decay strategy was used for protein identification directly on tissue. This strategy allowed tubulin protein isoforms distinction and identification, which is one of the main targets in cancer therapy. MALDI imaging coupled to ISD identified tumorigenesis proteins within tumor structures. Bevacizumab and temozolmide distribution was followed within brain tissue sections. For the first time a monoclonal antibody was deciphered on tissue. Finally, markers that predict therapy response were demonstrated by a comparison between protein expression profiles from tumors with and without chemotherapy treatment. These results highlight the interest of MALDI imaging for chemotherapy improvement and open the way for its use in the clinics

Mass spectrometry imaging is moving toward drug protein co-localization

Mass spectrometry (MS)-based technology provides label-free localization of molecules in tissue samples. Drugs, proteins, lipids and metabolites can easily be monitored in their environment. Resolution can be achieved down to the cellular level (10–20 mm) for conventional matrix-assisted laser desorption/ionization (MALDI) imaging, or even to the subcellular level for more complex technologies such as secondary ionization mass spectrometry (SIMS) imaging. One question remains: are we going to be able to investigate functional relationships between drugs and proteins and compare with localized phenomena? This review describes the various spatial levels of investigation offered by mass spectrometry imaging (MSI), and the advantages and disadvantages compared with other labeling technologies

MALDI imaging and in-source decay for top-down characterization of glioblastoma

International audienc

Monitoring therapeutic monoclonal antibodies in brain tumor

International audienc

Mass Spectrometry Imaging, Laser Capture Microdissection, and LC-MS/MS of the Same Tissue Section

Mass spectrometry imaging (MSI) is able to simultaneously record the distributions of hundreds of molecules directly from tissue. Rapid direct tissue analysis is essential for MSI in order to maintain spatial localization and acceptable measurement times. The absence of an explicit analyte separation/purification step means MSI lacks the depth of coverage of LC-MS/MS. In this work, we demonstrate how atmospheric pressure MALDI-MSI enables the same tissue section to be first analyzed by MSI, to identify regions of interest that exhibit distinct molecular signatures, followed by localized proteomics analysis using laser capture microdissection isolation and LC-MS/MS

Tubulin isoforms identified in the brain by MALDI in-source decay

Identification of biomarkers is a major issue for enhancement of chemotherapies. The molecular characterization of tissues necessitates the identification of thousands of biomolecules each participating in physiopathological processes. MALDI in-source decay (ISD) fragmentation has already been proven to be effective for protein characterization. However, the difficulty to identify proteins from complex mixtures such as tissue sections can limit the applications of this technique. In this study, we evidenced that tubulin has an unusual fragmentation pathway in the MALDI source. This striking property allowed the detecting of several mouse brain tubulin isotypes simultaneously by simply using laser fragmentation. Tubulin isoforms are consistent markers of a bad prognosis of solid tumors and could be the target of targeted chemotherapies. Such a direct molecular printout of tubulin in tissues is a milestone that should be useful either at preclinical or clinical stage

Mechanisms of innate events during skin reaction following intradermal injection of seasonal influenza vaccine

International audienceThe skin plays a crucial role in host defences against microbial attack and the innate cells must provide the immune system with sufficient information to organize these defences. This unique feature makes the skin a promising site for vaccine administration. Although cellular innate immune events during vaccination have been widely studied, initial events remain poorly understood. Our aim is to determine molecular biomarkers of skin innate reaction after intradermal (i.d.) immunization. Using an ex vivo human explant model from healthy donors, we investigated by NanoLC-MS/MS analysis and MALDI-MSI imaging, to detect innate molecular events (lipids, metabolites, proteins) few hours after i.d. administration of seasonal trivalent influenza vaccine (TIV). This multimodel approach allowed to identify early molecules differentially expressed in dermal and epidermal layers at 4 and 18 h after TIV immunization compared with control PBS. In the dermis, the most relevant network of proteins upregulated were related to cell-to-cell signalling and cell trafficking. The molecular signatures detected were associated with chemokines such as CXCL8, a chemoattractant of neutrophils. In the epidermis, the most relevant networks were associated with activation of antigen-presenting cells and related to CXCL10. Our study proposes a novel step-forward approach to identify biomarkers of skin innate reaction. SIGNIFICANCE: To our knowledge, there is no study analyzing innate molecular reaction to vaccines at the site of skin immunization. What is known on skin reaction is based on macroscopic (erythema, redness…), microscopic (epidermal and dermal tissues) and cellular events (inflammatory cell infiltrate). Therefore, we propose a multimodal approach to analyze molecular events at the site of vaccine injection on skin tissue. We identified early molecular networks involved biological functions such cell migration, cell-to-cell interaction and antigen presentation, validated by chemokine expression, in the epidermis and dermis, then could be used as early indicator of success in immunization

Enhanced Sensitivity Using MALDI Imaging Coupled with Laser Postionization (MALDI-2) for Pharmaceutical Research

Visualizing the distributions of drugs and their metabolites is one of the key emerging application areas of matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) within pharmaceutical research. The success of a given MALDI-MSI experiment is ultimately determined by the ionization efficiency of the compounds of interest, which in many cases are too low to enable detection at relevant concentrations. In this work we have taken steps to address this challenge via the first application of laser-postionisation coupled with MALDI (so-called MALDI-2) to the analysis and imaging of pharmaceutical compounds. We demonstrate that MALDI-2 increased the signal intensities for 7 out of the 10 drug compounds analyzed by up to 2 orders of magnitude compared to conventional MALDI analysis. This gain in sensitivity enabled the distributions of drug compounds in both human cartilage and dog liver tissue to be visualized using MALDI-2, whereas little-to-no signal from tissue was obtained using conventional MALDI. This work demonstrates the vast potential of MALDI-2-MSI in pharmaceutical research and drug development and provides a valuable tool to broaden the application areas of MSI. Finally, in an effort to understand the ionization mechanism, we provide the first evidence that the preferential formation of [M + H]+ ions with MALDI-2 has no obvious correlation with the gas-phase proton affinity values of the analyte molecules, suggesting, as with MALDI, the occurrence of complex and yet to be elucidated ionization phenomena

Integrated Human Evaluation of the Lysophosphatidic Acid Pathway as a Novel Therapeutic Target in Atherosclerosis

Variants in the PLPP3 gene encoding for lipid phosphate phosphohydrolase 3 have been associated with susceptibility to atherosclerosis independently of classical risk factors. PLPP3 inactivates lysophosphatidic acid (LPA), a pro-inflammatory, pro-thrombotic product of phospholipase activity. Here we performed the first exploratory analysis of PLPP3, LPA, and LPA receptors (LPARs 1–6) in human atherosclerosis. PLPP3 transcript and protein were repressed when comparing plaques versus normal arteries and plaques from symptomatic versus asymptomatic patients, and they were negatively associated with risk of adverse cardiovascular events. PLPP3 localized to macrophages, smooth muscle, and endothelial cells (ECs) in plaques. LPAR 2, 5, and especially 6 showed increased expression in plaques, with LPAR6 localized in ECs and positively correlated to PLPP3. Utilizing in situ mass spectrometry imaging, LPA and its precursors were found in the plaque fibrous cap, co-localizing with PLPP3 and LPAR6. In vitro, PLPP3 silencing in ECs under LPA stimulation resulted in increased expression of adhesion molecules and cytokines. LPAR6 silencing inhibited LPA-induced cell activation, but not when PLPP3 was silenced simultaneously. Our results show that repression of PLPP3 plays a key role in atherosclerosis by promoting EC activation. Altogether, the PLPP3 pathway represents a suitable target for investigations into novel therapeutic approaches to ameliorate atherosclerosis. Keywords: atherosclerosis, therapy, biobank profilin