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See detailNew mechanism of resistance to anti-angiogenic therapy.
Cimino, Jonathan ULg; Sounni, Nor Eddine ULg; Debois, Delphine ULg et al

Conference (2013, May 13)

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See detailTowards Lipidomics of Low-Abundant Species for Exploring Tumor Heterogeneity Guided by High-Resolution Mass Spectrometry Imaging
Cimino, Jonathan ULg; Calligaris, David; Far, Johann ULg et al

in International Journal of Molecular Sciences (2013), 14

Many studies have evidenced the main role of lipids in physiological and also pathological processes such as cancer, diabetes or neurodegenerative diseases. The identification and the in situ localization ... [more ▼]

Many studies have evidenced the main role of lipids in physiological and also pathological processes such as cancer, diabetes or neurodegenerative diseases. The identification and the in situ localization of specific low-abundant lipid species involved in cancer biology are still challenging for both fundamental studies and lipid marker discovery. In this paper, we report the identification and the localization of specific isobaric minor phospholipids in human breast cancer xenografts by FTICR MALDI imaging supported by histochemistry. These potential candidates can be further confirmed by liquid chromatography coupled with electrospray mass spectrometry (LC-ESI-MS) after extraction from the region of interest defined by MALDI imaging. Finally, this study highlights the importance of characterizing the heterogeneous distribution of low-abundant lipid species, relevant in complex histological samples for biological purposes. [less ▲]

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See detailMALDI In-Source Decay, from sequencing to imaging
Debois, Delphine ULg; Smargiasso, Nicolas ULg; Demeure, Kevin ULg et al

in Topics in Current Chemistry (2013), 331

MALDI is now a mature method allowing the identification and, more challenging, the quantification of biopolymers (proteins, nucleic acids, glycans…). MALDI spectra show mostly intact singly charged ions ... [more ▼]

MALDI is now a mature method allowing the identification and, more challenging, the quantification of biopolymers (proteins, nucleic acids, glycans…). MALDI spectra show mostly intact singly charged ions. To obtain fragments, the activation of singly charged precursors is necessary, but not efficient above 3.5 kDa thus making MALDI MS/MS difficult for large species. In-source decay (ISD) is a prompt fragmentation reaction that can be induced thermally or by radicals. As fragments are formed in the source, precursor ions cannot be selected; however, the technique is not limited by the mass of the analyzed compounds and pseudo MS/MS can be performed on intense fragments. The discovery of new matrices that enhance the ISD yield, combined with the high sensitivity of MALDI mass spectrometers, and software development, opens new perspectives. We first review the mechanisms involved in the ISD processes, then discuss ISD applications like top-down sequencing and post-translational modifications studies, and finally review MALDI-ISD tissue imaging applications. [less ▲]

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See detailSelected Protein Monitoring in Histological Sections by Targeted MALDI-FTICR in-source decay Imaging.
Calligaris, David ULg; Longuespée, Rémi ULg; Debois, Delphine ULg et al

in Analytical Chemistry (2013), 85(4), 2117-26

MALDI mass spectrometry imaging (MALDI MSI) is a rapidly growing method in biomedical research allowing molecular mapping of proteins on histological sections. The images can be analyzed in terms of ... [more ▼]

MALDI mass spectrometry imaging (MALDI MSI) is a rapidly growing method in biomedical research allowing molecular mapping of proteins on histological sections. The images can be analyzed in terms of spectral pattern to define regions of interest. However, the identification and the differential quantitative analysis of proteins require off line or in situ proteomic methods using enzymatic digestion. The rapid identification of biomarkers holds great promise for diagnostic research but the major obstacle is the absence of rapid and direct method to detect and identify with a sufficient dynamic range a set of specific biomarkers. In the current work, we present a proof of concept for a method allowing identifying simultaneously a set of selected biomarkers on histological slices with minimal sample treatment using in-source decay (ISD) MSI and MALDI-Fourier transform ion cyclotron resonance (FTICR). In the proposed method, known biomarkers are spotted next to the tissue of interest, the whole MALDI plate being coated with 1,5-DAN matrix. The latter enhances MALDI radical-induced ISD, providing large tags of the amino acid sequences. Comparative analysis of ISD fragments between the reference spots and the specimen in imaging mode allows for unambiguous identification of the selected biomarker while preserving full spatial resolution. Moreover, the high resolution/high mass accuracy provided by FTICR mass spectrometry allows the identification of proteins. Well-resolved peaks and precise measurements of masses and mass differences allow the construction of reliable sequence tags for proteins identification. The method will allow the use MALDI-FTICR MSI as method for rapid targeted biomarker detection in complement to classical histology. [less ▲]

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See detailSéminaire des chercheurs Télévie 2013
Cimino, Jonathan ULg; Sounni, Nor Eddine ULg; Calligaris, David ULg et al

Poster (2012, December 10)

Séminaire des chercheurs Télévie 2013

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See detailNew Advances for In Situ Protein Identification by MALDI In-Source Decay FTMS Imaging
Calligaris, David; Longuespée, Rémi ULg; Zimmerman, Tyler et al

Poster (2012, November)

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See detailDetermination of the molecular players of adaptation to anti-angiogenic therapy in breast cancer by quantitative proteomic and high molecular MALDI Imaging.
Cimino, Jonathan ULg; Sounni, Nor Eddine ULg; Calligaris, David ULg et al

Poster (2012, October 13)

Breast carcinoma is the most common and second leading cause of cancer mortality in women. The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive ... [more ▼]

Breast carcinoma is the most common and second leading cause of cancer mortality in women. The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive pre-clinical researches on angiogenesis and finally the approval of VEGF-neutralizing antibodies (bevacizumab) and VEGF receptor tyrosine kinase inhibitors (RTKs:Sunitinib). The Sunitinib has been used clinically in patients with breast cancer refractory to other therapeutic agents. Unfortunately, like the cytotoxic therapies, these drugs do not produce lasting effects and resistance to treatment appeared clinically. Questions have emerged about the failure of anti-angiogenic therapy in clinic and the limitations of predictive preclinical models, and also about the molecular assessment of all stages of tumor adaptation and me<x>tastatic disease. To this end, we applied quantitative proteomics and imaging mass spectrometry tools to visualize and study the profiles of proteins and small molecules associated with tumor treated or not with Sunitinib using a novel preclinical model of breast carcinoma cells. In this project, we first developed a reproducible model of resistance to Sunitinib of human triple negative breast cancer MDA-MB-231 cells expressing luciferase gene. Cells were subcutaneously injected into mice RAG1-/- and divided into four experimental groups including, control mice treated with vehicle or Sunitinib for 30 days and sacrificed 1 days after treatment withdrawal or when tumor reached a volume of 300 mm3. In the second step. Tumors were analyzed using a nanoAcquity UPLC Synapt TM HDMS TM G1 (Waters, Manchester,UK) and Mass Spectrometry Imaging. For quantitative proteomic analyses of tumors, a bioinformatics analysis was used with the Protein lynx global server 2.2.5 software. Imaging mass spectrometry was performed on tissue sections of tumors and organs subsequently colonized by me<x>tastases. Matrix sublimation was used to coat tumor sections (14 µm-tick) with 1.5 Diaminonaphthalene for lipids analysis and Sinapinic acid for entire proteins analysis. Ion cartographies were recorded with a Solarix 9.4T FTMS instrument for lipids and with an Ultraflex II TOF-TOF instrument for entire proteins (Bruker Daltonics, Germany) with a spatial resolution of 100 µm. Global protemic revealed different protein profiles between tumor treated or not with Sunitinib. The Mass Spectrometry Imaging detected differences in intensity and location of some proteins and lipids are also associated with some histological features including inflammatory, necrotic and angiogenic areas. Bioinformatics analysis will be applied to ensure the integration of all data in order to provide the basis for identifying molecular pathways activated during the acquisition of refractoriness to drug treatments. [less ▲]

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See detailDistribution and identification of molecular interactions between tomato roots and bacterial biofilms
Debois, Delphine ULg; Jourdan, Emmanuel ULg; Ongena, Marc ULg et al

Conference (2012, September 05)

Some non pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in ... [more ▼]

Some non pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in soil (1). To initiate both phenomena leading to biocontrol activity, microorganisms use plant exudates to grow on roots and to produce in-situ active compounds. In Bacilli, cyclic lipopeptides of the surfactin, iturin and fengycin families represent important antibiotics involved in biocontrol (2). Recent studies in microbiology allowed a better understanding of plant microorganism interactions but few has been done at the molecular level. In this study, MALDI MS imaging has been used to study the nature of the secreted lipopeptide molecules, their relative quantity and their distribution in the root’s environment. Disinfected tomato seeds were first germinated at 28°C in sterile conditions for germination. Seedlings were then placed in Petri dish on ITO glass slide recovered with a thin layer of plant nutritive solution (Hoagland) containing 1,75% of agar and treated with freshly-grown cells of Bacillus amyloliquefaciens S499. Petri dishes were finally incubated vertically in phytotron at 28°C with a 16h photoperiod. Different root age / time of incubation were studied: 13 / 3; 13 / 7; 21 / 14 and 39 / 32. Control tomato root (without bacterial treatment) of the same ages were also analyzed (13 / 0; 21 / 0 and 42 / 0. For MALDI imaging experiments, the ITO slide was removed from the agar and dried in a dessiccator under vacuum. The matrix solution (α-cyano-hydroxycinnamic acid, 5mg/mL in ACN/0.2% TFA 70/30) was applied with an ImagePrep automated sprayer (Bruker Daltonics). An UltraFlex II TOF/TOF and a Solarix FT-ICR mass spectrometers were used to record molecular cartographies. The average mass spectra recorded around the tomato root (2-3 mm on both sides of the root) showed that lipopeptides were major compounds detected on the agar. The relative intensity of lipopeptides families varied with respect to the age of the root/biofilm system. In the 13/3 system, 3 homologues of surfactins were essentially detected (C13, C14 and C15), with very few iturins and fengycins. Their localizations were identical, whatever the considered homologue. Then the production of iturin and fengycin families increases in older systems (13/7 and 21/14) and a novel homologue of surfactin is detected (C12). Some variations in localizations within families may be observed (around the root or at the close vicinity of it in function of the considered homologue or alkali adduct). Then for the oldest system we studied, iturins and fengycins are not detected anymore and the localization of surfactins is less precise. In the 39/32 system, we also detected unknown compounds at 986.6, 1000.6, 1014.7 and 1028.7 m/z. The mass range of these compounds allied to the mass difference between two consecutive ion peaks let us think that these unknown compounds could be a new lipopeptide family. Investigations are in progress to identify these new secondary metabolites of Bacillus amyloliquefaciens. [less ▲]

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See detailStudy  of  breast  cancer  adaptation  to  anti-­angiogenic  therapies  by   molecular  imaging  on  tissue  slides
Cimino, Jonathan ULg; Calligaris, David ULg; Debois, Delphine ULg et al

Conference (2012, September 04)

Breast   carcinoma   is   the   most   common   and   second   leading   cause   of   cancer   mortality   in   women1.   The   ␣␣␣␣␣␣␣␣␣␣␣␣ ␣␣␣ ␣␣␣␣ ␣␣␣␣␣␣␣␣␣␣␣␣ ␣␣␣␣␣␣␣␣ ␣␣␣ ␣␣ ␣␣␣␣-­‐limiting   ... [more ▼]

Breast   carcinoma   is   the   most   common   and   second   leading   cause   of   cancer   mortality   in   women1.   The   ␣␣␣␣␣␣␣␣␣␣␣␣ ␣␣␣ ␣␣␣␣ ␣␣␣␣␣␣␣␣␣␣␣␣ ␣␣␣␣␣␣␣␣ ␣␣␣ ␣␣ ␣␣␣␣-­‐limiting   secondary   step   in   tumorigenesis   led   to   extensive   pre-­‐clinical   researches   on   angiogenesis   and   finally   the   approval   of   VEGF-­‐neutralizing   antibodies   (bevacizumab)  and  VEGF  receptor  tyrosine  kinase  inhibitors  (RTKs:sunitinib).  The  Sunitinib  has  been  used   clinically   in   patients   with   breast   cancer   refractory   to   other   therapeutic   agents2.   Unfortunately,   like   the   cytotoxic   therapies,   these   drugs   do   not   produce   lasting   effects   and   resistance   to   treatment   appeared   clinically3.   Recently,   independent   laboratories   have   reported   experimental   data   demonstrating   that   anti-­‐ angiogenic   treatments   inhibit   tumor   growth,   but   also   stimulate   the   formation   of   lung   metastases   after   treatment   discontinuation4.   The   field   of   imaging   mass   spectrometry   provides   new   tools   to   visualize   and   study  the  profiles  of  proteins  and  small  molecules  associated  with  biomedical  problems5.   To  this  aim,  we  conducted  a  series  of  experiments  to  setup  a  reproductible  model  of  resistance  to  sunitinib.   The   cells   MDA-­‐MB-­‐231   triple   negative,   from   human   breast   cancer   and   expressing   luciferase   are   injected   subcutaneously  into  mice  RAG1-­‐/-­‐.  The  mice  were  divided  into  four  experimental  groups  including,  on  the   one  hand,  control  mice  treated  with  placebo  (Carboxymethyl  cellulose,  CMC)  sacrificed  on  day  30  (group  1)   or  when  the  tumor  reached  a  volume  of  300  mm3  (group  2).    On  the  other  hand,  Sunitinib-­‐treated  mice  (LC   Laboratories,   40mg/kg/day),   sacrificed   at   day   30   (group   3),   or   when   the   tumor   reached   a   volume   of   300   mm3  (group  4).  MALDI  mass  spectrometry  imaging  was  performed  on  tissue  sections  of  tumors  and  organs   subsequently   colonized   by   metastases.   Matrix   sublimation   was   used   to   coat   tumor   sections   (14   μm-­‐tick)   with   1.5   Diaminonaphthalene   (1.5   DAN)   for   lipids   analysis   and   Sinapinic   acid   (SA)   for   entire   proteins   analysis.   Ion   cartographies   were   recorded   with   a   Solarix9.4T   FTMS   instrument   for   lipids   and   with   an   Ultraflex   II   TOF-­‐TOF   instrument   for   entire   proteins   (BrukerDaltonics,   Bremen,   Germany)   with   a   spatial   resolution  of  100  μm.     The  analysis  of  differential  protein/lipid  profiles  with  high  mass  accuracy  and  broadband  resolution  allows   detection   of   intense   signals   from   lipid   families   such   as   Phosphatidylcholine   (PC),   Triglyceride   (TAG),   Sphingomyelin   (SM)   and   precise   lipid   droplets   or   tumor   cells   differentiated   location   in   the   Sunitinib   resistant   tumor   cells   compared   to   control   cells.The   protein   profiles   of   the   4   groups   of   mice   show   differences   in   intensity   and   location,   enabling   a   correlation   to   inflammatory   (highlighted   by   histological   staining)  and  angiogenic  phenomenon.   [less ▲]

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See detailAnalysis of the Biocompatibility of Different Intraocular Lens (IOL) Material Using Mass Spectrometry Tisssue Imaging
Bertrand, Virginie ULg; Debois, Delphine ULg; Calligaris, David ULg et al

Conference (2012, September 04)

The cataract corresponds to the total or partial opacification of the lens of the eye preventing the passage of the light. At present, the surgery is the only effective treatment to overcome the cataract ... [more ▼]

The cataract corresponds to the total or partial opacification of the lens of the eye preventing the passage of the light. At present, the surgery is the only effective treatment to overcome the cataract. The surgical intervention consists in removing the cloudy lens and to replace it by an artificial intraocular lens (IOL). The in vivo implantation of these synthetic lenses involves the evaluation of several factors as their physico-chemical properties, their capacities to interact with lens epithelial cells and proteins, as well as their biocompatibility. During a previous study, we demonstrated major differences concerning the tackiness (atomic force microscopy), the cellular adhesion and the protein adsorption of various polymer disks intended for the manufacturing of intraocular lenses. The aim of this work was to correlate a histological analysis to a mass spectrometry imaging analysis performed on the same sample. To estimate the biocompatibility of the biomaterials, an animal testing was realized in rabbits. The various polymers were implanted subcutaneously. After one month, the 2 cm x 3 cm pieces of rabbit skin and underlying muscle with a 2 cm thickness were removed, fixed with formaldehyde 10% during six days, treated for the paraffin inclusion and stored at room temperature until use. Slices of 5 µm thickness were performed using a microtome. Paraffin was removed and tissue sections were washed in graded ethanol baths. The slices were then stained with the hematoxylin and eosin dyes. The analysis of stained sections showed different histo-morphological features according to the implanted polymer. For MALDI MSI purposes, on tissue protein digestion was performed using trypsin (1) and the MALDI matrix (α-cyanohydroxycinnamic acid, 5 mg/mL in ACN/0.2% TFA 70:30) was deposited using an ImagePrep automated sprayer (Bruker Daltonics, Bremen, Germany). Experiments were carried out using an UltraFlex II TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany). MALDI imaging can show the detection of different proteomic profiles according to the tested biomaterials, which may be considered as biocompatibility markers. The MALDI images of these markers are then correlated with the histo-morphological profiles. Consequently, mass spectrometry imaging can become a powerful tool in the evaluation of the biocompatibility of artificial implants in biomedical application. [less ▲]

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See detailDistribution and identification of molecular interactions between tomato roots and bacterial biofilms
Debois, Delphine ULg; Jourdan, Emmanuel ULg; Smargiasso, Nicolas ULg et al

Conference (2012, September)

Some non pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in ... [more ▼]

Some non pathogenic microorganisms evolving in the root micro-environment can trigger a positive effect on plant, increasing host defense against disease or/and directly inhibiting growth of pathogen in soil (1). To initiate both phenomena leading to biocontrol activity, microorganisms use plant exudates to grow on roots and to produce in-situ active compounds. In Bacilli, cyclic lipopeptides of the surfactin, iturin and fengycin families represent important antibiotics involved in biocontrol (2). Recent studies in microbiology allowed a better understanding of plant microorganism interactions but few has been done at the molecular level. In this study, MALDI MS imaging has been used to study the nature of the secreted lipopeptide molecules, their relative quantity and their distribution in the root’s environment. Disinfected tomato seeds were first germinated at 28°C in sterile conditions for germination. Seedlings were then placed in Petri dish on ITO glass slide recovered with a thin layer of plant nutritive solution (Hoagland) containing 1,75% of agar and treated with freshly-grown cells of Bacillus amyloliquefaciens S499. Petri dishes were finally incubated vertically in phytotron at 28°C with a 16h photoperiod. Different root age / time of incubation were studied: 13 / 3; 13 / 7; 21 / 14 and 39 / 32. Control tomato root (without bacterial treatment) of the same ages were also analyzed (13 / 0; 21 / 0 and 42 / 0. For MALDI imaging experiments, the ITO slide was removed from the agar and dried in a dessiccator under vacuum. The matrix solution (α-cyano-hydroxycinnamic acid, 5mg/mL in ACN/0.2% TFA 70/30) was applied with an ImagePrep automated sprayer (Bruker Daltonics). An UltraFlex II TOF/TOF and a Solarix FT-ICR mass spectrometers were used to record molecular cartographies. The average mass spectra recorded around the tomato root (2-3 mm on both sides of the root) showed that lipopeptides were major compounds detected on the agar. The relative intensity of lipopeptides families varied with respect to the age of the root/biofilm system. In the 13/3 system, 3 homologues of surfactins were essentially detected (C13, C14 and C15), with very few iturins and fengycins. Their localizations were identical, whatever the considered homologue. Then the production of iturin and fengycin families increases in older systems (13/7 and 21/14) and a novel homologue of surfactin is detected (C12). Some variations in localizations within families may be observed (around the root or at the close vicinity of it in function of the considered homologue or alkali adduct). Then for the oldest system we studied, iturins and fengycins are not detected anymore and the localization of surfactins is less precise. In the 39/32 system, we also detected unknown compounds at 986.6, 1000.6, 1014.7 and 1028.7 m/z. The mass range of these compounds allied to the mass difference between two consecutive ion peaks let us think that these unknown compounds could be a new lipopeptide family. Investigations are in progress to identify these new secondary metabolites of Bacillus amyloliquefaciens. [less ▲]

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See detailMolecular imaging through in combinaison with quantitative proteomic approaches unraveling the molecular players of breast cancer adaptation to anti-angiogenic therapy.
Cimino, Jonathan ULg; Sounni, Nor Eddine ULg; Calligaris, David ULg et al

Poster (2012, June 22)

Breast carcinoma is the most common and second leading cause of cancer mortality in women. The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive ... [more ▼]

Breast carcinoma is the most common and second leading cause of cancer mortality in women. The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive pre-clinical researches on angiogenesis and finally the approval of VEGF-neutralizing antibodies (bevacizumab) and VEGF receptor tyrosine kinase inhibitors (RTKs:Sunitinib). The Sunitinib has been used clinically in patients with breast cancer refractory to other therapeutic agents. Unfortunately, like the cytotoxic therapies, these drugs do not produce lasting effects and resistance to treatment appeared clinically. Questions have emerged about the failure of anti-angiogenic therapy in clinic and the limitations of predictive preclinical models, and also about the molecular assessment of all stages of tumor adaptation and metastatic disease. To this end, we applied quantitative proteomics and imaging mass spectrometry tools to visualize and study the profiles of proteins and small molecules associated with tumor treated or not with Sunitinib using a novel preclinical model of breast carcinoma cells. In this project, we first developed a reproducible model of resistance to Sunitinib of human triple negative breast cancer MDA-MB-231 cells expressing luciferase gene. Cells were subcutaneously injected into mice RAG1-/- and divided into four experimental groups including, control mice treated with vehicle or Sunitinib for 30 days and sacrificed 1 days after treatment withdrawal or when tumor reached a volume of 300 mm3. In the second step. Tumors were analyzed using a nanoAcquity UPLC Synapt TM HDMS TM G1 (Waters, Manchester,UK) and Mass Spectrometry Imaging. For quantitative proteomic analyses of tumors, a bioinformatics analysis was used with the Protein lynx global server 2.2.5 software. Imaging mass spectrometry was performed on tissue sections of tumors and organs subsequently colonized by metastases. Matrix sublimation was used to coat tumor sections (14 µm-tick) with 1.5 Diaminonaphthalene for lipids analysis and Sinapinic acid for entire proteins analysis. Ion cartographies were recorded with a Solarix 9.4T FTMS instrument for lipids and with an Ultraflex II TOF-TOF instrument for entire proteins (Bruker Daltonics, Germany) with a spatial resolution of 100 µm. Global protemic revealed different protein profiles between tumor treated or not with Sunitinib. The Mass Spectrometry Imaging detected differences in intensity and location of some proteins and lipids are also associated with some histological features including inflammatory, necrotic and angiogenic areas. Bioinformatics analysis will be applied to ensure the integration of all data in order to provide the basis for identifying molecular pathways activated during the acquisition of refractoriness to drug treatments. [less ▲]

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See detailA Promising Perspective for Pathologies Diagnosis by MALDI In-Source Decay Imaging with a FTMS System.
Calligaris, David ULg; Debois, Delphine ULg; Turtoi, Andrei ULg et al

Poster (2012, May 23)

Introduction MALDI imaging mass spectrometry has proven to be effective for the discovery and the monitoring of disease-related proteins. With this technique a molecular diagnosis could be done directly ... [more ▼]

Introduction MALDI imaging mass spectrometry has proven to be effective for the discovery and the monitoring of disease-related proteins. With this technique a molecular diagnosis could be done directly on tissue sections in the environment of the diseased area. The use of in-source decay (ISD), that does allow fast and reliable sequences assignments of proteins termini, is a crucial tool for the identification of known biomarkers during MALDI imaging experiments. Combined with ultra-high mass resolution and high mass measurement accuracy of Fourier transform ion-cyclotron (FTICR) mass spectrometry, it is possible to unambiguously assign sequences of proteins present in tissue slices. In this study, we have shown that FTICR mass spectrometry could be a powerful tool to diagnose pathologies by MALDI-ISD imaging. Methods All measurements were carried out on a SolariX FTMS (9.4 tesla) equipped with a Dual Source including smartbeamTMII laser which includes a robust solid state 1 kHz laser with advanced optics for molecular imaging (Bruker Daltonics). Lysozyme (14.3-kDa) or Human Serum Albumin (66.3-kDa) solution (1 mg/ml in 0.1 % TFA) was mixed with 1,5-diaminonaphthalene (DAN) and analyzed by MALDI-ISD and MALDI-ISD imaging. Mouse brain and rabbit eye tissue slices were washed (fixed) to obtain optimal sensitivity and high-quality ion. Before DAN application with an ImagePrep (Bruker Daltonics) and MALDI-ISD imaging analyzes, spots of myelin and crystalline were deposited near mouse brain or rabbit eye tissues, respectively. Results were interpreted using BioToolsTM 3.2 in combination with MascotTM (Matrix Science) for ISD spectra and FlexImagingTM 2.1 for MALDI-ISD imaging experiments. α Preliminary data The studies were carried out by MALDI-ISD and MALDI-ISD imaging analyses to evidence the interest on FTICR mass spectrometer for proteins identification in the field of biomarkers characterization. It is demonstrated that protein ISD leads to the same pattern of fragmentation observed during MALDI-TOF analyzes. Fragmentation generates cn- and zn-series ions of lysozyme and HSA in presence of DAN. Supplementary an-, bn-, xn- and yn-series ions can also be observed. The internal calibration of all the data provides a mass accuracy neighboring 2.5 ppm over the m/z range of interest (300-2500 Da) and a mass resolution of 70000 at m/z 400 Da. It allows the assignment of ISD fragments of proteins, in the low mass range (m/z between 300 and 900), whether from pure solutions or included in tissue slices. Moreover, spots of pure proteins solution (myelin or crystalline) near tissue slices allows to unambiguously validate the proteins identification during MALDI ISD imaging experiments. Novel aspect This study evidences the main input of FTICR mass spectrometer for pathologies diagnosis based on biomarkers localization and identification by MALDI-ISD imaging. [less ▲]

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See detailApplication of molecular imaging in combination with quantitative proteomic approaches to determine the molecular players of adaptation to anti-angiogenic therapy in breast cancer.
Cimino, Jonathan ULg; Sounni, Nor Eddine ULg; Calligaris, David ULg et al

Poster (2012, May 04)

The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive pre-clinical researches on angiogenesis and finally the approval of VEGF-neutralizing ... [more ▼]

The recognition of the “angiogenic switch” as a rate-limiting secondary step in tumorigenesis led to extensive pre-clinical researches on angiogenesis and finally the approval of VEGF-neutralizing antibodies (bevacizumab) and VEGF receptor tyrosine kinase inhibitors (RTKs:Sunitinib). The Sunitinib has been used clinically in patients with breast cancer refractory to other therapeutic agents. Unfortunately, like the cytotoxic therapies, these drugs do not produce lasting effects and resistance to treatment appeared clinically. Questions have emerged about the failure of anti-angiogenic therapy in clinic and the limitations of predictive preclinical models, and also about the molecular assessment of all stages of tumor adaptation and metastatic disease. To this end, we applied a quantitative proteomics and imaging mass spectrometry tools to visualize and study the profiles of proteins and small molecules associated with tumor treated or not with Sunitinib using a novel preclinical model of breast carcinoma cells. [less ▲]

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See detailImaging Guided Proteomics Unveils Heterogeniety in Colorectal Carcinoma Liver Metastases – Implications for Targeted Therapies.
blomme, Arnaud; Turtoi, Andrei ULg; Delvaux, David ULg et al

in Proceedings Giga Day 2012 (2012, May 04)

Detailed reference viewed: 47 (22 ULg)
See detailIn situ protein identification in imaging mass spectrometry
Calligaris, David ULg; Debois, Delphine ULg; De Pauw, Edwin ULg

Scientific conference (2012, May 04)

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is an emerging tool for clinical research. MALDI MSI can be used to elucidate the relative abundance and spatial ... [more ▼]

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is an emerging tool for clinical research. MALDI MSI can be used to elucidate the relative abundance and spatial localization of peptides and proteins throughout a tissue section. For this, a matrix is applied on the tissue in either a spotted array or a homogenous coating. Acquisition of mass spectra is then carried out by performing a raster with a laser across the tissue section in a defined pattern. The spectra acquired from each position on the tissue section contain molecular weight and intensity information representative of the biomolecules at that position. One can plot the intensity of any measured ion as a function of individual pixel locations to generate m/z specific images. But, if protein desorption/ionization and subsequent MS analyses provides a measurement of molecular weight, no protein identification is performed. To achieve this, several methods have been developed. In this talk, I will first present the methods inspired by classical proteomics techniques that are regularly used to identify proteins. Bottom-up and top-down approaches have been used directly from a tissue slice, leading to the identification of some of the most abundant proteins present within the tissue slice. Then, I will present the new developments led in our lab for imaging and especially for in situ protein identification. The first example will deal with the exceptional features of FT-ICR mass spectrometry for in-source decay (ISD)-based protein identification. The benefit of mass accuracy and high mass resolution allow unequivocal assignment of ISD fragments of proteins, in the low mass range (m/z between 400 and 900), whether from pure solutions or from tissue slices. The next example is the use of a matrix “cleaning” software that reduce/remove matrix peaks thus facilitating ISD spectra analyses. Finally, proteins identification by localization and MALDI-ISD profile matching will also be a really simplistic and interesting method that will complement the immunohistological techniques commonly used to validate expression of known biomarkers within diseased tissues. [less ▲]

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See detailINTRA-TUMORAL HETEROGENEITY AND RATIONAL SELECTION OF ANTIGENS FOR TARGETED THERAPY OF LIVER METASTASES
Turtoi, Andrei ULg; Blomme, Arnaud ULg; Delvaux, David ULg et al

in Acta Chirurgica Belgica (2012, May), 112(3), 8953

Objectives: Targeted therapies of liver metastases are gaining a major stake in current and future treatment options. However, the malignant lesions are heterogeneous in nature offering niches for cancer ... [more ▼]

Objectives: Targeted therapies of liver metastases are gaining a major stake in current and future treatment options. However, the malignant lesions are heterogeneous in nature offering niches for cancer cells causing treatment resistance and relapse. Therefore, a rational strategy is needed to select targetable antigens that would overcome this intra-tumoral heterogeneity. Methods: After ethical committee approval, 48 fresh liver metastases of colorectal origin were prospectively collected from patients undergoing liver resection. Here we macroscopically divided the lesion in different zones and generated a unique quantitative picture of the proteome heterogeneity in colorectal carcinoma liver metastases. Particular focus was laid on accessible proteins, a protein subclass comprising cell membrane associated and extracellular proteins. Accordingly, the tissues were ex-vivo biotinylated, affinity purified and analyzed for each zone separately using nano-UPLC-MSe proteomics technique. In total over 1500 unique proteins were statistically divided into different patterns of expression. Results: We have generated a quantitative picture of the proteome heterogeneity in colorectal carcinoma liver metastases. The study offers insight into novel targets but also antigens against which the antibodies are already involved in clinical trials or treatment of liver metastases. Extensive clustering and validation experiments highlight novel markers that offer the potential to homogeneously cover the metastatic lesion and become better targets. Conclusions: Two such antigens, LTBP2 and TGFBI were selected for functional analysis in colorectal carcinoma cells. In vitro and in vivo experiments showed that in particular TGFBI is relevant for migration and proliferation capacity of colorectal cancer cells. The suppression of this protein led to significant inhibition of tumor growth, crystalizing it as bona fide target for the development of anti-metastases therapies. [less ▲]

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