Séminaire des chercheurs Télévie 2013

Poster (2012, December 10)

Séminaire des chercheurs Télévie 2013

Determination of the molecular players of adaptation to anti-angiogenic therapy in breast cancer by quantitative proteomic and high molecular MALDI Imaging.

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 ▲]

Molecular imaging through in combinaison with quantitative proteomic approaches unraveling the molecular players of breast cancer adaptation to anti-angiogenic therapy.

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 ▲]

Contribution of high mass resolution and accuracy of FTMS to molecular imaging

Conference (2012, April 04)

Since its first implementation in 1997, MALDI Mass Spectrometry Imaging (MALDI MSI) has become an important tool in the proteomic arsenal, especially for biomarker hunting. First dedicated to high ... [more ▼]

Since its first implementation in 1997, MALDI Mass Spectrometry Imaging (MALDI MSI) has become an important tool in the proteomic arsenal, especially for biomarker hunting. First dedicated to high molecular weight, MALDI MSI is more and more used to map the distribution of small molecules too (lipids, drugs and metabolites,…). Last developments tend to improve the sample treatments to obtain the best spatial resolution as possible. From this perspective, great efforts have been made on the MALDI matrix deposition methods. Now, one of the remaining challenges for MALDI-MSI users consists of identification of detected molecules. For high molecular weight, methods inspired by classical proteomics techniques, are regularly used. Bottom-Up (PMF obtained after in situ trypsin digestion) and Top-Down (in situ In-Source Decay) approaches have been used directly from a tissue slice, leading to the identification of some of the most abundant proteins present at the surface of the tissue. When small molecules are analyzed, the identification is more straightforward. Indeed, tandem mass spectrometry can easily be used, leading to the fragmentation of the detected compounds which allows their unambiguous identification. This identification is even more reliable when high resolution exact mass measurements can be performed. In this talk, I will present how in our lab, we profit of the exceptional features of FT-ICR mass spectrometry for imaging and especially for identification purposes. The first example will deal with the benefit of high mass accuracy and high mass resolution for ISD-based protein identification. The mass accuracy and high mass resolution coupled with the use of a “cleaning” software allow unequivocal assignment of ISD fragments of proteins, in the low mass range (m/z between 300 and 900), whether from pure solutions or from tissue slices. The next examples will deal with the imaging of small molecules. The identification of drugs and their metabolites is facilitated with high mass accuracy. In our lab, we work on the localization of methadone and its first metabolite, EDDP in necrophagous fly larvae. In the mass range of these compounds (278-310 m/z), many matrix ion peaks are detected and the unique features of FT-ICR allows for unambiguous identification thanks to exact mass measurements. We also use MALDI Imaging to map the messenger molecules between plant roots and beneficial bacteria. The comparison of spectra recorded with a TOF/TOF instrument and with a FT-ICR demonstrates that high resolution allows for detecting molecules which could have been missed otherwise. It also allows to distinguish unknown compounds from alkali adducts of known molecules. [less ▲]