MMP-Mediated Collagen Remodeling and Vessel Functions
Noël, Agnès ; Sounni, Nor Eddine
in Brix, Klaudia; Stocker, Walter (Eds.) Proteases: Structure and Function (2013)Detailed reference viewed: 2 (1 ULg)
Towards Lipidomics of Low-Abundant Species for Exploring Tumor Heterogeneity Guided by High-Resolution Mass Spectrometry Imaging
Cimino, Jonathan ; ; Far, Johann 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 ▲]Detailed reference viewed: 39 (8 ULg)
Laser-induced choroidal neovascularization model to study age-related macular degeneration in mice.
LAMBERT, Vincent ; Lecomte, Julie ; Hansen, Sylvain et al
in Nature Protocols (2013), 8(11), 2197-2211
The mouse model of laser-induced choroidal neovascularization (CNV) has been used extensively in studies of the exudative form of age-related macular degeneration (AMD). This experimental in vivo model ... [more ▼]
The mouse model of laser-induced choroidal neovascularization (CNV) has been used extensively in studies of the exudative form of age-related macular degeneration (AMD). This experimental in vivo model relies on laser injury to perforate Bruch's membrane, resulting in subretinal blood vessel recruitment from the choroid. By recapitulating the main features of the exudative form of human AMD, this assay has served as the backbone for testing antiangiogenic therapies. This standardized protocol can be applied to transgenic mice and can include treatments with drugs, recombinant proteins, antibodies, adenoviruses and pre-microRNAs to aid in the search for new molecular regulators and the identification of novel targets for innovative treatments. This robust assay requires 7-14 d to complete, depending on the treatment applied and whether immunostaining is performed. This protocol includes details of how to induce CNV, including laser induction, lesion excision, processing and different approaches to quantify neoformed vasculature. [less ▲]Detailed reference viewed: 79 (32 ULg)
MicroRNA-146a is a therapeutic target and biomarker for peripartum cardiomyopathy.
Halkein, Julie ; ; et al
in Journal of Clinical Investigation (2013), 123(5), 2143-54
Peripartum cardiomyopathy (PPCM) is a life-threatening pregnancy-associated cardiomyopathy in previously healthy women. Although PPCM is driven in part by the 16-kDa N-terminal prolactin fragment (16K PRL ... [more ▼]
Peripartum cardiomyopathy (PPCM) is a life-threatening pregnancy-associated cardiomyopathy in previously healthy women. Although PPCM is driven in part by the 16-kDa N-terminal prolactin fragment (16K PRL), the underlying molecular mechanisms are poorly understood. We found that 16K PRL induced microRNA-146a (miR-146a) expression in ECs, which attenuated angiogenesis through downregulation of NRAS. 16K PRL stimulated the release of miR-146a-loaded exosomes from ECs. The exosomes were absorbed by cardiomyocytes, increasing miR-146a levels, which resulted in a subsequent decrease in metabolic activity and decreased expression of Erbb4, Notch1, and Irak1. Mice with cardiomyocyte-restricted Stat3 knockout (CKO mice) exhibited a PPCM-like phenotype and displayed increased cardiac miR-146a expression with coincident downregulation of Erbb4, Nras, Notch1, and Irak1. Blocking miR-146a with locked nucleic acids or antago-miRs attenuated PPCM in CKO mice without interrupting full-length prolactin signaling, as indicated by normal nursing activities. Finally, miR-146a was elevated in the plasma and hearts of PPCM patients, but not in patients with dilated cardiomyopathy. These results demonstrate that miR-146a is a downstream-mediator of 16K PRL that could potentially serve as a biomarker and therapeutic target for PPCM. [less ▲]Detailed reference viewed: 88 (29 ULg)
Conditioned Medium from Bone marrow-derived Mesenchymal Stem Cells improves recovery after Spinal Cord Injury in rats: an original strategy to avoid cell transplantation.
CANTINIEAUX, Dorothée ; ; BLACHER, Silvia et al
in PLoS ONE (2013), 8(8), 69515
Spinal cord injury triggers irreversible loss of motor and sensory functions. Numerous strategies aiming at repairing the injured spinal cord have been studied. Among them, the use of bone marrow-derived ... [more ▼]
Spinal cord injury triggers irreversible loss of motor and sensory functions. Numerous strategies aiming at repairing the injured spinal cord have been studied. Among them, the use of bone marrow-derived mesenchymal stem cells (BMSCs) is promising. Indeed, these cells possess interesting properties to modulate CNS environment and allow axon regeneration and functional recovery. Unfortunately, BMSC survival and differentiation within the host spinal cord remain poor, and these cells have been found to have various adverse effects when grafted in other pathological contexts. Moreover, paracrine-mediated actions have been proposed to explain the beneficial effects of BMSC transplantation after spinal cord injury. We thus decided to deliver BMSC-released factors to spinal cord injured rats and to study, in parallel, their properties in vitro. We show that, in vitro, BMSC-conditioned medium (BMSC-CM) protects neurons from apoptosis, activates macrophages and is pro-angiogenic. In vivo, BMSC-CM administered after spinal cord contusion improves motor recovery. Histological analysis confirms the pro-angiogenic action of BMSC-CM, as well as a tissue protection effect. Finally, the characterization of BMSC-CM by cytokine array and ELISA identified trophic factors as well as cytokines likely involved in the beneficial observed effects. In conclusion, our results support the paracrine-mediated mode of action of BMSCs and raise the possibility to develop a cell-free therapeutic approach. [less ▲]Detailed reference viewed: 60 (11 ULg)
Myoferlin is a key regulator of EGFR activity in breast cancer.
Turtoi, Andrei ; Blomme, Arnaud ; Bellahcene, Akeila et al
in Cancer Research (2013)
Myoferlin is a member of the ferlin family of proteins that participate in plasma membrane fusion, repair and endocytosis. While some reports have implicated myoferlin in cancer, the extent of its ... [more ▼]
Myoferlin is a member of the ferlin family of proteins that participate in plasma membrane fusion, repair and endocytosis. While some reports have implicated myoferlin in cancer, the extent of its expression in and contributions to cancer are not well established. In this study, we show that myoferlin is overexpressed in human breast cancers and that it is has a critical role in controlling degradation of the EGFR after its activation and internalization in breast cancer cells. Myoferlin depletion blocked EGF-induced cell migration and epithelial-to-mesenchymal transition. Both effects were induced as a result of impaired degradation of phosphorylated EGFR via dysfunctional plasma membrane caveolae and alteration of caveolin homooligomerization. In parallel, myoferlin depletion reduced tumor development in a chicken chorioallantoic membrane xenograft model of human breast cancer. Considering the therapeutic significance of EGFR targeting, our findings identify myoferlin as an novel candidate function to target for future drug development. [less ▲]Detailed reference viewed: 74 (10 ULg)
Aspects biologiques de l'angiogenèse dans le myélome multiple
Otjacques, Eléonore ; Binsfeld, Marilène ; Beguin, Yves et al
in Oncohématologie (2013), 7(2), 6-12
Le myélome multiple (MM) est une maladie hématologique caractérisée par la prolifération excessive de plasmocytes cancéreux au sein de la moelle osseuse. Une des caractéristiques principales de cette ... [more ▼]
Le myélome multiple (MM) est une maladie hématologique caractérisée par la prolifération excessive de plasmocytes cancéreux au sein de la moelle osseuse. Une des caractéristiques principales de cette maladie est l’interaction entre les cellules myélomateuses et les cellules voisines situées dans la moelle. De par l’activation des cellules endothéliales, l’angiogenèse joue un rôle essentiel dans le développement du MM. Dans cet article, les processus permettant la progression du phénomène d’angiogenèse dans le MM seront abordés. Entre autres, nous identifierons les cellules interagissant avec les plasmocytes cancéreux, les cytokines influençant l’angiogenèse ou encore les protéases responsables de la dégradation de la matrice extracellulaire impliquées dans la pathologie. Finalement, l’influence du phénomène d’hypoxie (via l’expression de la protéine hypoxia-inducible factor-1) et le rôle de l’activation constitutive du Nuclear Factor-kB dans la néovascularisation seront mis en avant. [less ▲]Detailed reference viewed: 42 (12 ULg)
Selected Protein Monitoring in Histological Sections by Targeted MALDI-FTICR in-source decay Imaging.
Calligaris, David ; Longuespée, Rémi ; Debois, Delphine 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 ▲]Detailed reference viewed: 73 (16 ULg)
Mithramycin Exerts an Anti-Myeloma Effect and Displays Anti-Angiogenic Effects through Up-Regulation of Anti-Angiogenic Factors.
Otjacques, Eléonore ; Binsfeld, Marilène ; Rocks, Natacha et al
in PLoS ONE (2013), 8(5), 62818
Mithramycin (MTM), a cytotoxic compound, is currently being investigated for its anti-angiogenic activity that seems to be mediated through an inhibition of the transcription factor SP1. In this study we ... [more ▼]
Mithramycin (MTM), a cytotoxic compound, is currently being investigated for its anti-angiogenic activity that seems to be mediated through an inhibition of the transcription factor SP1. In this study we evaluated its anti-myeloma effects in the syngenic 5TGM1 model in vitro as well as in vivo. In vitro, MTM inhibited DNA synthesis of 5TGM1 cells with an IC50 of 400 nM and induced an arrest in cell cycle progression at the G1/S transition point. Western-blot revealed an up-regulation of p53, p21 and p27 and an inhibition of c-Myc, while SP1 remained unaffected. In rat aortic ring assays, a strong anti-angiogenic effect was seen, which could be explained by a decrease of VEGF production and an up-regulation of anti-angiogenic proteins such as IP10 after MTM treatment. The administration of MTM to mice injected with 5TGM1 decreased 5TGM1 cell invasion into bone marrow and myeloma neovascularisation. These data suggest that MTM displays anti-myeloma and anti-angiogenic effects that are not mediated by an inhibition of SP1 but rather through c-Myc inhibition and p53 activation. [less ▲]Detailed reference viewed: 34 (5 ULg)
Inhibition of Tumor Angiogenesis and Growth by a Small-Molecule Multi-FGF Receptor Blocker with Allosteric Properties.
; ; et al
in Cancer Cell (2013), 23(4), 477-88
Receptor tyrosine kinases (RTK) are targets for anticancer drug development. To date, only RTK inhibitors that block orthosteric binding of ligands and substrates have been developed. Here, we report the ... [more ▼]
Receptor tyrosine kinases (RTK) are targets for anticancer drug development. To date, only RTK inhibitors that block orthosteric binding of ligands and substrates have been developed. Here, we report the pharmacologic characterization of the chemical SSR128129E (SSR), which inhibits fibroblast growth factor receptor (FGFR) signaling by binding to the extracellular FGFR domain without affecting orthosteric FGF binding. SSR exhibits allosteric properties, including probe dependence, signaling bias, and ceiling effects. Inhibition by SSR is highly conserved throughout the animal kingdom. Oral delivery of SSR inhibits arthritis and tumors that are relatively refractory to anti-vascular endothelial growth factor receptor-2 antibodies. Thus, orally-active extracellularly acting small-molecule modulators of RTKs with allosteric properties can be developed and may offer opportunities to improve anticancer treatment. [less ▲]Detailed reference viewed: 43 (16 ULg)
Deletion of cysteine cathepsins B or L yields differential impacts on murine skin proteome and degradome
; ; et al
in Molecular & Cellular Proteomics (2013), 12(3), 611-25
Numerous studies highlight that concerted proteolysis is essential for skin morphology and function. The cysteine protease cathepsin L (Ctsl) has been implicated in epidermal proliferation and ... [more ▼]
Numerous studies highlight that concerted proteolysis is essential for skin morphology and function. The cysteine protease cathepsin L (Ctsl) has been implicated in epidermal proliferation and desquamation as well as in hair cycle regulation. In stark contrast, mice deficient for cathepsin B (Ctsb) do not display an overt skin phenotype. To understand the systematic consequences of deleting Ctsb or Ctsl, we determined protein abundances of > 1300 proteins and proteolytic cleavage events in skin samples of wild-type, Ctsb-/- and Ctsl-/- mice by mass spectrometry based proteomics. Both protease deficiencies revealed distinct quantitative changes in proteome composition. Ctsl-/- skin revealed increased levels of the cysteine protease inhibitors cystatin B and cystatin M/E, increased cathepsin D and accumulation of the extracellular glycoprotein periostin. Immunohistochemistry located periostin predominantly in hypodermal connective tissue of Ctsl-/- skin. Proteomic identification of proteolytic cleavage sites within skin proteins revealed numerous processing sites that are underrepresented in Ctsl-/- or Ctsb-/- samples. Notably, few of the affected cleavage sites shared the canonical Ctsl or Ctsb specificity, providing further evidence for a complex proteolytic network in the skin. Novel processing sites in proteins such as dermokine and Notch-1 were detected. Simultaneous analysis of acetylated protein N-termini showed prototypical mammalian N-alpha acetylation. These results illustrate an influence of both Ctsb and Ctsl on the murine skin proteome and degradome with the phenotypic consequences of the absence of either protease differing considerably. [less ▲]Detailed reference viewed: 25 (7 ULg)
New prospects in the roles of the C-terminal domains of VEGF-A and their cooperation for ligand binding, cellular signaling and vessels formation.
Delcombel, Romain ; Janssen, Lauriane ; et al
in Angiogenesis (2013), 16(2), 353-71
VEGF-A is a crucial growth factor for blood vessel homeostasis and pathological angiogenesis. Due to alternative splicing of its pre-mRNA, VEGF-A is produced under several isoforms characterized by the ... [more ▼]
VEGF-A is a crucial growth factor for blood vessel homeostasis and pathological angiogenesis. Due to alternative splicing of its pre-mRNA, VEGF-A is produced under several isoforms characterized by the combination of their C-terminal domains, which determines their respective structure, availability and affinity for co-receptors. As controversies still exist about the specific roles of these exon-encoded domains, we systematically compared the properties of eight natural and artificial variants containing the domains encoded by exons 1-4 and various combinations of the domains encoded by exons 5, 7 and 8a or 8b. All the variants (VEGF(111)a, VEGF(111)b, VEGF(121)a, VEGF(121)b, VEGF(155)a, VEGF(155)b, VEGF(165)a, VEGF(165)b) have a similar affinity for VEGF-R2, as determined by Surface plasmon resonance analyses. They strongly differ however in terms of binding to neuropilin-1 and heparin/heparan sulfate proteoglycans. Data indicate that the 6 amino acids encoded by exon 8a must be present and cooperate with those of exons 5 or 7 for efficient binding, which was confirmed in cell culture models. We further showed that VEGF(165)b has inhibitory effects in vitro, as previously reported, but that the shortest VEGF variant possessing also the 6 amino acids encoded by exon 8b (VEGF(111)b) is remarkably proangiogenic, demonstrating the critical importance of domain interactions for defining the VEGF properties. The number, size and localization of newly formed blood vessels in a model of tumour angiogenesis strongly depend also on the C-terminal domain composition, suggesting that association of several VEGF isoforms may be more efficient for treating ischemic diseases than the use of any single variant. [less ▲]Detailed reference viewed: 30 (12 ULg)
Hyperglycosylated human chorionic gonadotropin stimulates angiogenesis through TGF-beta receptor activation.
; Blacher, Silvia ; Munaut, Carine et al
in FASEB Journal (2013), 27(4), 1309-21
Embryo implantation requires extensive angiogenesis at the maternal-fetal interface. Hyperglycosylated human chorionic gonadotropin (hCG-H), a trophoblast invasive signal produced by extravillous ... [more ▼]
Embryo implantation requires extensive angiogenesis at the maternal-fetal interface. Hyperglycosylated human chorionic gonadotropin (hCG-H), a trophoblast invasive signal produced by extravillous cytotrophoblasts and by choriocarcinoma, was evaluated for its angiogenic role. hCG-H was purified by HPLC from choriocarcinoma supernatant, and the glycosylation pattern was determined by 2D gel analysis. Angiogenesis models used were aortic ring assay with wild-type and LHCGR-knockout mice, endothelial and mural cell proliferation, and migration assays. The TGF-beta signaling pathway was studied by coimmunoprecipitation, competitive binding, TGF-beta reporter gene assays, and Smad immunoblotting. hCG-H displayed a potent angiogenic effect [3.2-fold increase of number of vessel intersections in wild-type aortic rings (11.406 to 36.964)]. hCG-H-induced angiostimulation was independent of the classic hCG signaling pathway since it persisted in LHCGR-knockout mice [4.73-fold increase of number of vessel intersections (10.826 to 51.288)]. Using TGF-beta signaling inhibitors, Tbeta-RII was identified as the hCG-H receptor responsible for its angiogenic switch. hCG-H exposure enhanced phosphorylation of Smad 2 in endothelial and mural cells and genomic activation of Smad-responsive elements. Interaction between hCG-H and Tbeta-RII was demonstrated by coimmunoprecipitation and binding competition with (125)I-TGF-beta. This new paracrine interaction between trophoblast and endothelial cells through the hCG-H and the TGF-beta receptor complex plays a key role in angiogenesis associated with placental development and tumorigenesis. [less ▲]Detailed reference viewed: 47 (8 ULg)
MiR-210 promotes a hypoxic phenotype and increases radioresistance in human lung cancer cell lines.
; ; et al
in Cell Death & Disease (2013), 4
The resistance of hypoxic cells to radiotherapy and chemotherapy is a major problem in the treatment of cancer. Recently, an additional mode of hypoxia-inducible factor (HIF)-dependent transcriptional ... [more ▼]
The resistance of hypoxic cells to radiotherapy and chemotherapy is a major problem in the treatment of cancer. Recently, an additional mode of hypoxia-inducible factor (HIF)-dependent transcriptional regulation, involving modulation of a specific set of micro RNAs (miRNAs), including miR-210, has emerged. We have recently shown that HIF-1 induction of miR-210 also stabilizes HIF-1 through a positive regulatory loop. Therefore, we hypothesized that by stabilizing HIF-1 in normoxia, miR-210 may protect cancer cells from radiation. We developed a non-small cell lung carcinoma (NSCLC)-derived cell line (A549) stably expressing miR-210 (pmiR-210) or a control miRNA (pmiR-Ctl). The miR-210-expressing cells showed a significant stabilization of HIF-1 associated with mitochondrial defects and a glycolytic phenotype. Cells were subjected to radiation levels ranging from 0 to 10 Gy in normoxia and hypoxia. Cells expressing miR-210 in normoxia had the same level of radioresistance as control cells in hypoxia. Under hypoxia, pmiR-210 cells showed a low mortality rate owing to a decrease in apoptosis, with an ability to grow even at 10 Gy. This miR-210 phenotype was reproduced in another NSCLC cell line (H1975) and in HeLa cells. We have established that radioresistance was independent of p53 and cell cycle status. In addition, we have shown that genomic double-strand breaks (DSBs) foci disappear faster in pmiR-210 than in pmiR-Ctl cells, suggesting that miR-210 expression promotes a more efficient DSB repair. Finally, HIF-1 invalidation in pmiR-210 cells removed the radioresistant phenotype, showing that this mechanism is dependent on HIF-1. In conclusion, miR-210 appears to be a component of the radioresistance of hypoxic cancer cells. Given the high stability of most miRNAs, this advantage could be used by tumor cells in conditions where reoxygenation has occurred and suggests that strategies targeting miR-210 could enhance tumor radiosensitization. [less ▲]Detailed reference viewed: 30 (4 ULg)
Targeting the tumor microenvironment for cancer therapy.
Sounni, Nor Eddine ; Noël, Agnès
in Clinical Chemistry (2013), 59(1), 85-93
BACKGROUND: With the emergence of the tumor microenvironment as an essential ingredient of cancer malignancy, therapies targeting the host compartment of tumors have begun to be designed and applied in ... [more ▼]
BACKGROUND: With the emergence of the tumor microenvironment as an essential ingredient of cancer malignancy, therapies targeting the host compartment of tumors have begun to be designed and applied in the clinic. CONTENT: The malignant features of cancer cells cannot be manifested without an important interplay between cancer cells and their local environment. The tumor infiltrate composed of immune cells, angiogenic vascular cells, lymphatic endothelial cells, and cancer-associated fibroblastic cells contributes actively to cancer progression. The ability to change these surroundings is an important property by which tumor cells are able to acquire some of the hallmark functions necessary for tumor growth and metastatic dissemination. Thus in the clinical setting the targeting of the tumor microenvironment to encapsulate or destroy cancer cells in their local environment has become mandatory. The variety of stromal cells, the complexity of the molecular components of the tumor stroma, and the similarity with normal tissue present huge challenges for therapies targeting the tumor microenvironment. These issues and their interplay are addressed in this review. After a decade of intensive clinical trials targeting cellular components of the tumor microenvironment, more recent investigations have shed light on the important role in cancer progression played by the noncellular stromal compartment composed of the extracellular matrix. SUMMARY: A better understanding of how the tumor environment affects cancer progression should provide new targets for the isolation and destruction of cancer cells via interference with the complex crosstalk established between cancer cells, host cells, and their surrounding extracellular matrix. [less ▲]Detailed reference viewed: 81 (11 ULg)
Use of Sheep Ovarian Tissue as a Model to Restore Fertility in Young Cancerous Women
Fransolet, Maïté ; HENRY, Laurie ; Rozet, Eric et al
Poster (2012, December 10)Detailed reference viewed: 62 (14 ULg)
Study of the molecular players and drastic changes in metabolic pathways of breast cancer adaptation to anti-angiogenic therapy with Molecular Imaging and quantitative proteomic approaches.
Cimino, Jonathan ; Sounni, Nor Eddine ; Calligaris, David et al
Poster (2012, November)Detailed reference viewed: 63 (14 ULg)
Determination of the molecular players of adaptation to anti-angiogenic therapy in breast cancer by quantitative proteomic and high molecular MALDI Imaging.
Cimino, Jonathan ; Sounni, Nor Eddine ; Calligaris, David 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 ▲]Detailed reference viewed: 84 (10 ULg)
Study of breast cancer adaptation to anti-angiogenic therapies by molecular imaging on tissue slides
Cimino, Jonathan ; Calligaris, David ; Debois, Delphine 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 ▲]Detailed reference viewed: 93 (8 ULg)