Dataset Information

Proteomic analysis of neuroblastoma-derived exosomes LC-MS

ABSTRACT: Neuroblastoma (NB) is the most common extracranial pediatric solid tumor. At diagnosis, around 70% of patients with metastatic disease present bone-marrow (BM) infiltration; however, the mechanism underlying this specific tropism has to be elucidated. Tumor-derived exosomes may support metastatic progression in several tumors by interacting with the microenvironment, and may serve as tumor biomarkers. The main objective of this study was to identify an exosomal signature associated with NB metastatic BM dissemination. Therefore, we characterized the proteomic cargo of exosomes isolated from NB cell lines derived from primary tumor (PT) and BM metastasis. The comparison among all exosomal proteins showed 15 proteins exclusively present in PT, mainly involved in neuronal development, and 6 proteins in BM metastasis-derived exosomes related to cancer progression. Significant proteins obtained with t-test analysis, performed between the 2 groups (PT and BM metastasis), revealed that PT exosomes contain a higher level of proteins involved in extra-cellular matrix (ECM) assembly and adhesion, as well as in neuronal development. Exosomes isolated from BM metastasis showed proteins involved in ameboidal cell migration and mitochondrial activity. This work shows that proteomic profiling of NB-derived exosomes reflects the advanced tumor stage and may be considered as potential tumor biomarker

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

DISEASE(S): Neuroblastoma

SUBMITTER: Andrea Petretto

LAB HEAD: Andrea Petretto

PROVIDER: PXD005864 | Pride | 2018-07-05

REPOSITORIES: Pride

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Publications

Proteomic Analysis of Neuroblastoma-Derived Exosomes: New Insights into a Metastatic Signature.

Colletti Marta M Petretto Andrea A Galardi Angela A Di Paolo Virginia V Tomao Luigi L Lavarello Chiara C Inglese Elvira E Bruschi Maurizio M Lopez Ana Amor AA Pascucci Luisa L Geoerger Birgit B Peinado Hector H Locatelli Franco F Di Giannatale Angela A

Proteomics 20170912 23-24

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor. Around 70% of patients with metastatic disease at diagnosis present bone-marrow infiltration, which is considered a marker of poor outcome; however, the mechanism underlying this specific tropism has to be elucidated. Tumor-derived exosomes may support metastatic progression in several tumors by interacting with the microenvironment, and may serve as tumor biomarkers. The main objective of this study is to identify an exos ...[more]

PMID: 28722341

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Project description:Exososmes, potent intercellular communicators, are supposed to contribute to metastasis formation, which we confirmed for exosomes of the metastatic rat pancreatic adenocarcinoma line BSp73ASML that promote metastatic settlement in lymph nodes and lung of poorly metastatic BSp73ASML cells with a selective CD44v4-v7 (BSp73ASML-CD44vkd) knockdown. To define the molecular pathway(s), whereby exosomes contribute to premetastatic niche preparation, we profiled mRNA miRNA of BSp73ASMLwt and BSp73ASML-CD44vkd- exosomes and evaluated the impact on potential target cells. BSp73ASML exosomes are recovered in the draining lymph node after subcutaneous injection. In vitro, they preferentially bind and are taken-up by lymph node stroma cells (LnStr) and lung fibroblasts (LuFb) that were chosen as exosome targets. BSp73ASMLwt and BSp73ASML-CD44kd exosomes contain a restricted repertoire of mRNA and miRNA, hwere the lattter differe significantly between the two lines and even more pronounced, exosomes derived thereof with a not yet explored dominance of tumor-suppressor miRNA in ASML-CD44kd cells and exosomes. Both, exosomal mRNA and miRNA are recovered in target cells and exosome-uptake is accompanied by significant changes in gene expression. We didn't observe a correlation between exosomal mRNA and changes in target cell mRNA or proteins. Instead transferred miRNA significantly affected target cell mRNA translation as demonstrated for selected, most abundant ASML exosomal miRNA besides others, miR-494 known target MAL (myelin and lymphocytes protein)/cadherin17, and miR-542-3p which targets TRAF/cadherin17. Furthermore, MMP transcription suggested to accompany cadherin17 dwon-regulation was upregulated in miR-494 or miR542-3p transfected or exosome co-cultured LnStr. Taken together, tumor exosomes target in vivo non-transformed cells in premetastatic organs. Exosome uptake induced altered target celll gene expression is strongly promoted by exosomal miRNA where we demonstrate for the first time that exosomes/exosomal miRNA from a metastasizing tumor line can modulate stroma cells from premetastatic organs. Endothelial cells lines were treated with pancreatic adenocarcinoma (AS) derived exosomes or pancreatic adenocarcinoma derived exosomes expressing tetraspanin 8. Total RNA was isolated and used to perform the Agilent gene expression microarrays. In this assay a replicate of endothelial cell lines treated with ASTspan8 were also included. Moreover, total RNA from both base line expression of endothelial cells and rat endothelial fibroblasts were also used to perfrom gene expression microarrays. RNA isolated from Rat endothelial fibroblasts treated with the exosomes derived from rat pancreatic adenocarcinoma and exosomes derived from rat pancreatic adenocarcinoma expressing tetraspanin8 were individually used to perfrom gene expression microarrays. RNA isolated from exosomes derived from rat pancreatic adenocarcinoma cell lines expressing tetraspanin were used to peform gene expresiion to see the base line expression. Another replicate were also used. RNA isolated from base line or control of rat pancreatic adenocarcinoma wild type cells and also base line RNA isolated from rat pancreatic adenocarcinoma cells lines where CD44 was knock-down.

Project description:Neuroblastoma (NB) is the most common extracranial solid tumor during infancy, causing up to 10% of mortality in children. NB shows notable heterogeneity with regard to histology and clinical behavior, ranging from low-risk localized tumors (LR-NB) to high-risk disease (HR-NB), characterized by aggressive metastatic phenotype, resistance to treatment, and fatal relapse occurrence. Risk stratification demands high accuracy, as it will determine the therapeutic treatment. However, the current therapeutic stratification, based on clinical and molecular risk factors, does not allow to discriminate among patients with similar clinical-pathological parameters who receive the same treatment despite showing markedly different clinical courses. The need of novel diagnostic molecular tools in oncology has led to increasing interest in liquid biopsies as a source of biomarkers, as they provide a minimally invasive method. Body fluids are a source of exosomes, nanosized extracellular vesicles that can drive tumor growth and chemoresistance. We previously identified exosomal microRNAs (exo-miRNA) indicative of HR-NB patient sensitivity/resistance to chemotherapy. As exosomes from cancer patients carry proteins that reflect the surface and cytoplasm content of parental cancer cells, including immunosuppressive molecules known to be associated with cancer progression and/or response to therapy, we extended our study to the exosomal proteins (Exo-prot).The purpose of the study is to identify Exo-prot that (i) are specifically expressed in NB patients and (ii) are associated with tumor phenotype and disease stage, in order to improve risk stratification and refine diagnosis.We isolated exosomes from plasma specimens of 24 HR-NB patients and 24 LR-NB patients at diagnosis and of 24 age-matched healthy controls (CTRL). Exo-prot expression was measured by liquid chromatography-mass spectrometry. Missing value distribution and differential expression analysis (performed with two different imputation methods) were applied to identify the most relevant Exo-prot. ROC analysis assessed the diagnostic value of the identified Exo-prot. We demonstrated that NB patients have a different Exo-prot expression profile compared to CTRL. The deregulated Exo-prot in NB specimens act mainly in tumor-associated pathways and build a strictly connected network. Furthermore, HR-NB patients show a different Exo-miR expression profile compared to LR-NB subjects, with the modulation of molecules involved in cell migration, proliferation and metastasis. Importantly, we show that NCAM, NCL, LUM and VASP have a diagnostic value in discriminating NB patients from CTRL; while MYH9, FN1, CALR, AKAP12 and, with a lower performance, LTBP1 can differentiate HR-NB and LR-NB patients with high accuracy. We demonstrated that Exo-prot deregulated in NB vs CTRL subjects and in HR-NB vs LR-NB patients contribute to NB tumor development and to the aggressive metastatic NB phenotype, respectively. We identified Exo-prot with significant diagnostic value for NB patients and able to efficiently identify the HR-NB subset of patients, which can be employed to improve risk stratification. Our results highlight the applicability of Exo-prot evaluation for integrating NB diagnosis and risk assessment.

Dataset Information

Proteomic analysis of neuroblastoma-derived exosomes LC-MS

Publications

Proteomic Analysis of Neuroblastoma-Derived Exosomes: New Insights into a Metastatic Signature.

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