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Identification of small extracellular vesicle protein biomarkers of pediatric Ewing Sarcoma

ABSTRACT: Label-free 1DLC-MS analysis of cell lines and cell line derived small extracellular vesicles (sEVs) with duplicate injections. Goal to determine shared and distinct features of these tumor cells and their respective sEVs. Samples included analyzed EWS cells with different EWS-ETS fusions (EWS-FLI1 type I, II, and III and EWS-ERG) and their corresponding sEVs. Non-EWS controls included osteosarcoma, rhabdomyosarcoma, and benign cells, i.e., osteoid osteoma and mesenchymal stem cells.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Andrew K. Godwin

PROVIDER: MSV000090623 | MassIVE | Mon Oct 31 13:08:00 GMT 2022

REPOSITORIES: MassIVE

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Project description:Rhabdomyosarcoma (RMS) is the most common childhood sarcoma and is identified as either the embryonal or alveolar (ARMS) subtype. In approximately 75% of cases, ARMSs are characterized by specific chromosomal translocations that involve PAX and FKHR genes. ARMS gene expression signatures vary, depending on the presence or absence of the translocations. Insulin-like growth factor-binding protein 2 (IGFBP2) is strongly overexpressed in translocation-negative RMS. Because IGFBP2 is associated with tumorigenesis, we investigated its functional role in RMS. An analysis of IGFBP2 distribution in RMS cell lines revealed a strong accumulation in the Golgi complex, in which morphological characteristics appeared peculiarly modified. After silencing IGFBP2 expression, our microarray analysis revealed mostly cell cycle and actin cytoskeleton gene modulations. In parallel, IGFBP2-silenced cells showed reduced cell cycle and rates of invasion and decreased seeding in the lungs after tail vein injections in immunodeficient mice. An analysis of IGFBP2 mRNA and protein localization in human tumors showed abnormal protein accumulation in the Golgi complex, mostly in PAX/FKHR-negative RMS. Moreover, an analysis of patients with RMS revealed the presence of conspicuous circulating levels of IGFBP2 proteins in children with highly aggressive RMS tumors. Taken together, our data provide evidence that IGFBP2 contributes to tumor progression and that it could be used as a marker to better classify clinical and biological risks in RMS. Three independent silencing experiments on RH36, an embryonal rhabdomyosarcoma cell line, were performed to study the role of IGFBP2 oncogene in this childhood tumor. Each of 3 biological replicates has its own control which is represented by cell culture treated with a non-targeting siRNA (siCONTROL). 3 controls were used to produce a M-bM-^@M-^\control poolM-bM-^@M-^] that has been used as reference in all microarray experiments. Each microarray experiment consists of a competitive hybridization between test and reference RNA (e.g. silenced cells vs. control), labelled with Cy3 or Cy5. Two microarray experiments were performed for each of three silencing experiment (technical replicate). Each slide was scanned and the data was normalized and analyzed.

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Identification of small extracellular vesicle protein biomarkers of pediatric Ewing Sarcoma

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