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Cell-of-origin-specific proteomics of extracellular vesicles.

ABSTRACT: The ability to assign cellular origin to low-abundance secreted factors in extracellular vesicles (EVs) would greatly facilitate the analysis of paracrine-mediated signaling. Here, we report a method, named selective isolation of extracellular vesicles (SIEVE), which uses cell type-specific proteome labeling via stochastic orthogonal recoding of translation (SORT) to install bioorthogonal reactive groups into the proteins derived from the cells targeted for labeling. We establish the native purification of intact EVs from a target cell, via a bioorthogonal tetrazine ligation, leading to copurification of the largely unlabeled EV proteome from the same cell. SIEVE enables capture of EV proteins at levels comparable with those obtained by antibody-based methods, which capture all EVs regardless of cellular origin, and at levels 20× higher than direct capture of SORT-labeled proteins. Using proteomic analysis, we analyze nonlabeled cargo proteins of EVs and show that the enhanced sensitivity of SIEVE allows for unbiased and comprehensive analysis of EV proteins from subpopulations of cells as well as for cell-specific EV proteomics in complex coculture systems. SIEVE can be applied with high efficiency in a diverse range of existing model systems for cell-cell communication and has direct applications for cell-of-origin EV analysis and for protein biomarker discovery.

SUBMITTER: Kehrloesser S

PROVIDER: S-EPMC10119638 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Cell-of-origin-specific proteomics of extracellular vesicles.

Kehrloesser Sebastian S Cast Oliver O Elliott Thomas S TS Ernst Russell J RJ Machel Anne C AC Chen Jia-Xuan JX Chin Jason W JW Miller Martin L ML

PNAS nexus 20230403 4

The ability to assign cellular origin to low-abundance secreted factors in extracellular vesicles (EVs) would greatly facilitate the analysis of paracrine-mediated signaling. Here, we report a method, named selective isolation of extracellular vesicles (SIEVE), which uses cell type-specific proteome labeling via stochastic orthogonal recoding of translation (SORT) to install bioorthogonal reactive groups into the proteins derived from the cells targeted for labeling. We establish the native puri ...[more]

PMID: 37091541

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Project description:BackgroundCancer patients have an increased risk of developing venous thromboembolism, with up to 30% dying within a month of their development. Some cancer cells are known to induce platelet aggregation, and this interaction is understood to contribute to thrombosis and haematogenous metastasis. Many researchers have reported on extracellular vesicles (EVs) released from platelets. However, less is known about how cancer cells' EVs may affect platelet function. Here EVs released by triple-negative breast cancer (TNBC) cell line variants were extensively investigated in this regard.MethodsEVs were separated from conditioned media of TNBC Hs578T and Hs578Ts(i)8 cells using filtration and ultracentrifugation and were characterised by nanoparticle tracking analysis, immunoblots, and transmission electron microscopy. Blood samples from consenting donors were procured, and their platelets collected by differential centrifugation. Light transmission aggregometry and optical microscopy evaluated the potential interaction of TNBC cells and their EVs with platelets. Global proteomic analysis was performed on the EVs, by in-solution digestion and mass spectrometry. Data analysis included the use of Perseus, FunRich, and Vesiclepedia. Immunoblotting was used as a secondary method to investigate some key EV cargo proteins identified by the global proteomics approach.ResultsBoth TNBC cell variants induced platelet aggregation. Increasing cell numbers significantly reduced the time taken for platelet aggregation to occur. EVs released by the cells also resulted in platelet aggregation. The time to induce platelet aggregation was EV dose-dependent. Proteomics profiling and immunoblotting of the EVs' cargo identified candidate proteins (including uPAR and PDGFRβ) that may be involved during this process.ConclusionsTNBC cells induce platelet aggregation. Furthermore, the cell-free EVs induced this undesirable effect. A number of EV cargo proteins were identified that may be relevant as therapeutic targets.

Dataset Information

Cell-of-origin-specific proteomics of extracellular vesicles.

Publications

Cell-of-origin-specific proteomics of extracellular vesicles.

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