Proteomic profiling of NCI-60 extracellular vesicles uncovers common protein cargo and cancer type-specific biomarkers

Hurwitz, Stephanie N.; Rider, Mark A.; Bundy, Joseph L.; Liu, Xia; Singh, Rakesh Kumar; Meckes, David G.

doi:10.18632/oncotarget.13569

Cited by 244 publications

(292 citation statements)

References 71 publications

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“…EVs were resuspended in proportional volumes of PBS (see methods). The tetraspanin molecules CD9 and CD63 [8,25,26,38,41], the ESCRT-I associated tumour suppressor gene 101 (TSG101) [19,20,25,42–44], and the late-endosome/lipid raft protein Flotillin-2 [7,45–47] were selected as markers. All were present in EV obtained by these three methods, but not the intracellular control Tubulin (Figure 2(a)).…”

Section: Resultsmentioning

confidence: 99%

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Large‐scale, cross‐flow based isolation of highly pure and endocytosis‐competent extracellular vesicles

McNamara

Caro-Vegas

Costantini

et al. 2018

J of Extracellular Vesicle

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Isolation of extracellular vesicles (EVs) from cell culture supernatant or plasma can be accomplished in a variety of ways. Common measures to quantify relative success are: concentration of the EVs, purity from non-EVs associated protein, size homogeneity and functionality of the final product. Here, we present an industrial-scale workflow for isolating highly pure and functional EVs using cross-flow based filtration coupled with high-molecular weight Capto Core size exclusion. Through this combination, EVs loss is kept to a minimum. It outperforms other isolation procedures based on a number of biochemical and biophysical assays. Moreover, EVs isolated through this method can be further concentrated down or directly immunopurified to obtain discreet populations of EVs. From our results, we propose that cross-flow/Capto Core isolation is a robust method of purifying highly concentrated, homogenous, and functionally active EVs from industrial-scale input volumes with few contaminants relative to other methods.

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Section: Resultsmentioning

confidence: 99%

“…As the EVs are immensely concentrated after purification additional steps e.g. antibody-based affinity purification or sucrose/iodixanol gradient flotation are easily added for selective enrichment of antigenically-defined or density-defined sub-populations of EV [8,24,26,56]. This method is applicable to human plasma, with similar results.…”

Section: Discussionmentioning

confidence: 99%

Large‐scale, cross‐flow based isolation of highly pure and endocytosis‐competent extracellular vesicles

McNamara

Caro-Vegas

Costantini

et al. 2018

J of Extracellular Vesicle

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“…Three publications have reported specifically on proteins identified in MCF-7 EVs [23–25]. Additionally, based on the results from 59 cancer cell lines, Hurwitz [23] compiled a list of 213 proteins with unique gene symbols found to be common to multiple cancer cell lines, including MCF-7.…”

Section: Resultsmentioning

confidence: 99%

Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein‐extraction methods

Joy

Ayre

Chute

et al. 2018

J of Extracellular Vesicle

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Sample amount is often a limiting factor for multi-parametric analyses that encompass at least three areas of ‘-omics’ research: genomics, transcriptomics and proteomics. Limited sample amounts are also an important consideration when these multi-parametric analyses are performed on extracellular vesicles (EVs), as the amount of EVs (and EV cargo) that can be isolated is often very low. It is well understood that a monophasic solution of phenol and guanidine isothiocyanate (i.e. TRIzol©) can simultaneously isolate DNA, RNA and proteins from biological samples; however, it is most commonly used for the extraction of RNA. Validation of this reagent for the isolation of multiple classes of biological molecules from EVs would provide a widely applicable method for performing multi-parametric analyses of EV material. In this report, we describe a comparison of proteins identified from EVs processed with either TRIzol© or the conventional Laemmli buffer protein-extraction reagents. EVs were isolated from 3 mL of cell-culture supernatant derived from MCF-10A, MCF-7 and MDA-MB-231 cells using the Vn96 EV capture technology. For the TRIzol© extraction protocol, proteins were precipitated with acetone from the organic phase and then re-solubilized in a mixture of 8M urea, 0.2% SDS and 1 M Tris-HCl pH 6.8, followed by dilution in 5× loading buffer prior to fractionation with 1D SDS-PAGE. NanoLC-MS/MS of the trypsin-digested proteins was used to generate proteomic profiles from EV protein samples extracted with each method. Of the identified proteins, 57.7%, 69.2% and 57.0% were common to both extraction methods for EVs from MCF-10A, MCF-7 and MDA-MB-231, respectively. Our results suggest that TRIzol© extraction of proteins from EVs has significant equivalence to the traditional Laemmli method. The advantage of using TRIzol© reagent is the ability to accumulate multi-parametric data (e.g., RNA and protein profiles) on the same limited EV sample while minimizing sample preparation and processing time.

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“…To our knowledge this is the first report on the presence of galectin mRNAs in trophoblast-derived EVs. However, mRNAs for all three gals were found in glioblastoma (Skog et al, 2008) and colorectal cancer cells (Hurwitz et al, 2016). The presence of gal mRNAs in EVs could indicate EVT cells' potential to contribute to gal synthesis in target cells with increased need for these lectins.…”

Section: Discussionmentioning

confidence: 98%

“…The findings presented here are novel for gal-3 and gal-8, since no literature data regarding their presence in EVs of trophoblast origin have been reported so far. However, both gal-3 and gal-8 were found in EVs from different cell types or body fluids (Ogawa et al, 2008;Wang et al, 2012;Webber et al, 2014;Hurwitz et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

Extracellular presence/release of galectins from HTR-8/SVneo extravillous trophoblast cells*

Ćujić¹,

Kosanović²,

Krivokuća³

et al. 2017

Turk J Biol

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Galectins (gals) are β-galactoside binding lectins, involved in many processes at the fetomaternal interface where they can exert their roles both in and out of cells. The aim of this work was to explore the extracellular presence/release of HTR-8/SVneo extravillous trophoblast cell line galectins. To that end, conditioned medium (CM) from HTR-8/SVneo cell culture was fractionated into a high molecular mass fraction (HMF) and low molecular mass fraction (LMF) using 100 kDa cut-off concentrators. In addition, extracellular vesicles (EVs) were isolated from CM by ultracentrifugation. Size and shape of the EVs were analyzed by transmission electron microscopy and their galectin mRNA content was determined by real-time PCR. The presence of galectins in fractions of HTR-8/SVneo CM and EVs was detected by western blot. All three galectins expressed by HTR-8/SVneo cells (gal-1, gal-3, and gal-8) were detected in the CM, HMF, and EVs, while only gal-1 was found in the LMF. In addition, EVs contained all three galectin mRNAs. These results reveal that free, complexed, and EV-associated forms of galectins were released from extravillous trophoblast cells, suggesting their potential to exert extracellular functions both in their immediate vicinity at the fetomaternal interface and distant locations.

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Proteomic profiling of NCI-60 extracellular vesicles uncovers common protein cargo and cancer type-specific biomarkers

Cited by 244 publications

References 71 publications

Large‐scale, cross‐flow based isolation of highly pure and endocytosis‐competent extracellular vesicles

Large‐scale, cross‐flow based isolation of highly pure and endocytosis‐competent extracellular vesicles

Proteome profiling of extracellular vesicles captured with the affinity peptide Vn96: comparison of Laemmli and TRIzol© protein‐extraction methods

Extracellular presence/release of galectins from HTR-8/SVneo extravillous trophoblast cells*

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