Differential Proteome Analysis of Extracellular Vesicles from Breast Cancer Cell Lines by Chaperone Affinity Enrichment

Abstract

The complexity of human tissue fluid precludes timely identification of cancer biomarkers by immunoassay or mass spectrometry. An increasingly attractive strategy is to primarily enrich extracellular vesicles (EVs) released from cancer cells in an accelerated manner compared to normal cells. The Vn96 peptide was herein employed to recover a subset of EVs released into the media from cellular models of breast cancer. Vn96 has affinity for heat shock proteins (HSPs) decorating the surface of EVs. Reflecting their cells of origin, cancer EVs displayed discrete differences from those of normal phenotype. GELFrEE LC/MS identified an extensive proteome from all three sources of EVs, the vast majority having been previously reported in the ExoCarta database. Pathway analysis of the Vn96-affinity proteome unequivocally distinguished EVs from tumorigenic cell lines (SKBR3 and MCF-7) relative to a non-tumorigenic source (MCF-10a), particularly with regard to altered metabolic enzymes, signaling, and chaperone proteins. The protein data sets provide valuable information from material shed by cultured cells. It is probable that a vast amount of biomarker identities may be collected from established and primary cell cultures using the approaches described here.

Keywords: GELFrEE; Vn96; breast cancer cell lines; extracellular vesicles; glycolysis; heat shock proteins.

Figure 1

Western blot analysis of fractions recovered from sucrose density gradient isolation of SKBR3 extracellular vesicles (EVs) reveals the presence of exosomal marker proteins TSG101 (A) and Alix (B). These proteins remain in the supernatant in the control sample following low speed centrifugation. With addition of Vn96, the low speed spin recovers the same proteins in the pelleted material.

Figure 2

TEM images showing the distribution of microvesicular bodies recovered from the extracellular media by precipitation with Vn96 peptide of (A) SKBR3; (B) MCF-7; (C) MCF-10a. The size distribution of the particles is consistent with small extracellular vesicles.

Figure 3

Immunoblots probing for exosomal marker proteins in the Vn96 pellets isolated from SKBR3 cell culture media. (A) A random peptide (SW) precipitates few proteins from the extracellular media, as evident from a total protein stain; (B) Marker proteins are observed in the immunoblots of the Vn96 pulldown following solubilization in SDS-containing buffer (USB); (C) Extracting the Vn96 pellet with mild detergent buffers, EB1 & EB2, from Millipore’s subcellular proteome extraction kit, S-PEK, will not release proteins from the Vn96 pellet, permitting incorporation of a wash step to improve purity. As controls, equivalent volumes of culture medium were processed by ultracentrifugation (UCF), the pellet was resuspended in electrophoresis buffer and loaded directly (Lane 11).

Figure 4

(A) SDS-PAGE of proteins recovered from Vn96 pellets reveal significantly more proteins from the cancerous cell lines SKBR3 and MCF-7 relative to the non-cancerous control MCF-10a; (B) GELFrEE fractionation resolves proteins according to molecular weight. Shown is the separation of SKBR-3 proteins from the EV pellet as obtained on the 8% (high molecular weight) gel cartridge; (C) Immunoblot of the identically resolved proteome as above, showing resolution of heat shock proteins (HSP70 and HSP90).

Figure 5

Venn diagrams summarize (A) the proteins and (B) peptides identified by mass spectrometry from each of the three cell lines. Detailed listings of identified proteins are provided as supplemental files.