In-Depth Proteome Profiling of Small Extracellular Vesicles Isolated from Cancer Cell Lines and Patient Serum

Abstract

Extracellular vesicle (EV) secretion has been observed in many types of both normal and tumor cells. EVs contain a variety of distinctive cargoes, allowing tumor-derived serum proteins in EVs to act as a minimally invasive method for clinical monitoring. We have undertaken a comprehensive study of the protein content of the EVs from several cancer cell lines using direct data-independent analysis. Several thousand proteins were detected, including many classic EV markers such as CD9, CD81, CD63, TSG101, and Syndecan-1, among others. We detected many distinctive cancer-specific proteins, including several known markers used in cancer detection and monitoring. We further studied the protein content of EVs from patient serum for both normal controls and pancreatic cancer and hepatocellular carcinoma. The EVs for these studies have been isolated by various methods for comparison, including ultracentrifugation and CD9 immunoaffinity column. Typically, 500-1000 proteins were identified, where most of them overlapped with the EV proteins identified from the cell lines studied. We were able to identify many of the cell-line EV protein markers in the serum EVs, in addition to the large numbers of proteins specific to pancreatic and HCC cancers.

Keywords: CD9; EV proteins; HCC; extracellular vesicles; immunoaffinity; pancreatic cancer; ultracentrifugation.

Figure 1.

Experimental workflow for the isolation, characterization, and processing of small EVs followed by direct DIA-MS analysis.

Figure 2.

Quantified expression of EV- and cancer-specific markers upon comparing EVs from cancer cell lines and their mix. (a) CD9 (dark blue), (b) CD81 (purple), (c) TSG101 (green), (d) CD29 (orange), (e) vimentin (red), (f) Platelet factor 4 (PF4) (mustard), (g) CD73 (yellow), (h) lysozyme (LYZ) (light blue), and (i) PARVB (black).

Figure 3.

DIA-MS performance evaluation for the analysis of extracellular vesicles. The identification number at the precursor, peptide and protein levels from four replicates of EVs from standard commercial samples extracted from PC3, HT29, and MCF7. Average identification number (a), and those quantified within 20 and 10% coefficient of variation (CV) for precursors (b), peptide (c), and protein groups (d). Violin plot of CV% for 1180 protein groups (e) with a median CV of 7.7% achieved. Quantitative correlation (R2) among these replicates (f). Protein identification overlap (g). 70% were identified across all four replicates while 16, 8, and 6% were detected in 3, 2, and 1 runs, respectively. Protein dynamic range varying from 2.9 to 6.1 in Log10 value with some EV markers shown.

Figure 4.

Quantified expression of EV and cancer markers upon comparing EVs from a standard mix of cancer cell lines with serum EVs obtained by UC. (a) CD9 (dark blue), (b) CD81 (purple), (c) TSG101 (green), (d) CD29 (orange), (e) vimentin (red), platelet factor 4 (PF4) (mustard), (g) CD73 (yellow), (h) lysozyme (LYZ) (light blue), and (i) PARVB (black).