Biochim Biophys Acta Proteins Proteom. 2018 Nov 28. pii: S1570-9639(18)30206-1. [Epub ahead of print]
Extracellular vesicles can be classified into two main classes - exosomes and shed microvesicles (sMVs). Whilst much is known about exosome cargo and functionality, sMVs are poorly understood. Here, we describe the large-scale purification of sMVs released from primary (SW480) and metastatic (SW620) human isogenic colorectal cancer (CRC) cell lines using a combination of differential ultracentrifugation and isopycnic iodixanol density centrifugation. The yield of SW480-sMVs and SW620-sMVs was 0.75 mg and 0.80 mg, respectively. Both SW480-/SW620-sMVs are heterogeneous in size (100-600 nm diameter) and exhibit identical buoyant densities (1.10 g/mL). In contrast to exosomes, sMVs are ALIX-, TSG101-, CD63- and CD9-. Quantitative mass spectrometry identified 1295 and 1300 proteins in SW480-sMVs and SW620-sMVs, respectively. Gene Ontology enrichment analysis identified 'cell adhesion' (CDH1, OCLN, CTN families), 'signalling pathway' (KRAS, NRAS, MAPK1, MAP2K1), and 'translation/RNA related' processes (EIF, RPL, HNRNP families) in both sMV types. Strikingly, SW480- and SW620-sMVs exhibit distinct protein signatures - SW480-sMVs enriched in ITGA/B, ANXA1, CLDN7, CD44 and EGFR/NOTCH signalling networks, while SW620-sMVs are enriched in PRKCA, MACC1, and FGFR4/MTOR/ MARCKS signalling networks. Both SW480- and SW620-sMVs are taken up by NIH3T3 fibroblasts, demonstrating similar cell invasion capability. This study provides, for the first time, molecular insights into sMVs and CRC biology.
Keywords: Colon cancer; Extracellular vesicles; Metastasis; Proteomics; Shed microvesicles