Proteomic profiling reveals key cancer progression modulators in shed microvesicles released from isogenic human primary and metastatic colorectal cancer cell lines

Wittaya Suwakulsiri; Alin Rai; Rong Xu; Maoshan Chen; David W Greening; Richard J Simpson

doi:10.1016/j.bbapap.2018.11.008

Proteomic profiling reveals key cancer progression modulators in shed microvesicles released from isogenic human primary and metastatic colorectal cancer cell lines

Biochim Biophys Acta Proteins Proteom. 2019 Dec;1867(12):140171. doi: 10.1016/j.bbapap.2018.11.008. Epub 2018 Nov 29.

Authors

Wittaya Suwakulsiri¹, Alin Rai¹, Rong Xu¹, Maoshan Chen¹, David W Greening¹, Richard J Simpson²

Affiliations

¹ Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia.
² Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia. Electronic address: Richard.Simpson@latrobe.edu.au.

PMID: 30502510
DOI: 10.1016/j.bbapap.2018.11.008

Abstract

Extracellular vesicles comprise two main classes - exosomes and shed microvesicles (sMVs). Whilst much is known about exosome cargo content 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 being enriched in ITGA/B, ANXA1, CLDN7, CD44 and EGFR/NOTCH signalling networks, while SW620-sMVs are enriched in PRKCA, MACC1, FGFR4 and MTOR/MARCKS signalling networks. Both SW480- and SW620-sMVs are taken up by NIH3T3 fibroblasts resulting in similar cell invasion capability. This study provides, for the first time, molecular insights into sMVs and CRC biology.

Keywords: Colon cancer; Exosomes; Extracellular vesicles; Metastasis; Proteomics; Shed microvesicles.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Line, Tumor
Cell-Derived Microparticles / metabolism*
Colorectal Neoplasms / metabolism*
Colorectal Neoplasms / pathology
Disease Progression
Humans
Mice
NIH 3T3 Cells
Proteome / metabolism*
Proteomics

Substances

Proteome