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

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posted on 2021-08-10, 23:49 authored by Wittaya SuwakulsiriWittaya Suwakulsiri, Alin Rai, Rong Xu, Maoshan Chen, David GreeningDavid Greening, Richard SimpsonRichard Simpson
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.


W.S., A.R., R.X., M.C., D.W.G., and R.J.S. acknowledge funding support from La Trobe University, Melbourne, Australia. W.S is supported by a La Trobe University Postgraduate Scholarship. We acknowledge the La Trobe University-Comprehensive Proteomics Platform for providing infrastructure.


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Biochimica et Biophysica Acta - Proteins and Proteomics





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