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 in the context of CRC biology.