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.

细胞外囊泡包括两个主要类别-外泌体和脱落微囊泡（sMVs）。尽管人们对外来货物的内容和功能了解很多，但对sMV的了解却很少。在这里，我们描述了使用差分超速离心和等渗碘克沙醇密度离心法相结合，从原发性（SW480）和转移性（SW620）人等基因结直肠癌（CRC）细胞系中释放的sMV的大规模纯化。SW480-sMV和SW620-sMV的产量分别为0.75 mg和0.80 mg。两种SW480- / SW620-sMV的尺寸均不相同（直径100-600 nm），并具有相同的浮力密度（1.10 g / mL）。相比于外来体，SMVS是ALIX ^-，TSG101 ^-，CD63 ^-和CD9 ^-。定量质谱法分别在SW480-sMV和SW620-sMV中鉴定出1295和1300种蛋白质。基因本体论富集分析确定了两者中的“细胞粘附”（CDH1，OCLN，CTN家族），“信号通路”（KRAS，NRAS，MAPK1，MAP2K1）和“翻译/ RNA相关”过程（EIF，RPL，HNRNP家族） sMV类型。令人惊讶的是，SW480-sMVs具有独特的蛋白质特征-SW480-sMVs富含ITGA / B，ANXA1，CLDN7，CD44和EGFR / NOTCH信号网络，而SW620-sMVs富含PRKCA，MACC1，FGFR4和MTOR / MARCKS信令网络。SW480-sMV和SW620-sMV均被NIH3T3成纤维细胞吸收，导致相似的细胞侵袭能力。这项研究首次提供了关于sMV和CRC生物学的分子见解。