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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| Affiliation: | 1. Molecular Proteomics Laboratory (MPL), Institute for Molecular Medicine, Medical Faculty, Heinrich Heine University Düsseldorf, Düsseldorf D-40225, Germany;2. Institute for Microbiology, Heinrich Heine University Düsseldorf, Düsseldorf D-40225, Germany;1. King Faisal Specialist Hospital & Research Centre (Jeddah), College of Medicine, Alfaisal University, PO Box 40047, MBC: J-64, Jeddah 21499, Saudi Arabia;2. Research Centre, King Faisal Specialist Hospital & Research Centre, Jeddah, Saudi Arabia;3. Pathology Department, King Faisal Specialist Hospital & Research Centre, Jeddah, Saudi Arabia;4. Department of Pathology & Laboratory Medicine, King Fahad Specialist Hospital, Dammam, Saudi Arabia;1. College of Laboratory Medicine, Dalian Medical University, Dalian 116044, Liaoning Province, China;2. Department of Clinical Laboratory, The First Affliated Hospital of Dalian Medical University, Dalian, Liaoning 116011, China;3. Cancer Center, Dalian Medical University, Dalian 116044, Liaoning Province, China;4. Department of Microbiology, Dalian Medical University, Dalian 116044, Liaoning Province, China;5. Department of Biochemistry, Dalian Medical University, Dalian 116044, Liaoning Province, China;1. Department of Laboratory Medicine and Pathology Mayo Clinic, Hilton 7, 200 1st Street SW, Rochester, MN 55905, USA;2. Department of Medicine, Mayo Clinic 200 1st Street SW, Rochester, MN 55905, USA;1. Department of Diagnostic Medicine and Public Health, , University of Modena and Reggio Emilia—Section of Pathology, University of Modena and Reggio Emilia, 41100 Modena, Italy;2. Department of Human Pathology, University of Messina, 98125 Messina, Italy |
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| 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. |
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