Biochemical and biological characterization of exosomes containing prominin-1/CD133

Abstract

Exosomes can be viewed as complex “messages” packaged to survive trips to other cells in the local microenvironment and, through body fluids, to distant sites. A large body of evidence indicates a pro-metastatic role for certain types of cancer exosomes. We previously reported that prominin-1 had a pro-metastatic role in melanoma cells and that microvesicles released from metastatic melanoma cells expressed high levels of prominin-1. With the goal to explore the mechanisms that govern proteo-lipidic-microRNA sorting in cancer exosomes and their potential contribution(s) to the metastatic phenotype, we here employed prominin-1-based immunomagnetic separation in combination with filtration and ultracentrifugation to purify prominin-1-expressing exosomes (prom1-exo) from melanoma and colon carcinoma cells. Prom1-exo contained 154 proteins, including all of the 14 proteins most frequently expressed in exosomes, and multiple pro-metastatic proteins, including CD44, MAPK4K, GTP-binding proteins, ADAM10 and Annexin A2. Their lipid composition resembled that of raft microdomains, with a great enrichment in lyso-phosphatidylcholine, lyso-phosphatidyl-ethanolamine and sphingomyelin. The abundance of tetraspanins and of tetraspanin-associated proteins, together with the high levels of sphingomyelin, suggests that proteolipidic assemblies, probably tetraspanin webs, might be the essential structural determinant in the release process of prominin-1 of stem and cancer stem cells. Micro-RNA profiling revealed 49 species of micro-RNA present at higher concentrations in prom1-exo than in parental cells, including 20 with cancer-related function. Extensive accumulation of prom1-exo was observed 3 h after their addition to cultures of melanoma and bone marrow-derived stromal cells (MSC). Short-term co-culture of melanoma cells and MSC resulted in heterologous prominin-1 transfer. Exposure of MSC to prom1-exo increased their invasiveness. Our study supports the concept that specific populations of cancer exosomes contain multiple determinants of the metastatic potential of the cells from which they are derived.

Human Prominin-1 (CD133) Is Detected in Both Neoplastic and Non-Neoplastic Salivary Gland Diseases and Released into Saliva in a Ubiquitinated Form

“…Given the presence of all three proteins ( prominin -1, CEA and MUC1) in secreted materials mixed with the circulatory system, it is tempting to speculate that prominin -1–containing membrane vesicles may become a biomarker of AdCC and PA. Actually, an elevated level of CEA and MUC1 in blood can be used as an indicator of advanced forms of certain cancers [40]-[42], and the analysis of prominin -1–containing membrane vesicles in cerebrospinal fluids for diagnostic purposes is under investigation in view of their differential levels in relation with neural diseases [25], [26]. The physiological function of prominin -1–containing membrane vesicles is currently unknown, but the presence of micro-RNAs in melanoma-derived prominin -1–containing exosomes [81] suggests that they might play a role in intercellular communication and/or cancer progression as proposed earlier [28].…”

Ceramide and Exosomes: A Novel Target in Cancer Biology and Therapy

“…A specific involvement of changes in the miRNA profile, either induced by CD44 or due to RNA transported by exosomes was not noted. Since CD44 and tetraspanins are RAPs that are abundant in exosomes it remains to be elucidated if they are also functional in mobile rafts (Rappa, Mercapide, Anzanello, Pope, & Lorico, 2013; Ronquist, Ronquist, Larsson, & Carlsson, 2010). …”

Thematic Review Series: Exosomes and Microvesicles: Lipids as Key Components of their Biogenesis and Functions, Cholesterol and the journey of extracellular vesicles

“…Indirect evidence for the elevated cholesterol content comes from the observation that EVs from cell line supernatant (15) and from human body fluids (16,17) contain cholesterol-binding by guest, on April 27, 2019 www.jlr.org Downloaded from 6 proteins such as prominin 1 (CD133). Membranes of EVs may also contain cholesterol-rich lipid rafts: EVs were bound by an antibody against clustered cholesterol (18) and they contained detergent-resistant membrane fractions featuring the raft marker stomatin and enhanced cholesterol levels (19).…”