Significance: Exosome-mediated intercellular transfer of proteins and nucleic acids has attracted considerable attention as exosomes may promote the development of cancer and other pathological conditions; however, the mechanism of exosome uptake by target cells and how this may be inhibited remain as important questions. We provide evidence that heparan sulfate proteoglycans (HSPGs) function as receptors of cancer cell-derived exosomes. Importantly, our data indicate that the HSPG-dependent uptake route is highly relevant for the biological activity of exosomes, and thus a potential target for inhibition of exosome-mediated tumor development. Given that several viruses have previously been shown to enter cells through HSPGs, our data implicate HSPG as a convergence point during cellular uptake of endogenous vesicles and virus particles.

Hypoxia, or low oxygen tension, is a major regulator of tumor development and aggressiveness. However, how cancer cells adapt to hypoxia and communicate with their surrounding microenvironment during tumor development remain important questions. Here, we show that secreted vesicles with exosome characteristics mediate hypoxia-dependent intercellular signaling of the highly malignant brain tumor glioblastoma multiforme (GBM). In vitro hypoxia experiments with glioma cells and studies with patient materials reveal the enrichment in exosomes of hypoxia-regulated mRNAs and proteins (e.g., matrix metalloproteinases, IL-8, PDGFs, caveolin 1, and lysyl oxidase), several of which were associated with poor glioma patient prognosis. We show that exosomes derived from GBM cells grown at hypoxic compared with normoxic conditions are potent inducers of angiogenesis ex vivo and in vitro through phenotypic modulation of endothelial cells. Interestingly, endothelial cells were programmed by GBM cell-derived hypoxic exosomes to secrete several potent growth factors and cytokines and to stimulate pericyte PI3K/AKT signaling activation and migration. Moreover, exosomes derived from hypoxic compared with normoxic conditions showed increased autocrine, promigratory activation of GBM cells. These findings were correlated with significantly enhanced induction by hypoxic compared with normoxic exosomes of tumor vascularization, pericyte vessel coverage, GBM cell proliferation, as well as decreased tumor hypoxia in a mouse xenograft model. We conclude that the proteome and mRNA profiles of exosome vesicles closely reflect the oxygenation status of donor glioma cells and patient tumors, and that the exosomal pathway constitutes a potentially targetable driver of hypoxia-dependent intercellular signaling during tumor development.

The role of exosomes in cancer can be inferred from the observation that they transfer tumor cell derived genetic material and signaling proteins, resulting in e.g. increased tumor angiogenesis and metastasis. However, the membrane transport mechanisms and the signaling events involved in the uptake of these virus-like particles remain ill-defined. We now report that internalization of exosomes derived from glioblastoma (GBM) cells involves nonclassical, lipid raft-dependent endocytosis. Importantly, we show that the lipid raft-associated protein caveolin-1 (CAV1), in analogy with its previously described role in virus uptake, negatively regulates the uptake of exosomes. We find that exosomes induce the phosphorylation of several downstream targets known to associate with lipid rafts as signaling and sorting platforms, such as extracellular signal-regulated kinase-1/2 (ERK1/2) and heat shock protein 27 (HSP27). Interestingly, exosome uptake appears dependent on unperturbed ERK1/2-HSP27 signaling, and ERK1/2 phosphorylation is under negative influence by CAV1 during internalization of exosomes. These findings significantly advance our general understanding of exosome-mediated uptake and offer potential strategies for how this pathway may be targeted through modulation of CAV1 expression and ERK1/2 signaling.

How various macromolecules are exchanged between cells and how they gain entry into recipient cells are fundamental questions in cell biology with important implications e.g. non-viral drug delivery, infectious disease, metabolic disorders, and cancer. The role of heparan sulfate proteoglycan (HSPG) as a cell-surface receptor of diverse macromolecular cargo has recently been manifested. Exosomes, cell penetrating peptides, polycation-nucleic acid complexes, viruses, lipoproteins, growth factors and morphogens among other ligands enter cells through HSPG-mediated endocytosis. Key questions that partially have been unraveled over recent years include the respective roles of HSPG core protein and HS chain structure specificity for macromolecular cargo endocytosis, the down-stream intracellular signaling events involved in HSPG-dependent membrane invagination and vesicle formation, and the biological significance of the HSPG transport pathway. Here, we discuss the intriguing role of HSPGs as a major entry pathway of macromolecules in mammalian cells with emphasis on recent in vitro and in vivo data that provide compelling evidence of HSPG as an autonomous endocytosis receptor.

Motivation: Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. Results: We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research. Availability and implementation: The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info. Contact:ysgho@postech.ac.kr