Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions

Anurag Purushothaman,Shyam Kumar Bandari,Jian Liu,James A Mobley,Elizabeth E Brown,Ralph D Sanderson

doi:10.1074/jbc.m115.686295

Abstract

Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression.

Highlights

Multiple myeloma remains incurable, despite recent treatment advances [1]
In analyzing exosomes from these aggressive and control cells, we found those secreted by the aggressive cells had higher levels of syndecan-1 (CD138), VEGF, and hepatocyte growth factor and were able to promote tumor cell
Because fibronectin is known to play an important role in adhesion via interactions with integrins, collagens, fibrin, and heparan sulfate proteoglycans, we hypothesized that it played a role in exosome-cell interactions

Summary

Introduction

Despite recent treatment advances [1]. There is mounting evidence that targeting intercellular communication between malignant plasma cells or between malignant cells and host cells may enhance therapeutic outcome. We discovered that fibronectin on the exosome surface mediates the interaction with heparan sulfate chains on target cells. We demonstrate that myeloma-derived exosomes have fibronectin bound on their surface and that, via its Hep-II domain, fibronectin facilitates exosome interaction with heparan sulfate chains present on the surface of target cells.

Results

Conclusion