Radiation increases the cellular uptake of exosomes through CD29/CD81 complex formation

Highlights

• Radiation increases cellular uptake of exosomes.

• Radiation induces colocalization of CD29 and CD81.

• Exosomes selectively bind the CD29/CD81 complex.

• Radiation increases the cellular uptake of exosomes through CD29/CD81 complex formation.

Abstract

Exosomes mediate intercellular communication, and mesenchymal stem cells (MSC) or their secreted exosomes affect a number of pathophysiologic states. Clinical applications of MSC and exosomes are increasingly anticipated. Radiation therapy is the main therapeutic tool for a number of various conditions. The cellular uptake mechanisms of exosomes and the effects of radiation on exosome–cell interactions are crucial, but they are not well understood. Here we examined the basic mechanisms and effects of radiation on exosome uptake processes in MSC. Radiation increased the cellular uptake of exosomes. Radiation markedly enhanced the initial cellular attachment to exosomes and induced the colocalization of integrin CD29 and tetraspanin CD81 on the cell surface without affecting their expression levels. Exosomes dominantly bound to the CD29/CD81 complex. Knockdown of CD29 completely inhibited the radiation-induced uptake, and additional or single knockdown of CD81 inhibited basal uptake as well as the increase in radiation-induced uptake. We also examined possible exosome uptake processes affected by radiation. Radiation-induced changes did not involve dynamin2, reactive oxygen species, or their evoked p38 mitogen-activated protein kinase-dependent endocytic or pinocytic pathways. Radiation increased the cellular uptake of exosomes through CD29/CD81 complex formation. These findings provide essential basic insights for potential therapeutic applications of exosomes or MSC in combination with radiation.

Introduction

Exosomes, bilipid membrane vesicles (30–100 nm in diameter) that originate in multi-vesicular bodies and are released into the extracellular milieu upon fusion with the plasma membrane, are attracting increased attention [1]. Exosome secretion is a cellular mechanism for delivering cargo to mediate intercellular communication and to affect biologic function by the exchange of proteins and lipids, or the delivery of genetic materials to recipient cells [2]. Exosomes are also involved in various other cellular functions and pathophysiologic states, and thus could potentially provide a new approach for detecting noninvasive disease and predicting disease progression [3]. Moreover, exosomes have properties that can be exploited for therapeutic interventions as a new drug delivery system and a novel therapeutic tool in various conditions, including cancer, inflammation, ischemia, and regeneration [4].

Tumor cells and the cancer-associated microenvironment, comprising fibroblast-like cells, extracellular matrix, and inflammatory cells, secrete exosomes between them, allowing for crosstalk that leads to the promotion or inhibition of tumor progression, but the precise mechanism of communication is poorly understood [5], [6]. Mesenchymal stem cells (MSC), clusters of multipotential fibroblast-like cells present in every organ as well as in the tumor stromal microenvironment, have regenerative and protective effects for injured tissues, and inhibit or promote tumor metastasis with their secreted exosomes, but the underlying mechanism is not clearly understood [6]. Potential applications of MSC and their secreted exosomes are currently attracting attention in a number of medical fields, such as oncology, immunology, and radiation therapy [7], [8].

Radiation and drug therapy are currently the main therapeutic tools for a number of diseases. Radiation therapy not only acts on target cells, but also affects the stromal microenvironment. Thus, understanding how radiation affects cellular uptake and the secretion of exosomes between target cells and stromal cells is crucial.

Recent studies of exosome biogenesis revealed that exosomes originate from endosomal proteins involved in membrane transport and fusion in processes requiring heat shock proteins, integrins, and tetraspanins, and that the source of exosomes defines their function [7]. For therapeutic application of exosomes, especially those derived from MSC, the target cells must effectively internalize the exosomes. Several mechanisms of exosome uptake involving their surface molecules have been described and two distinct modes of internalization have been suggested [1]. In monocytes and macrophages, exosome internalization depends on the actin cytoskeleton and phosphatidylinositol 3-kinase regulated by dynamin2, and non-phagocytic cells require an energy-dependent pathway, including caveolae, macropinocytosis, and clathrin-coated vesicles [9], [10]. The effects of radiation on exosome uptake processes, however, remain unknown. More detailed knowledge of the mechanisms of cellular uptake and the effects of radiation on these processes is needed to promote the effective use of exosomes and MSC as potential therapeutic tools. A better understanding of the processes involved will be instructive for modifying exosomes to be preferentially targeted in pathologic conditions by bioengineering. Here, we address several essential questions relating to the basic cellular uptake of exosomes and how radiation regulates that process, with a focus on target cell ligands. Our findings revealed that radiation leads to the colocalization of integrin (CD29) and tetraspanin (CD81) and increases the cellular uptake of exosomes.