In vivo imaging of long-term accumulation of cancer-derived exosomes using a BRET-based reporter

Tomoya Hikita,Mamiko Miyata,Chitose Oneyama,Risayo Watanabe

doi:10.1038/s41598-020-73580-5

Abstract

Monitoring of exosome dynamics in living organisms is essential to demonstrate the real functions of cancer-derived exosomes. Currently, these have been elucidated in vitro or under non-physiological conditions in vivo in most cases. To overcome these limitations, we developed an imaging method using Antares2-mediated bioluminescence resonance energy transfer (BRET) for observing long-term accumulation of exosomes in vivo. Ectopic expression of CD63-Antares2 effectively labeled exosomes with Antares2, which emitted intense, long-wavelength luminescence suitable for in vivo monitoring. Transplantation of CD63-Antares2-expressing prostate cancer cells into mice allowed determining the amount of cancer-derived exosomes released from primary tumors into the bloodstream and visualizing the long-term homing behavior of exosomes to their target organs or tissues. Interestingly, secreted exosome was decreased upon administration of low dose of dasatinib, an approved tyrosine-kinase inhibitor. The CD63-Antares2 xenograft mouse model will be useful for elucidating the dynamics of cancer-derived exosomes in vivo and evaluating the therapeutic efficacy and mechanism of exosome production inhibitors.

Highlights

Monitoring of exosome dynamics in living organisms is essential to demonstrate the real functions of cancer-derived exosomes
We evaluated the bioluminescence resonance energy transfer (BRET) system using the red-shifted reporter A ntares[225], a Nluc mutant teLuc fused with CyOFP1 (Fig. 1a,b)
We observed Nluc signal in bronchoepithelial cells in the lungs, lymphocyte and erythrocyte in the spleen, mesenteric lymph nodes, and adipocyte in the adipose tissues in the genital glands and intestine (Fig. 2f). These results indicated that the xenograft mouse model bearing CD63-Antares2expressing cancer cells allows monitoring long-term circulating cancer-derived exosomes quantitatively and in a time-dependent manner, and visualizing exosome-homing organs, tissues, and cells

Summary

Introduction

Monitoring of exosome dynamics in living organisms is essential to demonstrate the real functions of cancer-derived exosomes. To demonstrate the functions of cancer-derived exosomes in vivo, various imaging strategies have been developed to track them[13,14,15,16] In these approaches, chemically or genetically labeled exosomes are injected directly into animal’s circulation, which allows monitoring only for a short period of time (less than a few days). Exosomes prepared for exogenous injection may have a different heterogeneity from naturally secreted exosomes and contain other types of extracellular vesicles[18,19,20] It remains questionable whether the models used in these studies reflected the physiological dynamics of cancer-derived exosomes[12]. Ectopic expression of CD63-Antares[2] effectively labeled exosomes with long-wavelength bioluminescence suitable for in vivo visualization

Methods

Results

Conclusion