Sensitive and rapid quantification of exosomes by fusing luciferase to exosome marker proteins

Exosomes have emerged as important mediators of intercellular communication. Although their modes of action have been elucidated, the molecular mechanisms underlying their secretion, sorting of molecules, uptake into recipient cells, and biological distribution in vivo remain elusive.

Researchers from the Aichi Cancer Center Research Institute present a novel system for quantifying secreted exosomes by introducing ectopic or CRISPR/Cas9-mediated knock-in of luciferase-fusion exosome markers such as CD63. This luciferase-based method makes it possible to measure exosomes secreted into the culture medium with high linearity and wide dynamic range in a high-throughput manner. They demonstrate that data obtained by luminescent quantification are well correlated with data obtained by conventional nanoparticle tracking analysis under multiple conditions. In addition, this system is capable of evaluating the recipient cells or tissues that take up exosomes, as well as visualizing exosomes in vivo. The proposed system represents a powerful tool for understanding the molecular mechanisms underlying exosome production, uptake, and long-term distribution.

Ectopic Nluc-fused CD63 expression enables quantification of exosome production

(a) Western blot analysis of CD63 expression. (b) NanoLuc luciferase intensity in the culture medium of control (Mock) and Nluc-fused CD63-expressing HT29 and HCT116 cells (CD63Nluc). (c) Nluc intensity in the culture medium before and after ultracentrifugation (UC). (d) Western blot analysis of CD63 expression in exosomes secreted from control (Mock) and Nluc-fused CD63-expressing cells (CD63Nluc). ALIX was used as an exosome marker protein. (e) Correlation between luciferase intensity (in the culture medium) and cell number. The solid line shows the linearity of the fitted curve of luminescence vs. seeded cell number. (f) Correlation between luciferase intensity and exosome number. The solid line shows the linearity of the fitted curve luminescence vs. exosome number. (g) Detection limits of CD63Nluc-expressing HT29 and HCT116 cells in exosome quantification. Purified exosomes were adjusted to a concentration of 10¹¹ particles/mL, and a dilution series was prepared down to a concentration of 10⁵ particles/mL. Detection limits were decided by comparing luciferase intensities of the dilution series with those of buffer (20 mM HEPES, pH7.4). The number of purified exosomes was measured by NanoSight, and luminescence was measured by a luminometer. Results are expressed as means ± SD of three wells. All data are representative of at least three independent experiments. ***P < 0.001 by two-tailed Student’s t-test.