Assistant Professor University of Illinois at Urbana Champaign, United States
Introduction:: As a key mediator of cellular communication, nanosized extracellular vesicles (e.g., exosomes) have been widely explored for diagnostic and therapeutic applications. Here we report a facile and universal metabolic tagging technology that enables the installation of >3,000 chemical tags onto each cell-secreted exosome, 2-3 orders of magnitude higher than conventional protein expression methods. We demonstrate that this technology can be universally applied to exosomes secreted by cancer cells, stem cells, dendritic cells, and T cells, and the surface chemical tags enable quantitative conjugation of molecules via efficient click chemistry for in vitro and in vivo tracking and targeted modulation of exosomes. We further demonstrate that conjugation of thousands of toll-like receptor 9 (TLR9) agonists onto exosomes, as achieved by the metabolic tagging approach, can timely stimulate TLR9 on intracellular endosomes during the endocytosis of exosomes, resulting in dramatically improved activation of dendritic cells (175-fold) and presentation of exosome-encased antigens by dendritic cells. In therapeutic tumor studies, TLR9 agonist-conjugated tumor exosomes resulted in 80% tumor-free survival against E.G7 lymphoma and 33% tumor-free survival against B16F10 melanoma, arguably the best antitumor efficacy reported for tumor exosome vaccines. Our study yields a universal technology to generate chemically tagged exosomes from various types of cells, probe and modulate exosome-cell interactions, and develop next-generation exosome vaccines with superior T cell response and therapeutic efficacy.
Materials and Methods:: Azido-labelled exosomes from various cancer cells were isolated by incubating the cells in presence of azido-sugars for 3-4 days and collecting the cell culture medium containing exosomes via ultracentrifugation with an Amicon centrifugal filter (100 kDa). NanoSight NS300 was used to measure the concentration of exosomes. This azido labeled exosomes were conjugated with DBCO modified CpG oligonucleotide via efficient click chemistry. All cell analyses were performed using flow cytometry. C57BL/6 mice were used for prophylactic and therapeutic studies. All the statistical analyses were performed using GraphPad Prism v6 and v8.
Results, Conclusions, and Discussions:: To conclude, we report an exosome tagging technology that enables the generation of chemically tagged exosomes from various types of cells, and the development of next-generation exosome vaccines with superior T cell response and therapeutic efficacy. We demonstrated that metabolic glycan labeling of cells with azido-sugars resulted in the secretion of azido-labeled exosomes, which enabled the conjugation of DBCO-molecules via efficient click chemistry for in vitro and in vivo tracking and targeted modulation of exosomes. In the context of tumor exosomes, we showed that CpG-conjugated exosomes could dramatically improve the activation of DCs, processing and presentation of exosome-encased antigens by DCs, and subsequent priming of antigen-specific CD8+ T cells, leading to significantly improved CTL response and antitumor efficacy against lymphoma and melanoma. The exosome tagging and targeting technology will greatly facilitate the development of exosome-based therapies, and provides a facile platform to manipulate exosome-cell interactions and decipher the role of exosomes in different diseases.
