A new bioluminescent reporter system to study the biodistribution of systematically injected tumor-derived bioluminescent extracellular vesicles in mice

Abstract

In vivo biodistribution and fate of extracellular vesicles (EVs) are still largely unknown and require reliable in vivo tracking techniques. In this study, in vivo bioluminescence imaging (BLI) using Renilla luciferase (Rluc) was developed and applied to monitoring of EVs derived from thyroid cancer (CAL-62 cells) and breast cancer (MDA-MB-231) in nude mice after intravenous administration and was compared with a dye-based labeling method for EV derived from CAL-62 cells. The EVs were successfully labeled with Rluc and visualized by BLI in mice. In vivo distribution of the EVs, as measured by BLI, was consistent with the results of ex vivo organ analysis. EV-CAL-62/Rluc showed strong signals at lung followed by liver, spleen & kidney (P < 0.05). EV-MDA-MB-231/Rluc showed strong signals at liver followed by lung, spleen & kidney (P < 0.05). EV-CAL-62/Rluc and EV-MDA-MB-231/Rluc stayed in animal till day 9 and 3, respectively; showed a differential distribution. Spontaneous EV-CAL-62/Rluc shown distributed mostly to lung followed by liver, spleen & kidney. The new BLI system used to show spontaneous distribution of EV-CAL-62/Rluc in subcutaneous CAL-62/Rluc bearing mice. Dye (DiR)-labeled EV-CAL-62/Rluc showed a different distribution in vivo & ex vivo compared to EV-CAL-62/Rluc. Fluorescent signals were predominately detected in the liver (P < 0.05) and spleen (P < 0.05) regions. The bioluminescent EVs developed in this study may be used for monitoring of EVs in vivo. This novel reporter-imaging approach to visualization of EVs in real time is expected to pave the way for monitoring of EVs in EV-based treatments.

Advances in Analysis of Biodistribution of Exosomes by Molecular Imaging

“…On the other hand, genetically labeled exosomes have an advantage in comparing their in vivo distribution with and without additional labeling. Especially, genetically labeled exosomes can be utilized to monitor the effects of surface modifications, such as covalent binding or membrane integration, which may cause structural or functional changes in the membranes of exosomes [67]. [74,76] Labeling of exosomes by encapsulation has been performed with various labeling probes such as radioisotopes, nanoparticles, and fluorescent dyes ( Table 4).…”

“…Another study performed without covalent binding suggested that labeling exosomes with lipophilic dyes also slightly changes the biodistribution of exosomes. The researchers labeled exosomes containing luciferase, with a lipophilic fluorescent dye and compared the biodistribution of the exosomes with and without the lipophilic dyes [67]. The exosomes without the lipophilic dye, accumulated in the organs in the following order: lung > liver > spleen > kidney.…”

“…The most frequent accumulation tissues for exosomes after IV injection were reported as the liver, lung, spleen, and kidney (Table 4). Although the modification of surface proteins such as glycosylation may have affected the in vivo distribution of exosomes in a few reports [66,67], additional studies with more animals seems to be necessary for more accurate analysis. It was also reported that there was a difference in the biodistribution of exosomes according to the exosome-producing cells [68].…”

Extracellular vesicles enhance the targeted delivery of immunogenic oncolytic adenovirus and paclitaxel in immunocompetent mice

“…It has been reported that EVs have intrinsic cell targeting properties: cell surface structures, such as tetraspanins and integrins, may direct the EV uptake towards specific cells and protect therapeutic agents from opsonization and recognition by phagocytes, leading to a longer half-life and reduced side-effects in healthy cells and tissues [42,43]. EVs have been shown to be safe in several clinical trials [44,45], while their effectiveness in cancer therapy may strongly depends on their ability of EVs to target the tumor tissue: however, despite intense research in the field, still little is known about their in vivo biodistribution [35,[46][47][48][49].…”

<p>A novel strategy of transferring NIS protein to cells using extracellular vesicles leads to increase in iodine uptake and cytotoxicity</p>

“…Western blotting was carried out as previously described. 15 Cell or EV lysates were prepared in lysate RIPA buffer (Thermo Fisher Scientific), to which a cocktail of protease inhibitor was added (Sigma-Aldrich Co., St Louis, MO, USA). An equal amount of protein was loaded and run on a 10% SDS-PAGE gel.…”