Multiresolution Imaging Using Bioluminescence Resonance Energy Transfer Identifies Distinct Biodistribution Profiles of Extracellular Vesicles and Exomeres with Redirected Tropism

Abstract

Extracellular particles (EPs) including extracellular vesicles (EVs) and exomeres play significant roles in diseases and therapeutic applications. However, their spatiotemporal dynamics in vivo have remained largely unresolved in detail due to the lack of a suitable method. Therefore, a bioluminescence resonance energy transfer (BRET)-based reporter, PalmGRET, is created to enable pan-EP labeling ranging from exomeres (<50 nm) to small (<200 nm) and medium and large (>200 nm) EVs. PalmGRET emits robust, sustained signals and allows the visualization, tracking, and quantification of the EPs from whole animal to nanoscopic resolutions under different imaging modalities, including bioluminescence, BRET, and fluorescence. Using PalmGRET, it is shown that EPs released by lung metastatic hepatocellular carcinoma (HCC) exhibit lung tropism with varying distributions to other major organs in immunocompetent mice. It is further demonstrated that gene knockdown of lung-tropic membrane proteins, solute carrier organic anion transporter family member 2A1, alanine aminopeptidase/Cd13, and chloride intracellular channel 1 decreases HCC-EP distribution to the lungs and yields distinct biodistribution profiles. It is anticipated that EP-specific imaging, quantitative assays, and detailed in vivo characterization are a starting point for more accurate and comprehensive in vivo models of EP biology and therapeutic design.

An insight into small extracellular vesicles: Their roles in colorectal cancer progression and potential clinical applications

“…Some work has tried to give solutions to this issue, and they build their own quantification platforms based on antibodies, immunoassay, and imaging flow cytometry, which are able to selectively detect tumor‐derived EVs 151–153 . And in their work, compared to healthy donors, cancerous samples are able to release more EVs, and gene knockdown of organ‐tropic membrane proteins could decrease organ‐tropic EVs to specific organ 154 . This might indicate that increasing of EVs secretion promotes cancer progression.…”

“…[151][152][153] And in their work, compared to healthy donors, cancerous samples are able to release more EVs, and gene knockdown of organ-tropic membrane proteins could decrease organ-tropic EVs to specific organ. 154 This might indicate that increasing of EVs secretion promotes cancer progression. Besides the quantification of sEVs, how sEVs contents are regulated also spawn our attention.…”

Visualizing Extracellular Vesicles and Their Function in 3D Tumor Microenvironment Models

“…Alternatives to dye labeling methods include genetic modification of cells to express fluorescent or bioluminescent proteins fused with a transmembrane domain of a membrane protein, EV-associated protein, or a membrane-anchoring signal sequence [ 36 ]. Small ATP-independent luciferases such as Gaussia luciferase (Gluc) and NanoLuc have been used for tracking EVs in culture and living subjects [ 82 , 83 , 84 , 85 ]. Since EVs generally lack ATP and extracellular levels of ATP are negligible, the development of these bioluminescent reporters was critical to enabling bioluminescent imaging approaches, which have led to rapid and high-throughput EV screens [ 86 ].…”

“…One such method is the to use bioluminescence resonance energy transfer (BRET), which allows for both fluorescence and bioluminescence imaging. An example is the PalmGRET reporter utilizing GFP and Nanoluc bioluminescence reporter genes fused to a palmitoylation signal peptide, allowing the reporter to be trafficked to the cell membrane and expressed in EVs [ 84 ]. Mouse hepatocellular carcinoma cells (HCA1) stably expressing PalmGRET EVs were injected into mice and had greater distribution to liver and lungs.…”

Extracellular MicroRNAs as Intercellular Mediators and Noninvasive Biomarkers of Cancer

“…These newly discovered exomeres are the predominant extracellular particles released by most cancer cells studied. These three nanoparticles subpopulations (Exo-L, Exo-S, and exomeres) were isolated from the extracellular vesicle (EV) fraction of several cancer and normal cells using asymmetric-flow field-flow fractionation (AF4), showing diverse organ distribution patterns and cargo content, which suggest specific biological functions [ 100 , 130 ]. Zhang et al [ 100 ] investigated the organ biodistribution of B16-F10 melanoma-derived nanoparticles subsets in naïve mice, and found that Exo-L showed tropism for lymph nodes, whereas Exo-L, Exo-S, and exomeres were uptaken by hematopoietic organs (liver, spleen, and bone marrow), whereas lungs and kidneys showed less uptake and Exo-L, Exo-S, and exomeres were absent in the brain.…”