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.
Highlights
Extracellular vesicles (EVs) are nano-sized membrane-bound vesicles that are released from cells into the extracellular space
In vivo bioluminescence imaging (BLI) using Renilla luciferase (Rluc) was developed and applied to monitoring of extracellular vesicles (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
In order to study the distribution of EVs derived from cancer cells using the imaging reporter system, Rluc was stably transduced into a human anaplastic thyroid cancer cell line CAL-62 cells (CAL-62/Rluc) and a human breast cancer cell line MDA-MB-231 via lentiviral delivery of the Rluc gene (Supplementary Figure 1A)
Summary
Extracellular vesicles (EVs) are nano-sized membrane-bound vesicles that are released from cells into the extracellular space. Aside from healthy/nonmalignant cells, tumor cells release EVs into their microenvironment. Recent studies showed that tumor-derived EVs promote endothelial cell migration during angiogenesis in the tumor microenvironment via ERK1/2 and JNK signaling pathways [12]. Numerous studies have shown that tumor-derived EVs transfer oncogenic activity, promoting tumor progression [23, 24]. One of the main considerations when a fluorescent dye is used to label the cell membrane is that the dye can be released into the tissue; this situation can lead to the production of non-membraneassociated signals [32, 37, 38] In addition, labeling of EVs with exogenous signaling agents can result in changes to the characteristics of EVs, due to the labeling procedures used. There has been a steadily growing number of studies on dye-based labeling of EVs, but it is necessary to ensure that the results observed reflect reality. We selected Renilla luciferase (Rluc), which is a cofactorless, single-subunit, blue-light-emitting luciferase isolated from the marine anthozoan Renilla reniformis
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