Visualization and tracking of tumour extracellular vesicle delivery and RNA translation using multiplexed reporters

TLDR

Accurate spatiotemporal assessment of extracellular vesicle delivery and cargo RNA translation requires specific and robust live‑cell imaging technologies. The study engineers optical reporters to label multiple EV populations for visualization and tracking of tumour EV release, uptake, and exchange between cell populations in culture and in vivo. The authors fuse EGFP and tdTomato to palmitoylation signals for EV membrane labeling and tag PalmtdTomato transcripts with MS2 sequences, detecting them via an MS2 coat protein–EGFP fusion. Multiplexed fluorescent and bioluminescent reporters show rapid EV uptake and cargo mRNA translation within one hour, confirming dynamic, multidirectional EV‑mediated communication delivering functional mRNA.

Abstract

Abstract Accurate spatiotemporal assessment of extracellular vesicle (EV) delivery and cargo RNA translation requires specific and robust live-cell imaging technologies. Here we engineer optical reporters to label multiple EV populations for visualization and tracking of tumour EV release, uptake and exchange between cell populations both in culture and in vivo . Enhanced green fluorescence protein (EGFP) and tandem dimer Tomato (tdTomato) were fused at NH 2 -termini with a palmitoylation signal (PalmGFP, PalmtdTomato) for EV membrane labelling. To monitor EV-RNA cargo, transcripts encoding PalmtdTomato were tagged with MS2 RNA binding sequences and detected by co-expression of bacteriophage MS2 coat protein fused with EGFP. By multiplexing fluorescent and bioluminescent EV membrane reporters, we reveal the rapid dynamics of both EV uptake and translation of EV-delivered cargo mRNAs in cancer cells that occurred within 1-hour post-horizontal transfer between cells. These studies confirm that EV-mediated communication is dynamic and multidirectional between cells with delivery of functional mRNA.