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Visualization and Tracking of Tumour Extracellular Vesicle Delivery and RNA Translation Using Multiplexed Reporters


Visualization and Tracking of Tumour Extracellular Vesicle Delivery and RNA Translation Using Multiplexed Reporters

Charles P Lai et al. Nat Commun.


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 NH2-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.


Figure 1
Figure 1. Palmitoylated-GFP and tdTomato are trafficked to the plasma membrane and EVs.
(a) Schematic diagram of EV membrane labelling with palmitoylated GFP (PalmGFP) or tdTomato (PalmtdTomato). (b) Live-cell confocal microscopy of stable PalmGFP-expressing 293T cells releasing EVs. Left panel: 3D reconstruction of confocal Z-stack images demonstrating EV release from stable 293T-PalmGFP cells into surrounding regions. Right panel: enlarged image of boxed region, showing bud-like structure from the surface (arrows) as well as processes extending from cells (arrow heads). (c,d) Western blot analysis of protein extracted from fractions following sucrose-gradient centrifugation of isolated EVs. PalmGFP (c) and PalmtdTomato (d) were detected in fractions exhibiting the EV marker, Alix (95 kDa). (e,f) Transmission electron micrograph (TEM) showing PalmGFP and PalmtdTomato labelling of EVs on the membrane following immunolabelling with anti-GFP (e) or anti-tdTomato (f) and secondary gold-conjugated secondary antibodies. Scale bar, 100 nm. (g) Demonstration that PalmGFP and PalmtdTomato labels the inner membrane of EVs. PalmGFP- or PalmtdTomato EVs were dot blotted on nitrocellulose membrane in a dose range followed by immunoprobing with anti-GFP or anti-tdTomato, respectively, and horseradish peroxidase (HRP)-conjugated secondary antibodies in the presence or absence of detergent [0.1% (v/v) Tween20] for chemiluminescence detection.
Figure 2
Figure 2. PalmtdTomato labels different-sized EVs and co-labels with PKH.
(a) Dot blot detection of PalmtdTomato+ EV isolated from 0.22 or 0.8 μm filtered conditioned medium of 293T-PalmtdTomato cells. (b) Confocal microscopy of EVs isolated from 0.22 or 0.8 μm filtered conditioned medium of 293T-PalmtdTomato cells. Scale bar, 10 μm. (c) Digital display of PalmtdTomato+ EVs signals as spheres. Scale bar, 10 μm. (d) Quantification of PalmtdTomato+ EV signals per field in 0.22 or 0.8 μm filtered samples. *P<0.001 by two-tailed, t-test with 14 replicates. (e) Nanoparticle tracking analysis (NTA) of PalmtdTomato+ EVs isolated from 0.22 and 0.8 μm filtered samples. *P<0.05 by two-tailed t-test with six replicates. The results are presented as the mean ± s.e.m. (f) EV-depleted medium (top rows) or EVs isolated from 293T-PalmtdTomato cells (bottom rows) were stained with PKH67 followed by a 1 h wash in PBS at 100,000 g. Scale bar, 20 μm. (g,h) Semi-quantification of EV signals from PKH67-stained EV-depleted medium (g) or PalmtdTomato+ EVs co-labelling with PKH67 (h). *P<0.05 by repeated measures analysis of variance (ANOVA) followed by Tukey's post hoc test with four replicates. The results are presented as the mean ± s.e.m.
Figure 3
Figure 3. Live-cell imaging of EV exchange and uptake.
(a) Stably transduced 293T-PalmtdTomato and primary GBM-PalmGFP cells were co-cultured and imaged with confocal microscopy for cellular interactions and EV exchanges between the cells. Magnification of boxes in merged panels are shown in far right panels. (i) Multiple thin cellular projections were readily observed extending from both GBM (arrowhead) and 293T (arrow) cells. Notably, many GBM cellular projections exhibited vesicle-like structure at the tips of the projections. Vesicle-like structures were also detected on the surface of GBM cells (star). (ii) PalmGFP+ EVs from GBM cells are found inside of 293T-PalmtdTomato cells (arrows). (iii) PalmtdTomato+ EVs from 293T cells are detected inside GBM-PalmGFP cells (arrows). Scale bar, 20 μm. (b) 3D reconstruction of Z-stack images showing 293T-PalmGFP cells exposed to PalmtdTomato+ EVs for 1.5 h. (c) Time-lapsed confocal imaging of PalmtdTomato+ EV attachment on 293T-PalmGFP cells at 2.45 h. Images shown at an 18-s interval from 0 to 3 min followed by a 1-minute interval from 3 to 10 min. PalmtdTomato+ EVs were surrounded by 293T-PalmGFP cell membranes (arrows) within a minute, and the pattern lasted until the end of the experiment (10 min). Notably, a round-shaped, PalmGFP-labelled entity with a diameter ≥ 4 μm (arrowhead) in the extracellular space was also observed from 00:18 to 04:03 min. Scale bar, 10 μm.
Figure 4
Figure 4. Visualization of EVs and packaged mRNAs.
(a) Dual-function EV-RNA reporter system enabling EV and EV-RNA labelling; two constructs encoding either PalmtdTomato protein with RNA reporter transcripts containing repeat MS2 RNA binding sequences (MS24X; PalmtdTomato-MS24X; top), or MS2-GFP coat protein (MS2CP) with a nuclear localization signal (NLS) (bottom). (b) Diagram of vesicle RNA labelling process: [1] RNA reporter (blue bar) allows detection of EV-RNA reporter transcripts through binding of MS2 coat protein fused with GFP (MS2CP-GFP-NLS, green dewdrop) to PalmtdTomato-MS24X sequence in mRNA; [2] PalmtdTomato-MS24X:MS2CP-GFP RNA reporter complexes are packaged into EVs; [3] EVs are released from cells by budding from the cell membrane and/or fusion of multivesicular bodies with the cell membrane; and [4] nonspecific EV packaging of MS2CP-GFP-NLS is minimized by translocating the unbound RNA MS2CP-GFP reporter into the nucleus via the NLS signal. CMV, cytomegalovirus enhancer/promoter; HA, haemagglutinin tag; NLS, nuclear localization signal; UTR, untranslated region. (c) EVs were isolated from human Gli36 glioma cells expressing MS2CP-GFP with either PalmtdTomato-MS24X or PalmtdTomato alone (negative control for MS24X mRNA binding sites). Confocal microscopy imaging showed that only EVs isolated from cells co-expressing MS2CP-GFP and PalmtdTomato-MS24X exhibited co-localization between EVs (tdTomato) and MS2CP-GFP:MS24X mRNA complexes (GFP; top row). The negative control for MS24X (PalmtdTomato) showed only membrane labelled EVs (tdTomato). Scale bar, 10 μm.
Figure 5
Figure 5. In vivo visualization of EVs.
(a) PalmGFP-expressing EL4 tumours were implanted in pre-installed dorsal skinfold chambers (DSFCs) in C57BL/6 mice, and imaged 9 days later by MP-IVM. (b) Intravital micrograph from the central region of an EL4 tumour with regions characterized by the absence (subpanel 1), low (subpanel 2) and intermediate density (subpanel 3) of detectable PalmGFP+ vesicles. (c) Intravital micrograph of a peripheral region of a tumour with a high density of extracellular PalmGFP+ vesicles. Time-lapse recordings highlighting a tethered vesicle (subpanel 1) and a motile cluster of three vesicles (subpanel 2). Time is shown as min:s. (d) An identical experiment was performed as described for (ac), but EL4 tumour cells expressing soluble GFP instead of PalmGFP were used instead. Note the absence of small micron- and submicron-sized particles (e) Nanoparticle tracking analysis of vesicles purified from the supernatant of PalmGFP-expressing EL4 tumour cells. (f) Non-fluorescent EL4 tumours were implanted into DSFCs and purified PalmGFP+ vesicles directly injected into the tumour on day 9. (g) Intravital micrograph of an EL4 tumour 60 min after injection of GFP+ EVs.
Figure 6
Figure 6. EV uptake and EV-RNA translation.
(a) Schematic of two expression reporters used to detect EV uptake (PalmGFP) and translation of EV-delivered GlucB mRNA (luciferase activity). 293T cells (donors) were stably transduced with lentivirus vectors expressing these reporters. EVs were isolated from conditioned medium of these cells and added to Gli36-mCherry glioma cells followed by centrifugation at 4 °C to facilitate EV docking on cells while minimizing EV uptake. PalmGFP+GlucB+ EV-containing media was then replaced with EV-depleted media in the presence and absence of cycloheximide (CHX) and cells were incubated at 37 °C for 24 h. Medium and cell samples were collected at indicated time points to assess EV uptake (fluorescence and luciferase activity) and EV-mRNA translation (luciferase activity). CMV, cytomegalovirus enhancer/promoter; ss, signal sequence; BAPTM, biotin acceptor peptide with transmembrane domain of platelet-derived growth factor receptor. (b) Flow cytometry analysis of the percentage of Gli36-mCherry cells positive for PalmGFP+ EV uptake in the presence or absence of CHX. Cells that have taken up EVs exhibited signal for both PalmGFP (EVs) and mCherry (recipient cells), whereas cells without or with low levels of EVs showed only mCherry (see also Supplementary Fig. 5). (c) GlucB reporter assay was performed to detect translation of EV-delivered GlucB mRNA by Gli36-mCherry cells expressed as fold change. (d) Flow cytometry analysis of nascent protein synthesis in Gli36-mCherry cells revealed by Click-iT HPG labelling and Alexa Fluor488 conjugation expressed as percentage of cells positive for Alexa Fluor 488 (see also Supplementary Fig. 6).

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