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. 2014 Apr 15;323(1):178-188.
doi: 10.1016/j.yexcr.2014.01.014. Epub 2014 Jan 24.

Tumor exosomes induce tunneling nanotubes in lipid raft-enriched regions of human mesothelioma cells

Venugopal Thayanithy  1 , Victor Babatunde  2 , Elizabeth L Dickson  3 , Phillip Wong  1 , Sanghoon Oh  4 , Xu Ke  4 , Afsar Barlas  4 , Sho Fujisawa  4 , Yevgeniy Romin  4 , André L Moreira  5 , Robert J Downey  6 , Clifford J Steer  7 , Subbaya Subramanian  8 , Katia Manova-Todorova  4 , Malcolm A S Moore  2 , Emil Lou  9
Affiliations

Tumor exosomes induce tunneling nanotubes in lipid raft-enriched regions of human mesothelioma cells

Venugopal Thayanithy et al. Exp Cell Res. .

Abstract

Tunneling nanotubes (TnTs) are long, non-adherent, actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study, we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24-48 h; and this effect was most prominent in media conditions (low-serum, hyperglycemic medium) that support TnT formation (1.3-1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs, in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs, which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation, and also lipid raft formation as a potential biomarker for TnT-forming cells.

Keywords: Exosomes; Intercellular communication; Intercellular transfer; Lipid rafts; Mesothelioma; Tunneling nanotubes.

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Conflict of interest statement

Conflict-of-interest disclosures: The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. Confirmation and characterization of exosomes
A) SDS-PAGE analysis of VAMT exosomal proteins. Exosomes were isolated from the supernatant of VAMT sarcomatoid mesothelioma cells and analyzed by denaturing SDS-PAGE (10%) analysis. B) Western blot analysis of VAMT and Met5A exosomes. VAMT and Met5A exosomes were subjected to Western blot analysis with exosome-verified antibodies as described in the Methods section. C) ELISA confirm of exosome purification. Quantification of VAMT and Met5A exosomes was performed by ELISA, using the exosome standard provided by the manufacturer (System Biosciences) for comparison. Exosome specific rabbit anti-human-CD9 antibody were used for the assay. Exosome standards provided by the vendor were assayed using CD9 antibodies. Visual confirmation of exosomes using EM is shown in Figure 2.
Figure 2
Figure 2. Structural examination of exosomes in and around TnTs using scanning and transmission electron microscopy (EM)
A) Scanning EM of TnTs as long, thin tubular structures emerging directly from the plasma membrane (left panel). TnTs have characteristic bulges (right panel), representing putative cellular cargo in transit (inset photograph). The indicated TnT is non-adherent and passes over short, thicker, adherent cellular extensions consistent with microvilli, which are also characteristic of mesothelioma.B) Two different TnTs providing either an ‘intercellular bridge’ between two cells or a partially-formed TnT stemming from one of the cells. C & D) The middle and lower panels provide closer views of panel B. E) The enlarged image to the right demonstrates exosomes in the extracellular environment and around the base of the connected TnT (arrowheads). These images are representative of the findings from this study. The speckled appearance of mesothelioma exosomes is identical to that demonstrated in a previous study by Hegmans et al.[15]
Figure 3
Figure 3. Movement of internalized exogenous VAMT exosomes across cells via TnTs
MSTO-211H cells were co-cultured with DiI-labeled VAMT-derived exosomes and imaged by time-lapse microscopy. TnTs (arrows) and a moving DiI-labeled VAMT exosome (top arrow) are shown. The VAMT exosome was transported from one cell to another within 20-30 minutes (Supplementary Movie S1).
Figure 4
Figure 4. Tumor-derived exosomes stimulate increased rate of formation and early development of TnTs
The average number of TnTs per cells at 24, 48, and 72 hours are shown for cells treated with VAMT exosomes (A) or Met5A exosomes (B). Purified exosomes were added to MSTO-211H cells in culture under various media conditions in the presence or absence of exogenous VAMT or Met5A-derived exosomes. An Olympus IX70 inverted microscope was used to count TnTs in 10 fields per cell for each medium condition at regular time intervals (0, 24, 48, and 72 hours) using a 20x objective lens. The number of cells was also counted, and data were graphed and reported as average number of TnTs per cell (TnTs/cell). Experiments were performed in duplicate, with measurements averaged. Statistically significant differences were calculated and P values are indicated by asterisks.
Figure 5
Figure 5. Representative images of confocal microscopy showing enrichment of lipid rafts in TnT-forming VAMT and MSTO-211H cells
Cells were cultured for 48-72 hours in low-serum (2.5% FBS), hyperglycemic (50 mM glucose) RPMI medium. After microscopic confirmation of TnT formation, cells were fixed as described in “Methods”. Biotinylated-cholera toxin B was used to stain and identify lipid rafts. The top row demonstrates VAMT cells (panels A, B, & C); the bottom row demonstrates MSTO-211H cells (panels D, E, & F). Images were obtained using a Leica SP5 confocal microscope using either 20x/0.7 water immersion objective (panels A and B), 63x/1.4 oil immersion objective (panel C), or 40 x (panels D, E, & F) lenses.
Figure 6
Figure 6. Quantification of lipid raft fluorescence assessed by average CTCF/area of TnT-forming and non-TnT-forming cells
ImageJ software was used to measure the integrative density and area of cells and TnTs (measured as pixels). Using this information, we calculated the average CTCF/average area of TnT-forming cells and non-TnT forming cells in three mesothelioma (MSTO-211H, VAMT, H2052) cell lines and one mesothelial (Met5A) cell line.
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