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. 2017 Jan 18;6(1):1265291.
doi: 10.1080/20013078.2017.1265291. eCollection 2017.

TRAIL delivery by MSC-derived extracellular vesicles is an effective anticancer therapy

ZhengQiang Yuan  1 , Krishna K Kolluri  1 , Kate H C Gowers  1 , Sam M Janes  1
Affiliations

TRAIL delivery by MSC-derived extracellular vesicles is an effective anticancer therapy

ZhengQiang Yuan et al. J Extracell Vesicles. .

Abstract

Extracellular vesicles (EVs) are lipid membrane-enclosed nanoparticles released by cells. They mediate intercellular communication by transferring biological molecules and therefore have potential as innovative drug delivery vehicles. TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis of cancer cells. Unfortunately, the clinical application of recombinant rTRAIL has been hampered by its low bioavailability and resistance of cancer cells. EV-mediated TRAIL delivery may circumvent these problems. Mesenchymal stromal cells (MSCs) produce EVs and could be a good source for therapeutic EV production. We investigated if TRAIL could be expressed in MSC-derived EVs and examined their cancer cell-killing efficacy. EVs were isolated by ultracentrifugation and were membranous particles of 50-70 nm in diameter. Both MSC- and TRAIL-expressing MSC (MSCT)-derived EVs express CD63, CD9 and CD81, but only MSCT-EVs express surface TRAIL. MSCT-EVs induced apoptosis in 11 cancer cell lines in a dose-dependent manner but showed no cytotoxicity in primary human bronchial epithelial cells. Caspase activity inhibition or TRAIL neutralisation blocked the cytotoxicity of TRAIL-positive EVs. MSCT-EVs induced pronounced apoptosis in TRAIL-resistant cancer cells and this effect could be further enhanced using a CDK9 inhibitor. These data indicate that TRAIL delivery by MSC-derived EVs is an effective anticancer therapy.

Keywords: EV; MSC; TRAIL; cancer.

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Figures

Figure 1.
Figure 1.
Isolation and characterisation of MSC-derived extracellular vesicles (MSC-EVs). (a) Negative contrast micrograph of MSC-EVs examined and imaged by transmission electron microscopy (TEM), scale bar 100 nm. (b) EVs were labelled by the lipid membrane dye PKH67 (green) and taken up by MDAMB231 (M231) cells, followed by confocal microscopy examination; nuclei of M231 cells were labelled with DAPI (blue). (c) Flow cytometry analysis of EVs. EVs were labelled with a FITC-conjugated antibody against CD63 and compared with those labelled with a control FITC-isotype antibody. Top, a representative dot plot of at least three experiments showing the percentage of CD63-expressing EVs among the tested sample; bottom, quantitation of median fluorescent intensity (MFI) of labelled EVs.
Figure 2.
Figure 2.
Flow cytometry analysis of tetraspanin CD63, CD9 and CD81 expression on purified EVs, which were captured and purified with magnetic latex beads coated with mAb against CD63 and labelled with PE-conjugated antibodies; the labelling was done in detergent-free buffer aiming for EV surface protein labelling only. (a) Purified EV-bead complexes were stained with IgG-PE and IgG-AF647 isotype antibodies. (b) EV-bead complexes were labelled with CD63-AF647 and CD81-PE antibodies. (c) EV-bead complexes were labelled with CD63-AF647 and CD9-PE antibodies. (d) EV quantification showed that MSCTRAIL cells released more EVs than parental MSCs and empty vector lentivirus-transfected cells (MSCEV). EV number was determined by measuring the EV-enriched acetyl-CoA acetylcholinesterase (AChE) activity with a commercial EV quantification kit, values are mean ± SEM, n = 4; ** p < 0.01, by Student’s t-test.
Figure 3.
Figure 3.
Detection of TRAIL and tetraspanin expression in isolated MSC-EVs. (a) Measurement of TRAIL expression in EVs with a commercial ELISA kit. (b) Flow cytometry analyses of TRAIL and CD63 expression in purified EVs. MSC- and MSCT-EVs were purified with latex beads coated with CD63 antibody and labelled with IgG-PE, IgG-AF647 or TRAIL-PE and CD63-AF647 antibodies, respectively. (c) Western blotting detection of TRAIL and tetraspanin CD63, CD9 and CD81 in EVs and MSCTRAIL lysates; for each sample 30 µg of cellular proteins or 20 µg of EV proteins were analysed; EV preparation of medium without cell culture and 1 ng of rTRAIL (amino acids 114–281, PeproTech, USA) were used as negative and positive controls, respectively. (d) Comparison of TRAIL expression levels in MSCs, MSCTRAILs and MSCT-EVs using a commercial TRAIL-specific ELISA kit. Values are mean ± SEM, n = 4; ** p < 0.01.
Figure 4.
Figure 4.
MSCT-EVs induced apoptosis of cancer cells with high efficiency. Cells were cultured in 96-well plate and treated for 24 h with MSC-EVs or MSCT-EVs with concentrations varying from 1.0 to 100.0 µg/ml, or treated with 1.0–100.0 ng/ml rTRAIL, followed by cell harvesting and labelling with Annexin V-AF647 and DAPI for apoptosis assay by flow cytometry. (a) Treatment of primary human bronchial epithelial cells (HBECs). (b) Treatment of M231 cells. (c) Treatment of A549 cells. (d) Treatment of M231 cells with 100 ng/ml of TRAIL-neutralising monoclonal antibody (Ab) (T3067, Sigma-Aldrich) alone, 20 µmol/l of pan-caspase inhibitor Z-VAD-FMK (inhib) alone, or with MSCT-EVs (100 µg/ml) alone, or with MSCT-EVs in combination with Ab (MSCT-EV+Ab) or with inhibitor (MSCT-EV+inhib). Data represent averages ±S.E.M, n = 4. * p < 0.05, ** p < 0.01 compared with MSC-EV control, by one-way ANOVA followed by Dunnett’s post-test.
Figure 5.
Figure 5.
MSCT-EVs efficiently killed rTRAIL-resistant cancer cell lines. (a) Six rTRAIL-sensitive cancer cell lines (H2795, H2804, NCI-H460, NCI-H727, SHEP-TET and M231) were cultured in 96-well plates and treated with MSC-EVs (100 µg/ml), MSCT-EVs (100 µg/ml), rTRAIL (100 ng/ml) or with control medium for 24 h, followed by analysis by cell apoptosis assay. (b) Five rTRAIL-resistant cancer cell lines (H2810, H2818, HA7-RCC, RCC10 and A549) were treated with same agents like in (a) and analysed for cell apoptosis. (c) Caspase-8 activity was examined by flow cytometry in A549 cells, which were treated with MSC-EVs (100 µg/ml proteins), MSCT-EVs (100 µg/ml proteins) or with rTRAIL (100 ng/ml) for 24 h. Treated cells were labelled with the active caspase-8 binding dye Red-IETD-FMK and analysed by flow cytometry. (d) CDK9 inhibition by SNS032 drastically enhanced apoptosis induction in A549 cells by MSCT-EVs. Cell were treated with MSC-EV (10 µg/ml), MSCT-EV (10 µg/ml) or SNS032 (300 nmol/l) alone or with MSCT-EV (10 µg/ml) in combination with SNS032 (300 nmol/l) for 24 h, and analysed by cell apoptosis assay. Data represent averages ± SEM, n = 4. * p < 0.05, *** p < 0.001, analysed by Student’s t-test.
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References

    1. Lässer C, Alikhani VS, Ekström K, et al. Human saliva, plasma and breast milk exosomes contain RNA: uptake by macrophages. J Transl Med. 2011;9:1 pii:1479-5876-9-9 DOI:10.1186/1479-5876-9-9 - DOI - PMC - PubMed
    1. Valadi H, Ekström K, Bossios A, et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007;9(6):654–13. pii:ncb1596 DOI:10.1038/ncb1596 - DOI - PubMed
    1. Rivoltini L, Chiodoni C, Squarcina P, et al. TNF-related apoptosis-inducing ligand (TRAIL)-armed exosomes deliver proapoptotic signals to tumor site. Clin Cancer Res. 2016;22:3499–3512. pii:1078-0432.CCR-15-2170 DOI:10.1158/1078-0432.CCR-15-2170 - DOI - PubMed
    1. Stenqvist A-C, Nagaeva O, Baranov V, et al. Exosomes secreted by human placenta carry functional Fas ligand and TRAIL molecules and convey apoptosis in activated immune cells, suggesting exosome-mediated immune privilege of the fetus. J Immunol. 2013;191(11):5515–5523. pii:jimmunol.1301885 DOI:10.4049/jimmunol.1301885 - DOI - PubMed
    1. Ratajczak J, Miekus K, Kucia M, et al. Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery. Leukemia. 2006;20(5):847–856. pii:2404132 DOI:10.1038/sj.leu.2404132 - DOI - PubMed