Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Jul;17(7):885-96.
doi: 10.1016/j.jcyt.2015.03.603. Epub 2015 Apr 14.

Mesenchymal stromal cell delivery of full-length tumor necrosis factor-related apoptosis-inducing ligand is superior to soluble type for cancer therapy

ZhengQiang Yuan  1 , Krishna K Kolluri  1 , Elizabeth K Sage  1 , Kate H C Gowers  1 , Sam M Janes  2
Affiliations

Mesenchymal stromal cell delivery of full-length tumor necrosis factor-related apoptosis-inducing ligand is superior to soluble type for cancer therapy

ZhengQiang Yuan et al. Cytotherapy. 2015 Jul.

Abstract

Background aims: Mesenchymal stromal cell (MSC) delivery of pro-apoptotic tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is an attractive strategy for anticancer therapy. MSCs expressing full-length human TRAIL (flT) or its soluble form (sT) have previously been shown to be effective for cancer killing. However, a comparison between the two forms has never been performed, leaving it unclear which approach is most effective. This study addresses the issue for the possible clinical application of TRAIL-expressing MSCs in the future.

Methods: MSCs were transduced with lentiviruses expressing flT or an isoleucine zipper-fused sT. TRAIL expression was examined and cancer cell apoptosis was measured after treatment with transduced MSCs or with MSC-derived soluble TRAIL.

Results: The transduction does not adversely affect cell phenotype. The sT-transduced MSCs (MSC-sT) secrete abundant levels of soluble TRAIL but do not present the protein on the cell surface. Interestingly, the flT-transduced MSCs (MSC-flT) not only express cell-surface TRAIL but also release flT into medium. These cells were examined for inducing apoptosis in 20 cancer cell lines. MSC-sT cells showed very limited effects. By contrast, MSC-flT cells demonstrated high cancer cell-killing efficiency. More importantly, MSC-flT cells can overcome some cancer cell resistance to recombinant TRAIL. In addition, both cell surface flT and secreted flT are functional for inducing apoptosis. The secreted flT was found to have higher cancer cell-killing capacity than either recombinant TRAIL or MSC-secreted sT.

Conclusions: These observations demonstrate that MSC delivery of flT is superior to MSC delivery of sT for cancer therapy.

Keywords: TRAIL; apoptosis; cancer; mesenchymal stromal cell; tumor.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Expression of recombinant TRAIL by transduced MSCs. (A) Schematic description of TRAIL expression constructs. (B) Fluorescence-activated cell sorting analysis of lentivirus-transduced MSCs. (C) Long-term fluorescence-activated cell sorting analysis of TRAIL expression in MSCs transduced at passage 3 (p3), expanded and passaged every 7 days until p8. (D) Detection of TRAIL and α-tubulin expression by immunoblotting. Cont. represents MSC-GFP cell lysates or supernatant medium; flT and sT represent cell lysates or concentrated culture supernatant from MSC-flT and MSC-sT cells, respectively; rT represents 1 ng of purified recombinant human TRAIL (amino acids 114–281) produced from bacterial cells (PeproTech). (E) Levels of TRAIL in cell culture supernatants from MSCs transduced at p3 and expanded for one passage, measured by ELISA. Data presented as TRAIL released by 1 million cells per hour (ng/h/1 × 106 cells). Data represent averages ± SEM (n = 5).
Figure 2
Figure 2
Immunofluorescent detection of recombinant TRAIL (red) expressed by transduced MSCs. Phalloidin staining was used to show filamentous actin (green); nuclei were labeled with DAPI (blue). Top panel shows intracellular staining; bottom panel shows cell surface staining. Images are representative of at least three experiments for each staining condition. Magnification ×400.
Figure 3
Figure 3
TRAIL expression by lentiviral transduction does not affect MSC viability, proliferation, marker protein expression or differentiation potential. (A) Cell viability and proliferation were assessed with the use of the XTT assay for 7 days after transduction. (B) Phenotyping of cultured MSCs for expression of conventional MSC markers is shown. Dashed line shows isotype antibody control; solid line shows marker-specific antibody staining (red, MSC; blue, MSC-flT; green, MSC-sT). (C) MSC differentiation potential was assessed by means of real-time quantitative PCR for adipogenic marker gene PPARG and osteogenic gene BMP2 before and after differentiation period (Differ.) and immunochemistry. (D) High content screening lipidTOX green staining (green) for neutral lipid and DAPI staining for nuclei (blue) to show adipogenic differentiation; middle, alizarin red S staining (red) to show osteogenic differentiation; right, alcian blue staining (blue) to show chondrogenic differentiation. Magnification ×200 for adipogenesis and osteogenesis assays; magnification ×50 for chondrogenesis assay.
Figure 4
Figure 4
MSCs expressing TRAIL induce apoptosis in cancer cells. (A, B) Cancer cell apoptosis was measured by means of flow cytometry 24 h after co-culturing the breast adenocarcinoma cells MDAMB231 (M231) (A) or the lung adenocarcinoma cells A549 (B) with MSC-GFP, MSC-flT or MSC-sT cells, with an increasing ratio of MSCs to cancer cells in the co-culture system. (C) Activated caspase-8 levels in A549 cells were measured by means of flow cytometry after co-culture with MSC-GFP, MSC-sT or MSC-flT cells at a ratio of 4:10 (MSC: cancer cell). (D) MSC-flT–induced cancer cell apoptosis can be blocked by the 20 μmol/L pan-caspase inhibitor Z-VAD-FMK (zVAD) and 100 ng/mL TRAIL-neutralizing monoclonal Ab (T3067, Sigma-Aldrich). Data represent averages ± SEM. ∗∗P < 0.01 compared with MSC-flT co-culture by Student's t-test.
Figure 5
Figure 5
MSC-flT cells induce cancer cell apoptosis with a higher efficiency than MSC-sT cells. (A–D) Apoptosis of five highly TRAIL-sensitive cancer cell lines, Colo205, NCI-H460, H727, H2795 and H2804 (A); five moderately TRAIL-sensitive cancer cell lines, H2731, H226, H2869, PC9 and M231 (B); four cancer cell lines of low TRAIL sensitivity, HT29, H357, H2452 and RKO (C); and six TRAIL-resistant cancer cell lines, A549, NCI-H2052, H2810, NCI-H23, RCC10 and HA7-RCC (D) was determined after 24 h of culture with medium (control), 50 ng/mL of purified recombinant TRAIL (rTRAIL), MSC-GFP, MSC-flT or MSC-sT, with a ratio of 4 p4 MSC to 10 cancer cells. Data represent averages of three experiments with triplicate repeats for each cell line.
Figure 6
Figure 6
MSC-flT–derived cell surface and secreted TRAILs contribute to induction of apoptosis in cancer cells. (A, B) Apoptosis was measured by means of flow cytometry after exposure of M231 cells (A) or A549 cells (B) for 24 h to increasing doses of recombinant TRAIL (rTRAIL), supernatant TRAIL from MSC-sT (sTRAIL) or supernatant TRAIL from MSC-flT (flTRAIL). (C) Apoptosis was measured after 24-h co-culture of A549 cells with live or fixed MSC-GFP and MSC-flT cells, respectively. Data represent averages ± SEM (n = 3). ∗P < 0.05, ∗∗P < 0.01 compared with rTRAIL and MSC-GFP treatment, respectively, by Student's t-test.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Wiley S.R., Schooley K., Smolak P.J., Din W.S., Huang C.P., Nicholl J.K. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity. 1995;3:673–682. - PubMed
    1. Pitti R.M., Marsters S.A., Ruppert S., Donahue C.J., Moore A., Ashkenazi A. Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem. 1996;271:12687–12690. - PubMed
    1. Lemke J., von Karstedt S., Abd El Hay M., Conti A., Arce F., Montinaro A. Selective CDK9 inhibition overcomes TRAIL resistance by concomitant suppression of cFlip and Mcl-1. Cell Death Differ. 2014;21:491–502. - PMC - PubMed
    1. Walczak H., Miller R.E., Ariail K., Gliniak B., Griffith T.S., Kubin M. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med. 1999;5:157–163. - PubMed
    1. Ashkenazi A., Pai R.C., Fong S., Leung S., Lawrence D.A., Marsters S.A. Safety and antitumor activity of recombinant soluble Apo2 ligand. J Clin Invest. 1999;104:155–162. - PMC - PubMed

Publication types

MeSH terms

Substances