Assessment of therapeutic efficacy and fate of engineered human mesenchymal stem cells for cancer therapy

Abstract

The poor prognosis of patients with aggressive and invasive cancers combined with toxic effects and short half-life of currently available treatments necessitate development of more effective tumor selective therapies. Mesenchymal stem cells (MSCs) are emerging as novel cell-based delivery agents; however, a thorough investigation addressing their therapeutic potential and fate in different cancer models is lacking. In this study, we explored the engineering potential, fate, and therapeutic efficacy of human MSCs in a highly malignant and invasive model of glioblastoma. We show that engineered MSC retain their "stem-like" properties, survive longer in mice with gliomas than in the normal brain, and migrate extensively toward gliomas. We also show that MSCs are resistant to the cytokine tumor necrosis factor apoptosis ligand (TRAIL) and, when engineered to express secreted recombinant TRAIL, induce caspase-mediated apoptosis in established glioma cell lines as well as CD133-positive primary glioma cells in vitro. Using highly malignant and invasive human glioma models and employing real-time imaging with correlative neuropathology, we demonstrate that MSC-delivered recombinant TRAIL has profound anti-tumor effects in vivo. This study demonstrates the efficacy of diagnostic and therapeutic MSC in preclinical glioma models and forms the basis for developing stem cell-based therapies for different cancers.

Fig. 1.

Transduced human MSCs proliferate in culture and survive longer in mice bearing gliomas. (A) Light image of MSCs in culture. (B–D) MSCs were transduced with LV-GFP-Fluc and, 48 h later, were FACS-sorted. Photomicrograph of MSCs expressing GFP-Fluc (B). (C) Plot showing the percentage of transduced MSCs expressing GFP. (D) In vitro imaging shows the expression of Fluc in MSC-GFP-Fluc over time. (E) Genomic profiles of transduced MSCs. (F) Karyotype analysis of transduced MSCs. (G) Fluc bioluminescence intensities of MSC-GFP-Fluc implanted intraparenchymally either alone or mixed with Gli36-EGFRvIII human glioma cells. One representative image of mice with MSC-GFP-Fluc implanted with (+) or without (-) glioma cells is shown. (H and I) Photomicrographs on brain sections from mice 16 days after implantation shows presence of GFP-positive MSCs in normal brain (H) and the presence of Ki67-positive glioma cells (red) and GFP-positive MSCs in glioma bearing brains (I). (Original magnification: B, H, and I, ×20.)

Fig. 2.

Human MSCs do not influence glioma growth in mice. (A) Fluc bioluminescence intensities of intraparenchymally implanted mice with Gli36-EGFRvIII-FD human glioma cells or a mix of Gli36-EGFRvIII-FD and MSC-GFP. One representative image of mice with Gli36-EGFRvIII-FD implanted with (+) or without (-) MSC-GFP is shown. (B and C) Photomicrographs on brain sections from mice 16 days after implantation shows expression of DsRed2 in glioma cells (B) and the presence of GFP-positive MSCs in mice bearing gliomas (C). (Original magnification: B and C, ×20.)

Fig. 3.

Bystander effect and migration of MSCs. (A–F) Human Gli36-EGFRvIII glioma cells were labeled with DI1 and MSCs were labeled with calcein-AM, mixed, and FACS-sorted after 5 min and after 3 h. FACS-sorted plots and graphs reveal different populations of labeled cells after 5 min (A, C, and D) and 3 h (B, E, and F). (G and H) Photomicrographs of MSCs expressing tdTomato (G) and human Gli36-EGFRvIII glioma cells expressing GFP-Fluc (H). MSCs expressing tdTomato were implanted intracranially at a 1-mm distance from established human gliomas expressing GFP-Fluc. (I and J) Photomicrographs showing MSC-tdTomato (red) and gliomas (green) on day 2 (I) and day 10 (J) in brain sections and by intravital microscopy on day 10 after MSC implantation (K). (L–O) Immunohistochemistry on day-14 brain sections from Gli36-EGFRvIII-glioma bearing mice implanted with MSCs expressing tdTomato. Representative images of brain sections immunostained for Ki67 (L), nestin (M), GFAP (N), and MAP-2 (O). (Green, GFP expression; blue, nestin, Ki67, GFAP, or MAP-2 expression; original magnification: I and J, ×10; K–O, ×20.)

Fig. 4.

Pharmacodynamics and therapeutic efficacy of MSC-S-TRAIL. (A) MSCs were co-transduced with LV-Gluc-S-TRAIL and LV-GFP-Fluc, and cells and conditioned culture medium were imaged for Fluc and Gluc activity, respectively. Plots show correlation between the different concentrations of cells-Fluc activity and medium-Gluc activity within the ranges tested. (B) MSCs expressing Gluc-S-TRAIL and GFP-Fluc were mixed with Gli36-EGFRvIII glioma cells and implanted s.c. in nude mice. Mice were imaged for Fluc and Gluc activity every week for a period of 2 weeks. (C–H) Serial in vivo bioluminescence imaging of tumor growth following intracranial implantation of Gli36-EGFRvIII-FD glioma cells mixed with MSCs expressing S-TRAIL (MSC-S-TRAIL; D, F, and H) or GFP (MSC-GFP; C, E, and G). One representative mouse image from each group is shown. (I) Relative mean bioluminescent signal intensities after quantification of in vivo images. (J–O) Photomicrographs show presence of cleaved caspase-3 (J) and Ki67-positive cells (M) in brain sections from MSC-S-TRAIL-treated and control mice (K and N) 6 days after implantation. Plot shows the number of cleaved caspase-3 (L) and Ki67 (O) cells in MSC-S-TRAIL and MSC-GFP-treated tumors. (Green, MSCs; red, glioma cells; purple, Ki67 or cleaved caspase-3 expression; original magnification: J, K, M, and N, ×20.)

Fig. 5.

Molecular profiling of primary brain tumor cells and therapeutic efficacy of MSC-S-TRAIL in primary brain tumor cells. (A) Western blot analysis of the lysates of CD133-positive human primary glioma cell line (GBM8) and human Gli36 glioma line (B and C) GBM8 cells were incubated with the conditioned medium from MSC-S-TRAIL and, 18 h later, cells were stained with anti-cleaved caspase-3 antibody. Photomicrograph shows S-TRAIL-treated (B) and control-treated cells (C). (D) Cell viability of GBM cells incubated with different concentrations of S-TRAIL. (E and F) Photomicrographs of GFP-Fluc-expressing GBM8 cells stained with anti-nestin (E) and anti-GFAP antibodies (F). (G and H) Photomicrographs of day-14 brain sections stained with anti-human nuclei (G) and H & E (H) from mice bearing GBM8-GFP-Fluc gliomas. (I) Plots of photon intensities from MSC-S-TRAIL or MSC-DsRed2 established GBM8-GFP-Fluc glioma-treated tumors at weeks 3 and 5 of implantation are shown. (J) Survival curves of GBM8-GFP-Fluc-bearing mice treated with MSC-DsRed2 and MSC-S-TRAIL. (K–M) Photomicrographs showing presence of DsRed2 MSCs in brain sections from control mice (K) and Ki67 (L and M) and cleaved caspase-3 (O and P) cells in brain sections from control and MSC-S-TRAIL mice 2 weeks after the second MSC implantation. (N and Q) Plot shows the number of Ki67 (N) and cleaved caspase-3 (Q) cells in MSC-S-TRAIL- and MSC-DsRed2-treated tumors. (Original magnification: B, G, and H, ×10; E, F, K–M, O, and P, ×20.)