Human Glioblastoma-Derived Cancer Stem Cells: Establishment of Invasive Glioma Models and Treatment with Oncolytic Herpes Simplex Virus Vectors

Molecular Neurosurgery Laboratory, Brain Tumor Research Center, Department of Neurosurgery, Massachusetts General Hospital, Boston, MA 02114, USA.
Cancer Research (Impact Factor: 9.33). 05/2009; 69(8):3472-81. DOI: 10.1158/0008-5472.CAN-08-3886
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Glioblastoma, the most malignant type of primary brain tumor, is one of the solid cancers where cancer stem cells have been isolated, and studies have suggested resistance of those cells to chemotherapy and radiotherapy. Here, we report the establishment of CSC-enriched cultures derived from human glioblastoma specimens. They grew as neurospheres in serum-free medium with epidermal growth factor and fibroblast growth factor 2, varied in the level of CD133 expression and very efficiently formed highly invasive and/or vascular tumors upon intracerebral implantation into immunodeficient mice. As a novel therapeutic strategy for glioblastoma-derived cancer stem-like cells (GBM-SC), we have tested oncolytic herpes simplex virus (oHSV) vectors. We show that although ICP6 (UL39)-deleted mutants kill GBM-SCs as efficiently as wild-type HSV, the deletion of gamma34.5 significantly attenuated the vectors due to poor replication. However, this was significantly reversed by the additional deletion of alpha47. Infection with oHSV G47Delta (ICP6(-), gamma34.5(-), alpha47(-)) not only killed GBM-SCs but also inhibited their self-renewal as evidenced by the inability of viable cells to form secondary tumor spheres. Importantly, despite the highly invasive nature of the intracerebral tumors generated by GBM-SCs, intratumoral injection of G47Delta significantly prolonged survival. These results for the first time show the efficacy of oHSV against human GBM-SCs, and correlate this cytotoxic property with specific oHSV mutations. This is important for designing new oHSV vectors and clinical trials. Moreover, the new glioma models described in this study provide powerful tools for testing experimental therapeutics and studying invasion and angiogenesis.

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    • "The glioblastoma stem cell line MGG8-Fluc-Mcherry was established and characterized as previously described [24,25]. Tumor spheres were grown in DMEM-F12 supplemented with penicillin-streptomycin, B27, EGF (5µg/mL), FGF(5µg/mL) and heparin(5mg/mL). "
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    ABSTRACT: Oncolytic adenoviral vectors are a promising alternative for the treatment of glioblastoma. Recent publications have demonstrated the advantages of shielding viral particles within cellular vehicles (CVs), which can be targeted towards the tumor microenvironment. Here, we studied T-cells, often having a natural capacity to target tumors, for their feasibility as a CV to deliver the oncolytic adenovirus, Delta24-RGD, to glioblastoma. The Jurkat T-cell line was assessed in co-culture with the glioblastoma stem cell (GSC) line, MGG8, for the optimal transfer conditions of Delta24-RGD in vitro. The effect of intraparenchymal and tail vein injections on intratumoral virus distribution and overall survival was addressed in an orthotopic glioma stem cell (GSC)-based xenograft model. Jurkat T-cells were demonstrated to facilitate the amplification and transfer of Delta24-RGD onto GSCs. Delta24-RGD dosing and incubation time were found to influence the migratory ability of T-cells towards GSCs. Injection of Delta24-RGD-loaded T-cells into the brains of GSC-bearing mice led to migration towards the tumor and dispersion of the virus within the tumor core and infiltrative zones. This occurred after injection into the ipsilateral hemisphere, as well as into the non-tumor-bearing hemisphere. We found that T-cell-mediated delivery of Delta24-RGD led to the inhibition of tumor growth compared to non-treated controls, resulting in prolonged survival (p = 0.007). Systemic administration of virus-loaded T-cells resulted in intratumoral viral delivery, albeit at low levels. Based on these findings, we conclude that T-cell-based CVs are a feasible approach to local Delta24-RGD delivery in glioblastoma, although efficient systemic targeting requires further improvement.
    Viruses 08/2014; 2014(6):3080-3096. DOI:10.3390/v6083080 · 3.35 Impact Factor
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    • "The lack of effective therapies for glioblastomas has prompted the development of novel experimental approaches that utilize oncolytic viruses (OV) combined with gene therapy. OV, either naturally occurring or developed through genetic engineering, are being investigated as oncolytic agents for cancer treatment due to their ability to specifically infect and lyse neoplastic cells while sparing normal cells [14], [15], [16], [17]. The use of transgenic technology facilitates the creation of OV vectors, which deliver therapeutic anti-angiogenic transgenes. "
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    ABSTRACT: Viruses have demonstrated strong potential for the therapeutic targeting of glioblastoma stem cells (GSCs). In this study, the use of a herpes simplex virus carrying endostatin-angiostatin (VAE) as a novel therapeutic targeting strategy for glioblastoma-derived cancer stem cells was investigated. We isolated six stable GSC-enriched cultures from 36 human glioblastoma specimens and selected one of the stable GSCs lines for establishing GSC-carrying orthotopic nude mouse models. The following results were obtained: (a) VAE rapidly proliferated in GSCs and expressed endo-angio in vitro and in vivo 48 h and 10 d after infection, respectively; (b) compared with the control gliomas treated with rHSV or Endostar, the subcutaneous gliomas derived from the GSCs showed a significant reduction in microvessel density after VAE treatment; (c) compared with the control, a significant improvement was observed in the length of the survival of mice with intracranial and subcutaneous gliomas treated with VAE; (d) MRI analysis showed that the tumor volumes of the intracranial gliomas generated by GSCs remarkably decreased after 10 d of VAE treatment compared with the controls. In conclusion, VAE demonstrated oncolytic therapeutic efficacy in animal models of human GSCs and expressed an endostatin-angiostatin fusion gene, which enhanced antitumor efficacy most likely by restricting tumor microvasculature development.
    PLoS ONE 04/2014; 9(4):e95872. DOI:10.1371/journal.pone.0095872 · 3.23 Impact Factor
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    • "Medulloblastoma (MB) cell lines (UW426, R262, R300, DAOY, UW473) from American Type Culture Collection (ATCC) [21] were cultured in Dulbecco's modified Eagle's medium (DMEM) (Invitrogen/GIBCO, Grand Island, NY) supplemented with 10% fetal calf serum (Valley Biomedical Inc. Winchester, VA), 1% Penicillin/Streptomycin (Invitrogen, Carlsbad, CA), 2% sodium bicarbonate (GIBCO), 1% nonessential amino acids (Mediatech Inc, Manassas, VA), and 1% sodium pyruvate (Cellgro, Manassas, VA). Established human glioma lines (LN229, LN319, U138, U373, U87, U251 and Gli36) and primary human glioma lines (GBM4 and GBM8) were grown as previously described [18], [22]. Murine mesenchymal stem cells (MSC) (kindly provided by Dr. Darwin Prockop, University of Texas) were grown as previously described [18]. "
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    ABSTRACT: Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) induced apoptosis specifically in tumor cells. However, with approximately half of all known tumor lines being resistant to TRAIL, the identification of TRAIL sensitizers and their mechanism of action become critical to broadly use TRAIL as a therapeutic agent. In this study, we explored whether c-Met protein contributes to TRAIL sensitivity. We found a direct correlation between the c-Met expression level and TRAIL resistance. We show that the knock down c-Met protein, but not inhibition, sensitized brain tumor cells to TRAIL-mediated apoptosis by interrupting the interaction between c-Met and TRAIL cognate death receptor (DR) 5. This interruption greatly induces the formation of death-inducing signaling complex (DISC) and subsequent downstream apoptosis signaling. Using intracranially implanted brain tumor cells and stem cell (SC) lines engineered with different combinations of fluorescent and bioluminescent proteins, we show that SC expressing a potent and secretable TRAIL (S-TRAIL) have a significant anti-tumor effect in mice bearing c-Met knock down of TRAIL-resistant brain tumors. To our best knowledge, this is the first study that demonstrates c-Met contributes to TRAIL sensitivity of brain tumor cells and has implications for developing effective therapies for brain tumor patients.
    PLoS ONE 04/2014; 9(4):e95490. DOI:10.1371/journal.pone.0095490 · 3.23 Impact Factor
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