Research ReportGlioma stem cells targeted by oncolytic virus carrying endostatin–angiostatin fusion gene and the expression of its exogenous gene in vitro
Research highlights
► VAE can infect glioma stem cells (GSCs) and inhibit the viability of GSCs. ► The expression of Endo–Angio gene can be detected in GSCs after VAE infections. ► The exogenous Endo–Angio fusion protein inhibits HBMEC proliferation. ► Residual viable cells lost the ability of self-renewal and adherent differentiation. ► VAE can both lyse tumor cells and induce expression of Endo–Angio gene.
Introduction
Despite the currently available treatments for glioma, which include surgery, radiation and chemotherapy, gliomas, especially high-grade gliomas (WHO Classes III–IV), are typically fatal. Thus, there is an urgent need for an emergence of novel, improved treatments.
In recent years, a growing body of evidence has indicated that CSCs play a key role in tumor invasion, metastasis, recurrence and resistance to various treatments (Bonnet and Dick, 1997, Wulf et al., 2001). In human brain glioma, GSCs are isolated with neural stem or progenitor cells with similar properties, but whether GSCs arise from normal stem cells or more differentiated cells is not known. Nevertheless, GSCs resemble the normal stem or progenitor cells of the corresponding tissue of origin. Evidence suggests that normal neural stem cells exist in vascular niches (Schmidt et al., 2009, Shen et al., 2004). Tumor growth and metastasis require neovascularization, the process by which new blood vessels are formed from preexisting host vasculature (Folkman, 1990), and the biological properties of GSCs are closely related to the surrounding microvascular architecture (Gilbertson and Rich, 2007, Takakura, 2010). Data from many experiments support the hypothesis that vascular niches in brain tumors are abnormal and contribute directly to the generation of GSCs and tumor growth (Calabrese et al., 2007). Therefore, research targeting GSCs and their surrounding microvacular niche is truly significant.
Oncolytic viruses are natural or genetically modified viruses that, upon infection, selectively replicate and kill neoplastic cells while sparing normal cells (Selznick et al., 2008, Aghi and Martuza, 2005, Liu et al., 2007, Parato et al., 2005). These viruses, especially herpes simplex virus type 1 (HSV-1), are genetically engineered to restrict virus replication to tumor cells and to widen the therapeutic window. A direct comparison of oncolytic adenoviruses and oncolytic HSVs in glioma cell lines has shown oncolytic HSV to be more efficacious than adenovirus (Hoffmann and Wildner, 2007). Another advantage of HSV-1 is the capacity to incorporate large and/or multiple transgenes within the viral genome (Todo, 2008).
Angiostatin, a proteolytic fragment of plasminogen comprising the first four kringle domains, was first identified as a natural inhibitor of angiogenesis in the serum and urine of tumor-bearing mice (Folkman, 1995). Endostatin, a proteolytic fragment of collagen type XVIII identified after angiostatin, is a potent inhibitor of angiogenesis that was isolated from a mouse hemangioendothelioma cell line. Recombinant endostatin made in bacteria has been shown to inhibit the growth of tumors (De Bouard et al., 2003). Based on these anti-angiogenic properties, it is possible that inserting an endostatin and angiostatin (Endo–Angio) fusion gene into an engineered oncolytic virus may enhance the efficacy of tumor treatment.
To test this hypothesis, we examined the effects of VAE (γ34.5−, ICP6−, Endo–Angio+) on GSCs and HBMECs. VAE is an oncolytic virus of recombinant-HSV (r-HSV) modified by the insertion of an Endo–Angio fusion gene. The virus was engineered from wild-type HSV-1 strain F by deleting 1 kb within both copies of the γ34.5 gene and inserting the Endo–Angio fusion gene into the ICP6 coding region.
VAE should differ from other oncolytic viruses, which rely exclusively on oncolysis to combat tumors, in that it also expresses an exogenous therapeutic gene that may improve efficacy. One recent study reported that r-HSV may represent an efficacious agent against glioblastoma stem cells (GBM-SCs) (Wakimoto et al., 2009), thus highlighting the potential for viral gene therapy in treating glioblastoma and revealing a need for additional in-depth studies on ways to improve efficacy.
For these reasons, we investigated the efficacy of VAE in GSCs and the effects of exogenous Endo–Angio fusion proteins on microvascular endothelial cells in vitro.
Section snippets
Characterization of primary GSC cultures and human brain microvascular endothelial cell (HBMEC) cultures
We obtained surgical specimens from human high-grade gliomas to isolate and grow GSCs. All gliomas were classified as glioblastoma multiforme (GBM) by pathologic analysis. Of the 20 specimens collected, we were able to establish four stable cultures (G1, G2, G3 and G4) that could be passaged for more than 2 months. In the first 24 h, cells assembled into irregular cell clusters. After approximately 7 days of growth, the primary culture of tumor cells was observed under a light microscope. These
Discussion
Gliomas, the most common type of primary intracranial malignancy, are notorious for their highly invasive nature, multiplying ability and poor prognosis. The WHO classifies astrocytomas into four grades based on their histological features (Thurnher, 2009). Although comprehensive treatment is multifaceted and includes surgery, radiation and chemotherapy, the median survival of Grade III and Grade IV gliomas (high-grade gliomas) is only approximately 14 months (Stupp et al., 2005). Thus, there is
Isolation and culture of GSCs
Surgical specimens of high-grade gliomas were collected at Beijing Tiantan Hospital, Capital Medical University with approval by the institutional review board. Mechanically minced tissues were digested with 0.1% trypsin at 37 °C for 20 min. After washes, tissues were triturated and passed through a 74-μm cell strainer. Cells were plated in serum-free medium composed of DMEM/F12 (Gibco, USA) supplemented with B27 supplement (1:50) (Gibco, USA), 2 μg/ml heparin (Macgene, China), 20 ng/ml recombinant
Acknowledgment
This work was supported by a grant from the National Natural Science Foundation of China (No. 81071776)
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