Elsevier

Brain Research

Volume 1390, 16 May 2011, Pages 59-69
Brain Research

Research Report
Glioma stem cells targeted by oncolytic virus carrying endostatin–angiostatin fusion gene and the expression of its exogenous gene in vitro

https://doi.org/10.1016/j.brainres.2011.03.050Get rights and content

Abstract

The development of the cancer stem cell (CSCs) niche theory has provided a new target for the treatment of gliomas. Gene therapy using oncolytic viral vectors has shown great potential for the therapeutic targeting of CSCs. To explore whether a viral vector carrying an exogenous Endo–Angio fusion gene (VAE) can infect and kill glioma stem cells (GSCs), as well as inhibit their vascular niche in vitro, we have collected surgical specimens of human high-grade glioma (world health organization, WHO Classes III–VI) from which we isolated and cultured GSCs under conditions originally designed for the selective expansion of neural stem cells. Our results demonstrate the following: (1) Four lines of GSCs (isolated from 20 surgical specimens) could grow in suspension, were multipotent, had the ability to self-renew and expressed the neural stem cell markers, CD133 and nestin. (2) VAE could infect GSCs and significantly inhibit their viability. (3) The Endo–Angio fusion gene was expressed in GSCs 48 h after VAE infection and could inhibit the proliferation of human brain microvascular endothelial cells (HBMEC). (4) Residual viable cells lose the ability of self-renewal and adherent differentiation. In conclusion, VAE can significantly inhibit the activity of GSCs in vitro and the expression of exogenous Endo–Angio fusion gene can inhibit HBMEC proliferation. VAE can be used as a novel virus–gene therapy strategy for glioma.

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)

References (36)

  • J. Chou et al.

    Mapping of herpes simplex virus-1 neurovirulence to gamma 134.5, a gene nonessential for growth in culture

    Science

    (1990)
  • S. De Bouard et al.

    Antiangiogenic therapy against experimental glioblastoma using genetically engineered cells producing interferon-alpha, angiostatin, or endostatin

    Hum. Gene Ther.

    (2003)
  • N. Ferrara et al.

    Angiogenesis as a therapeutic target

    Nature

    (2005)
  • J. Folkman

    What is the evidence that tumors are angiogenesis dependent?

    J. Natl Cancer Inst.

    (1990)
  • J. Folkman

    Angiogenesis inhibitors generated by tumors

    Mol. Med.

    (1995)
  • R.J. Gilbertson et al.

    Making a tumour's bed: glioblastoma stem cells and the vascular niche

    Nat. Rev. Cancer

    (2007)
  • D. Hoffmann et al.

    Comparison of herpes simplex virus- and conditionally replicative adenovirus-based vectors for glioblastoma treatment

    Cancer Gene Ther.

    (2007)
  • R.L. Jensen

    Brain tumor hypoxia: tumorigenesis, angiogenesis, imaging, pseudoprogression, and as a therapeutic target

    J. Neurooncol.

    (2009)
  • Cited by (32)

    • Oncolytic HSV-1 suppresses cell invasion through downregulating Sp1 in experimental glioblastoma

      2023, Cellular Signalling
      Citation Excerpt :

      Some glioma stem cells preferentially grow along the white matter tract at the invasive edge of glioma tissue [47]. We have previously described the construction of an HSV-1 vector expressing an endostatin-angiostatin fusion, also known as VAE, which is highly sensitive to glioma-derived stem cells (GSCs) in vivo and in vitro, with good antitumor efficacy [48]. We also found that oHSV-1 significantly inhibits the invasive ability of glioma cells, suggesting that treatment with this oncolytic virus is appropriate for this aggressive form of glioma.

    • The enhanced efficacy of herpes simplex virus by lentivirus mediated VP22 and cytosine deaminase gene therapy against glioma

      2020, Brain Research
      Citation Excerpt :

      The membranes were incubated with the primary antibodies of CD (Abcam, ab35251, 1:100), EGFP (Abcam, ab111258, 1:200), and β-actin (Abcam, ab6276, 1:2000) at 4 °C for 12 h, followed by incubation with a HRP-conjugated second antibody (Santa Cruz Biotechnology, Sc-2005, USA, 1:5000) at room temperature for 2 h. The rHSV-1 (γ34.5–, ICP6–) virus was constructed by Prof. Willian Jia and stored in our laboratory (Zhu et al., 2011). To amplify the virus, vero cells were cultured to 80%-90% confluence and infected with rHSV-1 at a multiplicity of infection (MOI) of 0.1 and then cultured in DMEM supplemented with 2% FBS.

    • Synergistic combination of oncolytic virotherapy with CAR T-cell therapy

      2019, Progress in Molecular Biology and Translational Science
      Citation Excerpt :

      In addition, OVs may incorporate anti-angiogenic chemokines such as platelet factor 4,383 or induce or encode angiostatic chemokines including members of the C-X-C motif family such as CXCL-10.384 Alternatively, endogenous angiogenesis inhibitors such as angiostatin,385 endostatin,386 vasculostatin387 or Kringle 5 of human plasminogen,388 or novel agents such as GLAF-1 (a single chain antibody targeting murine and human VEGF)389 may be incorporated into OV vectors. Furthermore, such OVs are likely to exhibit pleiotropic effects in the TME that, when combined with ACT in the form of CAR T-cell therapy may outperform direct combination with VEGF-pathway targeting agents already present in the clinic such as the anti-VEGF-A mAb bevacizumab or the anti-VEGFR2 mAb, ramucirumab.

    View all citing articles on Scopus
    View full text