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

Abstract

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 serumfree 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 g34.5 significantly attenuated the vectors due to poor replication. However, this was significantly reversed by the additional deletion of a47. Infection with oHSV G47# (ICP6À ) not only killed GBMSCs 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 G47# 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. [Cancer Res 2009;69(8):3472-81]

Conversion of differentiated cancer cells into cancer stem-like cells in a glioblastoma model after primary chemotherapy

“…Most importantly, hematoxylin and eosin (H&E) analysis of DN glioma xenografts previously treated with TMZ showed a significantly increased number of invasive tumor foci in the DN non-GSC group treated with TMZ as compared with the DMSO-treated control (Po0.05). Such a property was very similar to DP GSC-implanted tumors that demonstrated invasive properties with or without TMZ treatment, a previously established characteristic of CSCs 25,26 (Figures 6d and f). The effects of TMZ on GSC frequency and malignant invasion can also be observed in Figure 6e Figure S6B) show an increase in both number and colocalization of GSC markers (CD133 and Sox2) and a decrease in differentiation markers (GFAP) in tumors treated with TMZ.…”

Girdin maintains the stemness of glioblastoma stem cells

“…Although the mechanisms underlying brain-tumor invasion have not been fully elucidated, the PI3K-Akt axis has been recognized as a contributor. CD133 þ cells also contribute to invasion and migration (Eyler et al, 2008;Gunther et al, 2008;Wakimoto et al, 2009). GBM stem cell transplantation generates tumors that phenocopy the infiltrative features of the parental human GBM tumor (Singh et al, 2004;Lee et al, 2006;Yuki et al, 2009).…”

Multiplexed RNAi therapy against brain tumor-initiating cells via lipopolymeric nanoparticle infusion delays glioblastoma progression

“…A panel of patient-derived GBM cells (GBM6, GBM12, GBM26, and GBM43, a gift from C. David James, Northwestern University; MGG8, a gift from M.L.S., Massachusetts General Hospital/Harvard Medical School; and MES83, a gift from Ichiro Nakano, University of Alabama at Birmingham and Shi-Yuan Cheng, Northwestern University) were maintained as neurospheres in serum-free Neurobasal or DMEM F12 medium with B27 and N2 supplements (Thermo Fisher Scientific), broad-spectrum antibiotics, L-glutamine, heparin (Millipore Sigma), EGF, and basic fibroblast growth factor (R&D Systems) (24,25,36).…”

“…To establish a suitable therapeutic target model using patient-derived GBM BTICs, we cultured GBM cell lines in serum-free Neurobasal medium supplemented for tumorsphere growth conditions (24,25,36). Cells then were collected for Western blotting and qRT-PCR analysis of the expression levels of four core TFs (for primer sequences see SI Appendix, Table S1).…”