Oncolytic measles virus strains have significant antitumor activity against glioma stem cells

Abstract

Glioblastoma (GBM) is the most common primary brain tumor in adults and has a dismal prognosis despite multimodality treatment. Given the resistance of glioma stem cells (GSC) to chemotherapy and radiation therapy, their eradication could prevent tumor recurrence. We sought to evaluate the antitumor activity of measles virus (MV) derivatives against GSC. We generated neurosphere cultures from patient-derived primary tumor GBM xenografts, and we characterized them for the GSC markers CD133, SOX2, Nestin, ATF5 and OLIG2. Using the MV-strains MV-GFP, MV-CEA and MV-NIS we demonstrated infection, viral replication and significant cytopathic effect in vitro against GSC lines. In tumorigenicity experiments, GBM44 GSC were infected with MV in vitro and subsequently implanted into the right caudate nucleus of nude mice: significant prolongation of survival in mice implanted with infected GSC was observed, compared with mock-infected controls (P=0.0483). In therapy experiments in GBM6 and GBM12 GSC xenograft models, there was significant prolongation of survival in MV-GFP-treated animals compared with inactivated virus-treated controls (GBM6 P=0.0021, GBM12 P=0.0416). Abundant syncytia and viral replication was demonstrated in tumors of MV-treated mice. Measles virus derivatives have significant antitumor activity against glioma-derived stem cells in vitro and in vivo.

Figure 1

Schematic representation of the viral strains: MV-CEA: MV strain expressing the human carcinoembryonic antigen; MV-NIS: MV strain expressing the sodium iodine symporter. N, nucleoprotein gene; MV-GFP: MV strain expressing the enhanced Green Fluorescent Protein; L, large protein gene; P, phosphoprotein gene; M, matrix protein gene; F, fusion protein gene; H, hemagglutinin gene; CEA, human carcinoembryonic antigen gene; NIS, sodium iodine symporter gene.

Figure 2

Neurospheres derived from GSC xenograft cultures express stem-cell markers (CD133, Nestin, SOX2, ATF5 and OLIG2). Representative data for GBM6, GBM12 and GBM38 glioma stem cell lines are shown.

Figure 3

Expression of measles virus receptor CD46 was examined by IHC and flow cytometry. Significant expression of CD46 was observed in all neurosphere lines tested. Representative ICH and FACS data in GBM34 and GBM22 GSC lines are presented.

Figure 4

A Infection with MV-GFP resulted in abundant GFP expression in infected GBM12 neurospheres. B, C, D: The cytopathic effect of MV-GFP, MV-NIS and MV-CEA was assessed in GSC neurosphere lines using trypan blue exclusion assays. Viral MOIs of 0.1 and 1 were used. Results are presented as percentage of uninfected controls. Representative results are shown for GBM 38 (B), GBM10 (C) and GBM12 (D). There was MOI dependent cytotoxicity with almost complete eradication of neurosphere cultures by day 6 post infection.

Figure 5

Viral replication was assessed by one-step viral growth curves. Representative results in GSC deriving from GBM 6, 10 and 12 are shown. Cells were infected with MV-NIS, MV-GFP or MV-CEA at an MOI of 1, harvested at different time points and titered in Vero cells. Results are expressed as TCID50 of harvested virus. All glioma stem cell lines effectively supported viral replication.

Figure 6

GBM44 GSC were either infected with MV-NIS (MOI 10) or mock-infected, and 3×10⁵ GSC cells were implanted into the right caudate nucleus of nude mice. Animals were followed for survival. There was significant prolongation of survival in mice which were implanted with cells infected with MV-NIS (p=0.04) with median survival of 68 days in the control group versus not having been reached at 100 days in mice implanted with infected GSC.

Figure 7

GBM6 (A) or GBM12 (B) derived GSC were orthotopically implanted into the right caudate nucleus of nude mice. Animals were treated with a total dose of 1.8×10⁶or 9×10⁵ TCID50 MV-GFP, respectively, or UV inactivated virus. In both orthotopic treatment models, significant prolongation in survival was observed (p=0.0021 and p=0.0416 respectively). Abundant syncytia were observed in MV-GFP treated mice in H and E stain (C, E). Syncytia expressed the stem cell marker CD133 (D). Viral replication was confirmed by in situ hybridization for measles virus N mRNA (F).