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. 2007 Feb;81(3):1479-91.
doi: 10.1128/JVI.01861-06. Epub 2006 Nov 15.

Variable deficiencies in the interferon response enhance susceptibility to vesicular stomatitis virus oncolytic actions in glioblastoma cells but not in normal human glial cells

Guido Wollmann  1 , Michael D Robek, Anthony N van den Pol
Affiliations

Variable deficiencies in the interferon response enhance susceptibility to vesicular stomatitis virus oncolytic actions in glioblastoma cells but not in normal human glial cells

Guido Wollmann et al. J Virol. 2007 Feb.

Abstract

With little improvement in the poor prognosis for humans with high-grade glioma brain tumors, alternative therapeutic strategies are needed. As such, selective replication-competent oncolytic viruses may be useful as a potential treatment modality. Here we test the hypothesis that defects in the interferon (IFN) pathway could be exploited to enhance the selective oncolytic profile of vesicular stomatitis virus (VSV) in glioblastoma cells. Two green fluorescent protein-expressing VSV strains, recombinant VSV and the glioma-adapted recombinant VSV-rp30a, were used to study infection of a variety of human glioblastoma cell lines compared to a panel of control cells, including normal human astrocytes, oligodendrocyte precursor cells, and primary explant cultures from human brain tissue. Infection rate, cell viability, viral replication, and IFN-alpha/beta-related gene expression were compared in the absence and presence of IFN-alpha or polyriboinosinic polyribocytidylic acid [poly(I:C)], a synthetic inducer of the IFN-alpha/beta pathway. Both VSV strains caused rapid and total infection and death of all tumor cell lines tested. To a lesser degree, normal cells were also subject to VSV infection. In contrast, IFN-alpha or poly(I:C) completely attenuated the infection of all primary control brain cells, whereas most glioblastoma cell lines treated with IFN-alpha or poly(I:C) showed little or no sign of protection and were killed by VSV. Together, our results demonstrate that activation of the interferon pathway protects normal human brain cells from VSV infection while maintaining the vulnerability of human glioblastoma cells to viral destruction.

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Figures

FIG. 1.
FIG. 1.
VSV action on four glioblastoma cell lines. Four different glioblastoma cultures were infected with VSV-rp30a at an MOI of 5 in the presence of IFN-α (100 IU/ml) or poly(I:C) (PIC) (20 μg/ml). Expression of GFP was used to monitor viral infection; EthD-1 was used to assess virus induced cell death. Representative photomicrographs were taken at 24 h postinfection (U-87 and U-118) or at 48 h postinfection (U-373 and A-172). The presence of IFN or PIC had only a modest impact on the oncolytic action of VSV-rp30a.
FIG. 2.
FIG. 2.
IFN and poly(I:C) protect normal glial cells, but not glioblastoma cells, from VSV infection. A mixed culture of four glioblastoma cell lines (see Fig. 1) and normal human astrocytes were infected with VSV-rp30a at an MOI of 5. Overnight pretreatment with IFN-α (100 IU/ml) or poly(I:C) (PIC) (20 μg/ml) did not protect tumor cells but did protect control cells from VSV infection and lysis. Viral infection was monitored by expression of GFP, and cell death was assessed by an ethidium homodimer assay. Panel displays representative images taken 24 h (tumor cultures) and 48 h (control cultures) after viral inoculation.
FIG. 3.
FIG. 3.
Effect of IFN on VSV infection. Using an MTT cell viability assay, the effect of IFN pretreatment on the action of VSV on U-87 cells and normal human astrocytes was assessed. IFN had little effect on glioblastoma cell destruction by VSV but completely protected normal astrocytes. Low (MOI, 0.1) and high (MOI, 5) concentrations of VSV and VSV-rp30a were used. Note the slightly stronger oncolysis by VSV-rp30a. Error bars indicate standard errors of the means from triplicate experiments. hpi, hours postinfection.
FIG. 4.
FIG. 4.
Effect of poly(I:C) on VSV infection. Using an MTT cell viability assay, the effect of poly(I:C) pretreatment in several concentrations was assessed. U-87 glioblastoma cells were effectively killed by virus in the absence or presence of poly(I:C). Note the slightly stronger oncolysis by VSV-rp30a. Noteworthy is that in the presence of poly(I:C), VSV action on U-87 cells was even stronger than in nonpretreated cultures, suggesting a synergistic effect of poly(I:C) with VSV/VSV-rp30a. In contrast, normal human astrocytes were completely protected from VSV infection. hpi, hours postinfection.
FIG. 5.
FIG. 5.
Effect of IFN-α and poly(I:C) on viral replication. Viral replication was assessed using plaque assays. U-87 glioblastoma cultures and normal human astrocytes were infected at an MOI of 1, washed, and incubated. Supernatant was collected 24 h later. In IFN-α- and poly(I:C) (PIC)-pretreated astrocyte cultures, no viral progeny could be detected. In contrast, viral replication in U-87 cells was mildly attenuated by IFN and unaffected by poly(I:C).
FIG. 6.
FIG. 6.
IFN protection of human brain tissue explant cultures and human oligodendrocyte precursor cells. Primary explant cultures from postsurgery human brain tissue (upper set) and human oligodendrocyte precursor cells (lower set) were infected with VSV-rp30a in the presence or absence of IFN-α. Normal morphology was seen in noninfected control cultures. The primary brain culture used here corresponds to control sample C in Fig. 7 and 8.
FIG. 7.
FIG. 7.
Differences in interferon-related gene expression upon VSV infection in glioblastoma and control brain cells. Cultures of five different human glioblastoma cell lines and three control cultures derived from postsurgery human brain tissue were either infected with VSV-rp30a (MOI of 2) or treated with human IFN-α (100 IU/ml) or poly(I:C) (25 μg/ml) for 6 h. Gene expression studies using quantitative RT-PCR revealed marked differences between glioblastoma and control cells for MxA (A) and IFN-β (B) but not for ISG15 (C). Data are expressed as fold induction relative to control untreated cultures. The level of expression was different for each tested gene (y axes have different scales). Expression levels are normalized to the cellular GAPDH gene and are presented as means of triplicates with standard errors of the means. n.d., none detected.
FIG. 8.
FIG. 8.
Complete protection of normal human brain cultures from VSV-rp30 infection by IFN-α and poly(I:C). The same set of cells used for the gene expression studies (see Fig. 7) was analyzed for cell viability after VSV-rp30a infection (MOI of 5) in the presence or absence of IFN-α and poly(I:C) over a 3-day time course. (A) IFN-α and poly(I:C) offer complete protection for all three control brain cultures. Even without pretreatment, viability was significantly higher after VSV-rp30a infection than in glioblastoma cells. (B) In contrast, all five glioblastoma cell lines were nearly completely killed by VSV-rp30a, and only one cell line showed a moderate response to protective IFN-α pretreatment. There was no protection with poly(I:C). Data represent means of triplicates and standard errors of the means.
FIG. 9.
FIG. 9.
Time-lapse recording of VSV-rp30a selectively infecting and killing glioblastoma cells in coculture with normal human astrocytes. Glioblastoma U-87 cells were stably transfected to express RFP and cocultured with normal human astrocytes. Cultures were pretreated with IFN-α (100 IU/ml) overnight. VSV-rp30a was added at an MOI of 5. The panel displays two representative experiments, one in control conditions without VSV (left) and one with addition of GFP-expressing VSV-rp30a (right). The center scale illustrates the increase of red glioblastoma cell number in control conditions and decrease in the virus-infected culture. Pictures for time-lapse recording were automatically taken every 6 min over a period of 50 h. For the displayed panels, every 100th frame was used, corresponding to 10-hour intervals.
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