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. 2010 Feb;17(2):281-7.
doi: 10.1038/gt.2009.132. Epub 2009 Nov 5.

Lister strain vaccinia virus, a potential therapeutic vector targeting hypoxic tumours

C T Hiley  1 , M Yuan, N R Lemoine, Y Wang
Affiliations

Lister strain vaccinia virus, a potential therapeutic vector targeting hypoxic tumours

C T Hiley et al. Gene Ther. 2010 Feb.

Abstract

Hypoxia contributes to the aggressive and treatment-resistant phenotype of pancreatic ductal adenocarcinoma. Oncolytic vaccinia virus has potential as an anti-tumour agent, but the ability to lyse hypoxic tumour cells is vital for clinical efficacy. We hypothesized that unique aspects of the poxvirus life cycle would protect it from attenuation in hypoxic conditions. We characterized and compared the viral protein production, viral replication, cytotoxicity and transgene expression of Lister strain vaccinia virus in a panel of pancreatic cancer cell lines after exposure to normoxic or hypoxic conditions. Viral protein production was not affected by hypoxia, and high viral titres were produced in both normoxic and hypoxic conditions. Interestingly, there was a 3.5-fold (P<0.001) and 20-fold (P<0.0001) increase in viral cytotoxicity for CFPac1 and MiaPaca2 cell lines, respectively, in hypoxic conditions. Cytotoxicity was equivalent in the remaining cell lines. Levels of transgene expression (luciferase reporter gene) from the vaccinia viral vector were comparable, regardless of the ambient oxygen concentration. The present study suggests that the vaccinia virus is a promising vector for targeting pancreatic cancer and potentially other hypoxic tumour types.

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Figures

Figure 1
Figure 1
Stabilisation and nuclear translocation of Hif-1α under hypoxia. Cell lines were incubated in normoxia (20% pO2) (lanes 1, 3, 5) or hypoxia (1% pO2) (lanes 2, 4, 6) for 16 hours before harvesting of nuclear extracts for immunoblotting. Lysates were probed for Hif-1α and proliferating cell nuclear antigen (PCNA) expression.
Figure 2
Figure 2
Viral gene expression of vaccinia virus in human pancreatic cancer after viral infection, and Hif-1α stabilisation and nuclear translocation in normoxia or hypoxia. A, Hypoxia does not affect viral gene expression of vaccinia virus. Cells were maintained in normoxia or hypoxia prior to and after viral infection. Cells were infected with VVLister at an MOI=1. Vaccinia virus protein was measured using an anti-vaccinia polyclonal antibody. Human PCNA was used as a loading control; B, Hif-1α stabilisation and nuclear translocation was demonstrated on nuclear lysates from MiaPaca2 cells in normoxia (20% pO2) or hypoxia (1% pO2) over a time course. β-actin was used as a loading control.
Figure 3
Figure 3
Viral replication of VVLister in normoxia (solid line) and hypoxia (dashed line) measured by TCID50 assay of viral burst assays. Cell lines were exposed to normoxia or hypoxia prior to and post infection with an MOI=1 of VVLister. Burst assay samples were collected at 24, 48, 72 and 96 hours post infection. TCID50 assays were performed on CV1 green monkey kidney cells. Experiments were performed in triplicate for each cell line, time point and condition. Results are presented as mean +/− standard deviation.
Figure 4
Figure 4
Effect of hypoxia on cytotoxicity of Vaccinia Virus Lister Strain. Pancreatic cancer cell lines were pre-treated with hypoxia or normoxia for at least 16 h, then infected with serial dilutions of VVLister and maintained under the same oxygen tension. The infected cells were assayed with MTS reagents at days 6 post infection in 37°C with 5% CO2 for 2 h. Viable cells were determined as a percentage of the non-infected controls and non linear regression analysis was used to draw dose response curves. Each assay contained six replicates and results are presented as mean +/− standard deviation of four independent experiments.
Figure 5
Figure 5
The effect of hypoxia on transgene expression from VVL15. Cells were infected with 1pfu/cell of VVL15 and luciferase activity measured at the time points indicated. All experiments were performed in triplicate and results represent the data from three separate experiments. Results are presented as mean +/− s.d. (Solid line & triangle = 20% pO2, Dashed line & triangle = 1% pO2, Solid line and square & mock infection. Light units = photons/second/cm2. * = P<0.05).
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