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Resistance to oncolytic myxoma virus therapy in nf1(-/-)/trp53(-/-) syngeneic mouse glioma models is independent of anti-viral type-I interferon.

Zemp FJ, McKenzie BA, Lun X, Maxwell L, Reilly KM, McFadden G, Yong VW, Forsyth PA - PLoS ONE (2013)

Bottom Line: Intracranial injection of MYXV failed to result in sustained viral replication or treatment efficacy, with minimal tumour infection that was completely resolved by 7 days post-infection.We hypothesized that the stromal production of Type-I interferons (IFNα/β) could explain the resistance seen in these models; however, we found that neither the cell lines in vitro nor the tumours in vivo produce any IFNα/β in response to MYXV infection.To confirm IFNα/β did not play a role in this resistance, we ablated the ability of tumours to respond to IFNα/β via IRF9 knockdown, and generated identical results.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Clark H. Smith Brain Tumor Center, University of Calgary, Tom Baker Cancer Centre, Southern Alberta Cancer Research Institute, Calgary, Alberta, Canada.

ABSTRACT
Despite promising preclinical studies, oncolytic viral therapy for malignant gliomas has resulted in variable, but underwhelming results in clinical evaluations. Of concern are the low levels of tumour infection and viral replication within the tumour. This discrepancy between the laboratory and the clinic could result from the disparity of xenograft versus syngeneic models in determining in vivo viral infection, replication and treatment efficacy. Here we describe a panel of primary mouse glioma lines derived from Nf1 (+/-) Trp53 (+/-) mice in the C57Bl/6J background for use in the preclinical testing of the oncolytic virus Myxoma (MYXV). These lines show a range of susceptibility to MYXV replication in vitro, but all succumb to viral-mediated cell death. Two of these lines orthotopically grafted produced aggressive gliomas. Intracranial injection of MYXV failed to result in sustained viral replication or treatment efficacy, with minimal tumour infection that was completely resolved by 7 days post-infection. We hypothesized that the stromal production of Type-I interferons (IFNα/β) could explain the resistance seen in these models; however, we found that neither the cell lines in vitro nor the tumours in vivo produce any IFNα/β in response to MYXV infection. To confirm IFNα/β did not play a role in this resistance, we ablated the ability of tumours to respond to IFNα/β via IRF9 knockdown, and generated identical results. Our studies demonstrate that these syngeneic cell lines are relevant preclinical models for testing experimental glioma treatments, and show that IFNα/β is not responsible for the MYXV treatment resistance seen in syngeneic glioma models.

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NPcis cell lines have variable susceptibility to MYXV infection and MYXV-mediated cell death in vitro.A – NPcis cell lines infected with 1.0 MOI vMyx-GFP (bottom row; 100× phase/contrast, 25× GFP inlay) or control (top row; 100× phase/contrast) at 48 hrs post-infection. B – Percent viability of NPcis cell lines at 48 hpi with vMyx-GFP as measured by Alamar blue. C – Viral recovery titred on BGMK cells from infected NPcis cell lines at 48 and 72 hpi. Input virus is vMyx-GFP seeded in wells with no cells. Error bars represent standard error and n = 4 for both experiments.
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pone-0065801-g001: NPcis cell lines have variable susceptibility to MYXV infection and MYXV-mediated cell death in vitro.A – NPcis cell lines infected with 1.0 MOI vMyx-GFP (bottom row; 100× phase/contrast, 25× GFP inlay) or control (top row; 100× phase/contrast) at 48 hrs post-infection. B – Percent viability of NPcis cell lines at 48 hpi with vMyx-GFP as measured by Alamar blue. C – Viral recovery titred on BGMK cells from infected NPcis cell lines at 48 and 72 hpi. Input virus is vMyx-GFP seeded in wells with no cells. Error bars represent standard error and n = 4 for both experiments.

Mentions: NPcis cell lines K1491, K1492, K1861 and K5001 were assayed for cell viability and viral replication after MYXV treatment in vitro. Despite these tumours being derived from the same genetic background and having the same TP53 and NF1 driving mutations [14], [15], [17], we found a large variability in MYXV infection and replication (Figure 1). K1492 and K1492 provided the most robust viral replication, producing high titres of 6.1×107 and 1.8×108 at 72 hour-post-infection (hpi). These lines were also the most sensitive to viral treatment, with 1 MOI resulting in 25% and 38% viability at 48 hpi, respectively. Conversely, K1861 was the most resistant to cell death with 74% viability at 1 MOI at 48 hpi, but was killed by the virus at higher MOIs (21% viability with 10MOI, 48 hpi). K5001 had intermediate sensitivity to viral mediated cell death. These more resistant cell lines markedly differed in their ability to replicate the virus, with K1861 and K5001 unable to reach the high titres seen in the other lines, but still able to produce functional virions following infection.


Resistance to oncolytic myxoma virus therapy in nf1(-/-)/trp53(-/-) syngeneic mouse glioma models is independent of anti-viral type-I interferon.

Zemp FJ, McKenzie BA, Lun X, Maxwell L, Reilly KM, McFadden G, Yong VW, Forsyth PA - PLoS ONE (2013)

NPcis cell lines have variable susceptibility to MYXV infection and MYXV-mediated cell death in vitro.A – NPcis cell lines infected with 1.0 MOI vMyx-GFP (bottom row; 100× phase/contrast, 25× GFP inlay) or control (top row; 100× phase/contrast) at 48 hrs post-infection. B – Percent viability of NPcis cell lines at 48 hpi with vMyx-GFP as measured by Alamar blue. C – Viral recovery titred on BGMK cells from infected NPcis cell lines at 48 and 72 hpi. Input virus is vMyx-GFP seeded in wells with no cells. Error bars represent standard error and n = 4 for both experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675064&req=5

pone-0065801-g001: NPcis cell lines have variable susceptibility to MYXV infection and MYXV-mediated cell death in vitro.A – NPcis cell lines infected with 1.0 MOI vMyx-GFP (bottom row; 100× phase/contrast, 25× GFP inlay) or control (top row; 100× phase/contrast) at 48 hrs post-infection. B – Percent viability of NPcis cell lines at 48 hpi with vMyx-GFP as measured by Alamar blue. C – Viral recovery titred on BGMK cells from infected NPcis cell lines at 48 and 72 hpi. Input virus is vMyx-GFP seeded in wells with no cells. Error bars represent standard error and n = 4 for both experiments.
Mentions: NPcis cell lines K1491, K1492, K1861 and K5001 were assayed for cell viability and viral replication after MYXV treatment in vitro. Despite these tumours being derived from the same genetic background and having the same TP53 and NF1 driving mutations [14], [15], [17], we found a large variability in MYXV infection and replication (Figure 1). K1492 and K1492 provided the most robust viral replication, producing high titres of 6.1×107 and 1.8×108 at 72 hour-post-infection (hpi). These lines were also the most sensitive to viral treatment, with 1 MOI resulting in 25% and 38% viability at 48 hpi, respectively. Conversely, K1861 was the most resistant to cell death with 74% viability at 1 MOI at 48 hpi, but was killed by the virus at higher MOIs (21% viability with 10MOI, 48 hpi). K5001 had intermediate sensitivity to viral mediated cell death. These more resistant cell lines markedly differed in their ability to replicate the virus, with K1861 and K5001 unable to reach the high titres seen in the other lines, but still able to produce functional virions following infection.

Bottom Line: Intracranial injection of MYXV failed to result in sustained viral replication or treatment efficacy, with minimal tumour infection that was completely resolved by 7 days post-infection.We hypothesized that the stromal production of Type-I interferons (IFNα/β) could explain the resistance seen in these models; however, we found that neither the cell lines in vitro nor the tumours in vivo produce any IFNα/β in response to MYXV infection.To confirm IFNα/β did not play a role in this resistance, we ablated the ability of tumours to respond to IFNα/β via IRF9 knockdown, and generated identical results.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Clark H. Smith Brain Tumor Center, University of Calgary, Tom Baker Cancer Centre, Southern Alberta Cancer Research Institute, Calgary, Alberta, Canada.

ABSTRACT
Despite promising preclinical studies, oncolytic viral therapy for malignant gliomas has resulted in variable, but underwhelming results in clinical evaluations. Of concern are the low levels of tumour infection and viral replication within the tumour. This discrepancy between the laboratory and the clinic could result from the disparity of xenograft versus syngeneic models in determining in vivo viral infection, replication and treatment efficacy. Here we describe a panel of primary mouse glioma lines derived from Nf1 (+/-) Trp53 (+/-) mice in the C57Bl/6J background for use in the preclinical testing of the oncolytic virus Myxoma (MYXV). These lines show a range of susceptibility to MYXV replication in vitro, but all succumb to viral-mediated cell death. Two of these lines orthotopically grafted produced aggressive gliomas. Intracranial injection of MYXV failed to result in sustained viral replication or treatment efficacy, with minimal tumour infection that was completely resolved by 7 days post-infection. We hypothesized that the stromal production of Type-I interferons (IFNα/β) could explain the resistance seen in these models; however, we found that neither the cell lines in vitro nor the tumours in vivo produce any IFNα/β in response to MYXV infection. To confirm IFNα/β did not play a role in this resistance, we ablated the ability of tumours to respond to IFNα/β via IRF9 knockdown, and generated identical results. Our studies demonstrate that these syngeneic cell lines are relevant preclinical models for testing experimental glioma treatments, and show that IFNα/β is not responsible for the MYXV treatment resistance seen in syngeneic glioma models.

Show MeSH
Related in: MedlinePlus