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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 are protected by exogenous mouse IFNβ and mouse IFNβ is non-functional on human glioma cell lines.A – NPcis cell lines infected with 1.0 MOI vMyx-GFP (top row; 100× phase/contrast, 25× GFP inlay) or 1.0 MOI MYXV-GFP pretreated with 1.0 units of mouse IFNβ (bottom row; 100× phase/contrast, 25× GFP inlay) at 48 hpi. B – Percent viability corresponding to controls (MYXV and DV to no treatment control; MYXV+IFN to IFN alone control) of NPcis cell lines at 48 hpi with 1.0 MOI vMyx-GFP or 1.0 MOI vMyx-GFP pretreated with 1.0 units mouse IFNβ as measured by Alamar blue. Error bars represent standard error and asterisks p<0.05 compared to control. C – Mouse or human IFNβ on K1492 or U87 lines which stably express an ISRE::FLUC construct. Graph is quantified from representative picture (left). Error bars represent standard deviation and asterisks p<0.05 between groups indicated by the line.
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pone-0065801-g003: NPcis cell lines are protected by exogenous mouse IFNβ and mouse IFNβ is non-functional on human glioma cell lines.A – NPcis cell lines infected with 1.0 MOI vMyx-GFP (top row; 100× phase/contrast, 25× GFP inlay) or 1.0 MOI MYXV-GFP pretreated with 1.0 units of mouse IFNβ (bottom row; 100× phase/contrast, 25× GFP inlay) at 48 hpi. B – Percent viability corresponding to controls (MYXV and DV to no treatment control; MYXV+IFN to IFN alone control) of NPcis cell lines at 48 hpi with 1.0 MOI vMyx-GFP or 1.0 MOI vMyx-GFP pretreated with 1.0 units mouse IFNβ as measured by Alamar blue. Error bars represent standard error and asterisks p<0.05 compared to control. C – Mouse or human IFNβ on K1492 or U87 lines which stably express an ISRE::FLUC construct. Graph is quantified from representative picture (left). Error bars represent standard deviation and asterisks p<0.05 between groups indicated by the line.

Mentions: We have previously shown that we can cure or significantly extended lifespan in CB17 SCID or Nude mice bearing human xenografts of conventional [8] or primary neurosphere cell cultures [28]. A preliminary experiment implanting K1492 into CB17 SCID mice showed no treatment efficacy (Figure S2C), suggesting to us that species-specific innate immune signalling could be involved in this mechanism. Type-I interferon (IFNα/β) is the quintessential antiviral cytokine [22]–[24], and we hypothesized that the production of IFNα/β, particularly by the tumour stroma, was responsible for in vivo treatment resistance. We first determined if the NPcis cell lines were protected by mIFNβ from MYXV viral infection in vitro, and found protection with as little as 1 U/mL of mIFNβ (Figure 3A, B). This demonstrated that mIFNα/β production from the tumour or stroma in syngeneic models had the potential to protect the tumour from viral infection. We confirmed that murine IFNα/β does not interact with the human IFNα/β receptors [25]–[27] in gliomas using the human U87 cell line and murine K1492 cell line (Figure 3C, S3A). This suggested to us that any IFNα/β made by the murine stroma in response to MYXV could protect the murine but not human glioma grafts, and explain the treatment discrepancy between xenografts and syngeneic models.


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 are protected by exogenous mouse IFNβ and mouse IFNβ is non-functional on human glioma cell lines.A – NPcis cell lines infected with 1.0 MOI vMyx-GFP (top row; 100× phase/contrast, 25× GFP inlay) or 1.0 MOI MYXV-GFP pretreated with 1.0 units of mouse IFNβ (bottom row; 100× phase/contrast, 25× GFP inlay) at 48 hpi. B – Percent viability corresponding to controls (MYXV and DV to no treatment control; MYXV+IFN to IFN alone control) of NPcis cell lines at 48 hpi with 1.0 MOI vMyx-GFP or 1.0 MOI vMyx-GFP pretreated with 1.0 units mouse IFNβ as measured by Alamar blue. Error bars represent standard error and asterisks p<0.05 compared to control. C – Mouse or human IFNβ on K1492 or U87 lines which stably express an ISRE::FLUC construct. Graph is quantified from representative picture (left). Error bars represent standard deviation and asterisks p<0.05 between groups indicated by the line.
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Related In: Results  -  Collection

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pone-0065801-g003: NPcis cell lines are protected by exogenous mouse IFNβ and mouse IFNβ is non-functional on human glioma cell lines.A – NPcis cell lines infected with 1.0 MOI vMyx-GFP (top row; 100× phase/contrast, 25× GFP inlay) or 1.0 MOI MYXV-GFP pretreated with 1.0 units of mouse IFNβ (bottom row; 100× phase/contrast, 25× GFP inlay) at 48 hpi. B – Percent viability corresponding to controls (MYXV and DV to no treatment control; MYXV+IFN to IFN alone control) of NPcis cell lines at 48 hpi with 1.0 MOI vMyx-GFP or 1.0 MOI vMyx-GFP pretreated with 1.0 units mouse IFNβ as measured by Alamar blue. Error bars represent standard error and asterisks p<0.05 compared to control. C – Mouse or human IFNβ on K1492 or U87 lines which stably express an ISRE::FLUC construct. Graph is quantified from representative picture (left). Error bars represent standard deviation and asterisks p<0.05 between groups indicated by the line.
Mentions: We have previously shown that we can cure or significantly extended lifespan in CB17 SCID or Nude mice bearing human xenografts of conventional [8] or primary neurosphere cell cultures [28]. A preliminary experiment implanting K1492 into CB17 SCID mice showed no treatment efficacy (Figure S2C), suggesting to us that species-specific innate immune signalling could be involved in this mechanism. Type-I interferon (IFNα/β) is the quintessential antiviral cytokine [22]–[24], and we hypothesized that the production of IFNα/β, particularly by the tumour stroma, was responsible for in vivo treatment resistance. We first determined if the NPcis cell lines were protected by mIFNβ from MYXV viral infection in vitro, and found protection with as little as 1 U/mL of mIFNβ (Figure 3A, B). This demonstrated that mIFNα/β production from the tumour or stroma in syngeneic models had the potential to protect the tumour from viral infection. We confirmed that murine IFNα/β does not interact with the human IFNα/β receptors [25]–[27] in gliomas using the human U87 cell line and murine K1492 cell line (Figure 3C, S3A). This suggested to us that any IFNα/β made by the murine stroma in response to MYXV could protect the murine but not human glioma grafts, and explain the treatment discrepancy between xenografts and syngeneic models.

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