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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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K1492 and K1861 form aggressive intracranial tumours and MYXV treatment results in no efficacy and minimal viral infection with no viral replication.A – Histopathology of 14 day K1492 and K1861 by H&E (first column 25×, second column 200×) and astrocytic markers S100b (200×) and GFAP (200×). 5×104 cells of K1492 (B) and K1861 (C) were intracranially implanted in C57Bl/6J mice and received 5×106 PFUs vMyx-FLuc (MYXV), UV-inactivated virus (DV), PBS, or no treatment (NT) on day 14. D – Luciferase measured (Total FLUX) from region-of-interest around the entire mouse skull following 5×106 PFUs vMyx-FLuc in K1492 (n = 8), K1861 (n = 10) or no tumour (n = 5). Error bars represent standard error. E – Viral recovery from K1492 (n = 4) and K1861 (n = 4) tumours following intracranial treatment with vMyx-FLuc. Input virus represents mice where virus was recovered 1 hour post-injection. Error bars represent standard error. F – Immunohistochemical staining for early MYXV protein MT-7e in 14 day K1492 at 1, 3 and 7 days post-treatment (Top row 25×; Bottom row 100×).
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pone-0065801-g002: K1492 and K1861 form aggressive intracranial tumours and MYXV treatment results in no efficacy and minimal viral infection with no viral replication.A – Histopathology of 14 day K1492 and K1861 by H&E (first column 25×, second column 200×) and astrocytic markers S100b (200×) and GFAP (200×). 5×104 cells of K1492 (B) and K1861 (C) were intracranially implanted in C57Bl/6J mice and received 5×106 PFUs vMyx-FLuc (MYXV), UV-inactivated virus (DV), PBS, or no treatment (NT) on day 14. D – Luciferase measured (Total FLUX) from region-of-interest around the entire mouse skull following 5×106 PFUs vMyx-FLuc in K1492 (n = 8), K1861 (n = 10) or no tumour (n = 5). Error bars represent standard error. E – Viral recovery from K1492 (n = 4) and K1861 (n = 4) tumours following intracranial treatment with vMyx-FLuc. Input virus represents mice where virus was recovered 1 hour post-injection. Error bars represent standard error. F – Immunohistochemical staining for early MYXV protein MT-7e in 14 day K1492 at 1, 3 and 7 days post-treatment (Top row 25×; Bottom row 100×).

Mentions: To establish which lines we would utilize for in vivo studies, we implanted 5.0×104 cells in the right striatum of C57Bl/6J mice. All mice succumbed to tumour burden, but varied in length of survival (Figure S1). Only K1492 and K1861 reproducibly produced intracranial tumours, whilst K1491 and K5001 both tended to produce large extracranial tumour masses with or without a corresponding intracranial tumour. K1492 and K1861 orthotopically implanted in C57Bl/6J mice produced aggressive tumours (Figure 2A). Despite having protruding tendrils into the adjacent brain parenchyma, these tumours appear to have a defined tumour border and overall lacked the single cell infiltrate pattern that can appear in astrocytic tumours. Microscopically, these tumours were composed of spindle cells arranged in bundles with a focal storiform pattern. Immunohistochemistry shows patchy expression of GFAP and S100b protein in tumour cells (Figure 2A), a feature seen in a some MG patients [32]. The histologic features and immunohistochemical profile are consistent with a gliosarcoma, a glioblastoma variant that has a similar evolution and prognosis. It is easy to speculate that this morphological change to a more mesenchymal phenotype was acquired through culture of the primary cells in serum [33]–[35], since it has been shown that the sarcomatous component in glioblastoma represents an aberrant differentiation of the glioblastoma cells [32]. As the main purpose of this study was to identify intracranial glioma models in immunocompetent C57Bl/6J mice for the pre-clinical modelling of OVs, we deemed K1492 and K1861 relevant for use in our subsequent experiments.


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)

K1492 and K1861 form aggressive intracranial tumours and MYXV treatment results in no efficacy and minimal viral infection with no viral replication.A – Histopathology of 14 day K1492 and K1861 by H&E (first column 25×, second column 200×) and astrocytic markers S100b (200×) and GFAP (200×). 5×104 cells of K1492 (B) and K1861 (C) were intracranially implanted in C57Bl/6J mice and received 5×106 PFUs vMyx-FLuc (MYXV), UV-inactivated virus (DV), PBS, or no treatment (NT) on day 14. D – Luciferase measured (Total FLUX) from region-of-interest around the entire mouse skull following 5×106 PFUs vMyx-FLuc in K1492 (n = 8), K1861 (n = 10) or no tumour (n = 5). Error bars represent standard error. E – Viral recovery from K1492 (n = 4) and K1861 (n = 4) tumours following intracranial treatment with vMyx-FLuc. Input virus represents mice where virus was recovered 1 hour post-injection. Error bars represent standard error. F – Immunohistochemical staining for early MYXV protein MT-7e in 14 day K1492 at 1, 3 and 7 days post-treatment (Top row 25×; Bottom row 100×).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675064&req=5

pone-0065801-g002: K1492 and K1861 form aggressive intracranial tumours and MYXV treatment results in no efficacy and minimal viral infection with no viral replication.A – Histopathology of 14 day K1492 and K1861 by H&E (first column 25×, second column 200×) and astrocytic markers S100b (200×) and GFAP (200×). 5×104 cells of K1492 (B) and K1861 (C) were intracranially implanted in C57Bl/6J mice and received 5×106 PFUs vMyx-FLuc (MYXV), UV-inactivated virus (DV), PBS, or no treatment (NT) on day 14. D – Luciferase measured (Total FLUX) from region-of-interest around the entire mouse skull following 5×106 PFUs vMyx-FLuc in K1492 (n = 8), K1861 (n = 10) or no tumour (n = 5). Error bars represent standard error. E – Viral recovery from K1492 (n = 4) and K1861 (n = 4) tumours following intracranial treatment with vMyx-FLuc. Input virus represents mice where virus was recovered 1 hour post-injection. Error bars represent standard error. F – Immunohistochemical staining for early MYXV protein MT-7e in 14 day K1492 at 1, 3 and 7 days post-treatment (Top row 25×; Bottom row 100×).
Mentions: To establish which lines we would utilize for in vivo studies, we implanted 5.0×104 cells in the right striatum of C57Bl/6J mice. All mice succumbed to tumour burden, but varied in length of survival (Figure S1). Only K1492 and K1861 reproducibly produced intracranial tumours, whilst K1491 and K5001 both tended to produce large extracranial tumour masses with or without a corresponding intracranial tumour. K1492 and K1861 orthotopically implanted in C57Bl/6J mice produced aggressive tumours (Figure 2A). Despite having protruding tendrils into the adjacent brain parenchyma, these tumours appear to have a defined tumour border and overall lacked the single cell infiltrate pattern that can appear in astrocytic tumours. Microscopically, these tumours were composed of spindle cells arranged in bundles with a focal storiform pattern. Immunohistochemistry shows patchy expression of GFAP and S100b protein in tumour cells (Figure 2A), a feature seen in a some MG patients [32]. The histologic features and immunohistochemical profile are consistent with a gliosarcoma, a glioblastoma variant that has a similar evolution and prognosis. It is easy to speculate that this morphological change to a more mesenchymal phenotype was acquired through culture of the primary cells in serum [33]–[35], since it has been shown that the sarcomatous component in glioblastoma represents an aberrant differentiation of the glioblastoma cells [32]. As the main purpose of this study was to identify intracranial glioma models in immunocompetent C57Bl/6J mice for the pre-clinical modelling of OVs, we deemed K1492 and K1861 relevant for use in our subsequent experiments.

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