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Microglia and astrocytes attenuate the replication of the oncolytic vaccinia virus LIVP 1.1.1 in murine GL261 gliomas by acting as vaccinia virus traps.

Kober C, Rohn S, Weibel S, Geissinger U, Chen NG, Szalay AA - J Transl Med (2015)

Bottom Line: In BV-2 and IMA 2.1 cells with M1 phenotype a further reduction of virus progeny and virus-mediated cell death was detected.By acting as VACV traps they further reduce efficient virus infection of the tumor cells.These findings demonstrate that glia cells need to be taken into account for successful GBM therapy development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Biocenter, University of Wuerzburg, Am Hubland, 97074, Würzburg, Germany. christina.kober@uni-wuerzburg.de.

ABSTRACT

Background: Oncolytic virotherapy is a novel approach for the treatment of glioblastoma multiforme (GBM) which is still a fatal disease. Pathologic features of GBM are characterized by the infiltration with microglia/macrophages and a strong interaction between immune- and glioma cells. The aim of this study was to determine the role of microglia and astrocytes for oncolytic vaccinia virus (VACV) therapy of GBM.

Methods: VACV LIVP 1.1.1 replication in C57BL/6 and Foxn1(nu/nu) mice with and without GL261 gliomas was analyzed. Furthermore, immunohistochemical analysis of microglia and astrocytes was investigated in non-, mock-, and LIVP 1.1.1-infected orthotopic GL261 gliomas in C57BL/6 mice. In cell culture studies virus replication and virus-mediated cell death of GL261 glioma cells was examined, as well as in BV-2 microglia and IMA2.1 astrocytes with M1 or M2 phenotypes. Co-culture experiments between BV-2 and GL261 cells and apoptosis/necrosis studies were performed. Organotypic slice cultures with implanted GL261 tumor spheres were used as additional cell culture system.

Results: We discovered that orthotopic GL261 gliomas upon intracranial virus delivery did not support replication of LIVP 1.1.1, similar to VACV-infected brains without gliomas. In addition, recruitment of Iba1(+) microglia and GFAP(+) astrocytes to orthotopically implanted GL261 glioma sites occurred already without virus injection. GL261 cells in culture showed high virus replication, while replication in BV-2 and IMA2.1 cells was barely detectable. The reduced viral replication in BV-2 cells might be due to rapid VACV-induced apoptotic cell death. In BV-2 and IMA 2.1 cells with M1 phenotype a further reduction of virus progeny and virus-mediated cell death was detected. Application of BV-2 microglial cells with M1 phenotype onto organotypic slice cultures with implanted GL261 gliomas resulted in reduced infection of BV-2 cells, whereas GL261 cells were well infected.

Conclusion: Our results indicate that microglia and astrocytes, dependent on their activation state, may preferentially clear viral particles by immediate uptake after delivery. By acting as VACV traps they further reduce efficient virus infection of the tumor cells. These findings demonstrate that glia cells need to be taken into account for successful GBM therapy development.

No MeSH data available.


Related in: MedlinePlus

Injection of VACV into mouse brain tissues with and without implanted gliomas. a C57BL/6 and Foxn1nu/nu mice with (w) and without (w/o) GL261 brain tumors were infected with 5 × 106 pfu LIVP 1.1.1 (i.t). For analysis of viral titers, brains were excised 1, 3 and 7 days after virus injection and analyzed by standard plaque assay [n = 4/5 per group (w) and n = 3 per group (w/o) tumor]. All samples were measured in duplicate (data are mean values ± SD; t test *p < 0.05). b Immunohistochemical staining for astrocytes (anti-GFAP, red) and VACV (anti-VACV, green) 1 dpi in GL261 implanted tumors. Shown are representative examples.
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Fig1: Injection of VACV into mouse brain tissues with and without implanted gliomas. a C57BL/6 and Foxn1nu/nu mice with (w) and without (w/o) GL261 brain tumors were infected with 5 × 106 pfu LIVP 1.1.1 (i.t). For analysis of viral titers, brains were excised 1, 3 and 7 days after virus injection and analyzed by standard plaque assay [n = 4/5 per group (w) and n = 3 per group (w/o) tumor]. All samples were measured in duplicate (data are mean values ± SD; t test *p < 0.05). b Immunohistochemical staining for astrocytes (anti-GFAP, red) and VACV (anti-VACV, green) 1 dpi in GL261 implanted tumors. Shown are representative examples.

Mentions: To analyze VACV progeny in orthotopic GL261 gliomas in vivo, two different mouse strains, immunocompetent C57BL/6 wild-type mice and immunodeficient athymic Foxn1nu/nu mice, were injected intratumorally with 5 × 106 pfu/brain LIVP 1.1.1. Mice of both strains without GL261 gliomas (w/o) were used as controls (Figure 1a). We found that the virus titers were reduced in C57BL/6 wild-type mice with and without GL261 tumors, from initially injected 5 × 106 pfu/brain to 1 × 106 pfu/g brain tissues 1 day post infection (dpi). The titer further decreased to 5 × 104 and 1 × 104 pfu/g brain tissues at 7 dpi, respectively. Strikingly, similar results were obtained in immunodeficient Foxn1nu/nu mice. Only very few LIVP 1.1.1 viral particles were detectable by immunohistochemistry within tumors at 1 dpi (Figure 1b). This data implicates that VACV LIVP 1.1.1 did not replicate efficiently in orthotopic GL261 gliomas nor did it in healthy brains of immunocompetent and immunodeficient mice. Only a very minor part of the viral inoculum remained viable within the brain for the 7 day time period.Figure 1


Microglia and astrocytes attenuate the replication of the oncolytic vaccinia virus LIVP 1.1.1 in murine GL261 gliomas by acting as vaccinia virus traps.

Kober C, Rohn S, Weibel S, Geissinger U, Chen NG, Szalay AA - J Transl Med (2015)

Injection of VACV into mouse brain tissues with and without implanted gliomas. a C57BL/6 and Foxn1nu/nu mice with (w) and without (w/o) GL261 brain tumors were infected with 5 × 106 pfu LIVP 1.1.1 (i.t). For analysis of viral titers, brains were excised 1, 3 and 7 days after virus injection and analyzed by standard plaque assay [n = 4/5 per group (w) and n = 3 per group (w/o) tumor]. All samples were measured in duplicate (data are mean values ± SD; t test *p < 0.05). b Immunohistochemical staining for astrocytes (anti-GFAP, red) and VACV (anti-VACV, green) 1 dpi in GL261 implanted tumors. Shown are representative examples.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4492094&req=5

Fig1: Injection of VACV into mouse brain tissues with and without implanted gliomas. a C57BL/6 and Foxn1nu/nu mice with (w) and without (w/o) GL261 brain tumors were infected with 5 × 106 pfu LIVP 1.1.1 (i.t). For analysis of viral titers, brains were excised 1, 3 and 7 days after virus injection and analyzed by standard plaque assay [n = 4/5 per group (w) and n = 3 per group (w/o) tumor]. All samples were measured in duplicate (data are mean values ± SD; t test *p < 0.05). b Immunohistochemical staining for astrocytes (anti-GFAP, red) and VACV (anti-VACV, green) 1 dpi in GL261 implanted tumors. Shown are representative examples.
Mentions: To analyze VACV progeny in orthotopic GL261 gliomas in vivo, two different mouse strains, immunocompetent C57BL/6 wild-type mice and immunodeficient athymic Foxn1nu/nu mice, were injected intratumorally with 5 × 106 pfu/brain LIVP 1.1.1. Mice of both strains without GL261 gliomas (w/o) were used as controls (Figure 1a). We found that the virus titers were reduced in C57BL/6 wild-type mice with and without GL261 tumors, from initially injected 5 × 106 pfu/brain to 1 × 106 pfu/g brain tissues 1 day post infection (dpi). The titer further decreased to 5 × 104 and 1 × 104 pfu/g brain tissues at 7 dpi, respectively. Strikingly, similar results were obtained in immunodeficient Foxn1nu/nu mice. Only very few LIVP 1.1.1 viral particles were detectable by immunohistochemistry within tumors at 1 dpi (Figure 1b). This data implicates that VACV LIVP 1.1.1 did not replicate efficiently in orthotopic GL261 gliomas nor did it in healthy brains of immunocompetent and immunodeficient mice. Only a very minor part of the viral inoculum remained viable within the brain for the 7 day time period.Figure 1

Bottom Line: In BV-2 and IMA 2.1 cells with M1 phenotype a further reduction of virus progeny and virus-mediated cell death was detected.By acting as VACV traps they further reduce efficient virus infection of the tumor cells.These findings demonstrate that glia cells need to be taken into account for successful GBM therapy development.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Biocenter, University of Wuerzburg, Am Hubland, 97074, Würzburg, Germany. christina.kober@uni-wuerzburg.de.

ABSTRACT

Background: Oncolytic virotherapy is a novel approach for the treatment of glioblastoma multiforme (GBM) which is still a fatal disease. Pathologic features of GBM are characterized by the infiltration with microglia/macrophages and a strong interaction between immune- and glioma cells. The aim of this study was to determine the role of microglia and astrocytes for oncolytic vaccinia virus (VACV) therapy of GBM.

Methods: VACV LIVP 1.1.1 replication in C57BL/6 and Foxn1(nu/nu) mice with and without GL261 gliomas was analyzed. Furthermore, immunohistochemical analysis of microglia and astrocytes was investigated in non-, mock-, and LIVP 1.1.1-infected orthotopic GL261 gliomas in C57BL/6 mice. In cell culture studies virus replication and virus-mediated cell death of GL261 glioma cells was examined, as well as in BV-2 microglia and IMA2.1 astrocytes with M1 or M2 phenotypes. Co-culture experiments between BV-2 and GL261 cells and apoptosis/necrosis studies were performed. Organotypic slice cultures with implanted GL261 tumor spheres were used as additional cell culture system.

Results: We discovered that orthotopic GL261 gliomas upon intracranial virus delivery did not support replication of LIVP 1.1.1, similar to VACV-infected brains without gliomas. In addition, recruitment of Iba1(+) microglia and GFAP(+) astrocytes to orthotopically implanted GL261 glioma sites occurred already without virus injection. GL261 cells in culture showed high virus replication, while replication in BV-2 and IMA2.1 cells was barely detectable. The reduced viral replication in BV-2 cells might be due to rapid VACV-induced apoptotic cell death. In BV-2 and IMA 2.1 cells with M1 phenotype a further reduction of virus progeny and virus-mediated cell death was detected. Application of BV-2 microglial cells with M1 phenotype onto organotypic slice cultures with implanted GL261 gliomas resulted in reduced infection of BV-2 cells, whereas GL261 cells were well infected.

Conclusion: Our results indicate that microglia and astrocytes, dependent on their activation state, may preferentially clear viral particles by immediate uptake after delivery. By acting as VACV traps they further reduce efficient virus infection of the tumor cells. These findings demonstrate that glia cells need to be taken into account for successful GBM therapy development.

No MeSH data available.


Related in: MedlinePlus