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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

Recruitment and infiltration of microglia and astrocytes into murine gliomas. a, b Recruitment of microglia and astrocytes 1 and 3 days after intracranial injection of GLV-2b372 into naïve Foxn1nu/nu mice stained with Hoechst (blue) to detect cell nuclei, IB4 (green) to stain microglia and GFAP to stain astrocytes (green). Virus was detected by expression of TurboFP635 (red). The VACV injection sites are marked by white arrows in the overlays. Immunohistochemical staining and analysis of GL261 gliomas in C57BL/6 mice untreated (w/o; c, f), injected intratumorally with PBS (d, g) or LIVP 1.1.1 (5 × 106 pfu) (e, h) at 1 dpi. Sections were stained for cell nuclei (blue), microglia (Iba-1+, red) and astrocytes (GFAP+, green). i, j Quantitative analysis of microglia and astrocytes 1 and 7 dpi (n = 3; analyzed in duplicate). Fluorescence intensity and tumor area were measured with ImageJ and calculated as ratio: Iba-1 or GFAP/tumor area. For box plot diagrams a template from Vertex42 LLC has been used.
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Fig2: Recruitment and infiltration of microglia and astrocytes into murine gliomas. a, b Recruitment of microglia and astrocytes 1 and 3 days after intracranial injection of GLV-2b372 into naïve Foxn1nu/nu mice stained with Hoechst (blue) to detect cell nuclei, IB4 (green) to stain microglia and GFAP to stain astrocytes (green). Virus was detected by expression of TurboFP635 (red). The VACV injection sites are marked by white arrows in the overlays. Immunohistochemical staining and analysis of GL261 gliomas in C57BL/6 mice untreated (w/o; c, f), injected intratumorally with PBS (d, g) or LIVP 1.1.1 (5 × 106 pfu) (e, h) at 1 dpi. Sections were stained for cell nuclei (blue), microglia (Iba-1+, red) and astrocytes (GFAP+, green). i, j Quantitative analysis of microglia and astrocytes 1 and 7 dpi (n = 3; analyzed in duplicate). Fluorescence intensity and tumor area were measured with ImageJ and calculated as ratio: Iba-1 or GFAP/tumor area. For box plot diagrams a template from Vertex42 LLC has been used.

Mentions: In a further step, the virus injection site in athymic Foxn1 nu/nu mice without brain tumors was analyzed by immunohistochemical stainings of brain sections 1 and 3 days post infection (dpi). For infection 5 × 106 pfu of the red fluorescent VACV strain GLV-2b372 was injected intracranially. A strong infiltration of immune cells to the injection site could be detected (Figure 2a, b): microglial cells stained with the microglial marker Iba-1 were already recruited 1 day after virus injection, whereas astrocytes stained with astrocytic marker GFAP were present after 3 days. VACV replication detected by expression of TurboFP635 was visible at the injection site (Figure 2a, b) but did not spread further through tissues.Figure 2


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)

Recruitment and infiltration of microglia and astrocytes into murine gliomas. a, b Recruitment of microglia and astrocytes 1 and 3 days after intracranial injection of GLV-2b372 into naïve Foxn1nu/nu mice stained with Hoechst (blue) to detect cell nuclei, IB4 (green) to stain microglia and GFAP to stain astrocytes (green). Virus was detected by expression of TurboFP635 (red). The VACV injection sites are marked by white arrows in the overlays. Immunohistochemical staining and analysis of GL261 gliomas in C57BL/6 mice untreated (w/o; c, f), injected intratumorally with PBS (d, g) or LIVP 1.1.1 (5 × 106 pfu) (e, h) at 1 dpi. Sections were stained for cell nuclei (blue), microglia (Iba-1+, red) and astrocytes (GFAP+, green). i, j Quantitative analysis of microglia and astrocytes 1 and 7 dpi (n = 3; analyzed in duplicate). Fluorescence intensity and tumor area were measured with ImageJ and calculated as ratio: Iba-1 or GFAP/tumor area. For box plot diagrams a template from Vertex42 LLC has been used.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Recruitment and infiltration of microglia and astrocytes into murine gliomas. a, b Recruitment of microglia and astrocytes 1 and 3 days after intracranial injection of GLV-2b372 into naïve Foxn1nu/nu mice stained with Hoechst (blue) to detect cell nuclei, IB4 (green) to stain microglia and GFAP to stain astrocytes (green). Virus was detected by expression of TurboFP635 (red). The VACV injection sites are marked by white arrows in the overlays. Immunohistochemical staining and analysis of GL261 gliomas in C57BL/6 mice untreated (w/o; c, f), injected intratumorally with PBS (d, g) or LIVP 1.1.1 (5 × 106 pfu) (e, h) at 1 dpi. Sections were stained for cell nuclei (blue), microglia (Iba-1+, red) and astrocytes (GFAP+, green). i, j Quantitative analysis of microglia and astrocytes 1 and 7 dpi (n = 3; analyzed in duplicate). Fluorescence intensity and tumor area were measured with ImageJ and calculated as ratio: Iba-1 or GFAP/tumor area. For box plot diagrams a template from Vertex42 LLC has been used.
Mentions: In a further step, the virus injection site in athymic Foxn1 nu/nu mice without brain tumors was analyzed by immunohistochemical stainings of brain sections 1 and 3 days post infection (dpi). For infection 5 × 106 pfu of the red fluorescent VACV strain GLV-2b372 was injected intracranially. A strong infiltration of immune cells to the injection site could be detected (Figure 2a, b): microglial cells stained with the microglial marker Iba-1 were already recruited 1 day after virus injection, whereas astrocytes stained with astrocytic marker GFAP were present after 3 days. VACV replication detected by expression of TurboFP635 was visible at the injection site (Figure 2a, b) but did not spread further through tissues.Figure 2

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