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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

Analysis of VACV infections in murine GL261, microglial BV-2 and astrocytic IMA2.1 cells in cell culture. a–c Viral replication in GL261, BV-2 and IMA2.1 cells infected with LIVP 1.1.1 at an MOI of 0.1 was analyzed by standard plaque assay. The red line separates active replication from no replication. d–f MTT assay was performed to detect the percentage of surviving cells after infection with LIVP 1.1.1 (MOI 1.0). g BV-2 and GL261 cells were cultured as direct co-cultures for 24 h in various concentrations. For standard plaque assay cells were infected at an MOI of 0.1 in triplicate. h Viral titers were related to the infection medium for a better comparison and illustrated as ratio (total virus titer/infection medium). Experiments were repeated in independent experiments. Two-sided t test with unequal variances was used for statistics (*p < 0.05).
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Fig3: Analysis of VACV infections in murine GL261, microglial BV-2 and astrocytic IMA2.1 cells in cell culture. a–c Viral replication in GL261, BV-2 and IMA2.1 cells infected with LIVP 1.1.1 at an MOI of 0.1 was analyzed by standard plaque assay. The red line separates active replication from no replication. d–f MTT assay was performed to detect the percentage of surviving cells after infection with LIVP 1.1.1 (MOI 1.0). g BV-2 and GL261 cells were cultured as direct co-cultures for 24 h in various concentrations. For standard plaque assay cells were infected at an MOI of 0.1 in triplicate. h Viral titers were related to the infection medium for a better comparison and illustrated as ratio (total virus titer/infection medium). Experiments were repeated in independent experiments. Two-sided t test with unequal variances was used for statistics (*p < 0.05).

Mentions: One explanation of reduced VACV replication in the mouse brain could be the preferential uptake of viral particles into microglia or astrocytes. We tested this hypothesis in cell culture, using two specific cell lines, BV-2 and IMA2.1, with similar growth characteristics as primary cells. To determine LIVP 1.1.1 virus progeny in these cell lines in vitro, cells were infected at an MOI of 0.1 followed by standard plaque assay performed at different time points. Data was compared with infection of GL261 glioma cells under the same culture conditions (Figure 3a–c). We confirmed that LIVP 1.1.1 replicated efficiently in GL261 cells, determined by increasing cell-associated virus titers from 2 to 48 h post infection (hpi) and a plateau phase reached at 48 hpi at a virus dose of 1.5 × 106 ± 2.1 × 105 pfu/ml. Already, 24 hpi virus titers were higher than the infection medium (1.6 × 104 ± 2.3 × 103 pfu/ml). Virus titers in the supernatant steadily increased over time, reaching a maximum at 96 hpi (2.9 × 105 ± 5.1 × 104 pfu/ml) (Figure 3a). In contrast, the virus titer in BV-2 cells remained slightly below the infection medium at all times that were analyzed except for the 48 hpi time point (Figure 3b). Surprisingly, in IMA2.1 cells, the virus titer was far below the infection medium during the whole time course of the experiment (Figure 3c). No virus spreading occurred as indicated by the complete lack of viral particles in the supernatant of BV-2 and IMA2.1 infected cells. Cell viability after virus infection was examined by MTT assay at an MOI of 1.0. The analysis showed that GL261 and BV-2 cells were lysed upon infection with LIVP 1.1.1, whereas IMA2.1 cells were intact with a slight decrease in the amount of living cells (Figure 3d–f).Figure 3


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)

Analysis of VACV infections in murine GL261, microglial BV-2 and astrocytic IMA2.1 cells in cell culture. a–c Viral replication in GL261, BV-2 and IMA2.1 cells infected with LIVP 1.1.1 at an MOI of 0.1 was analyzed by standard plaque assay. The red line separates active replication from no replication. d–f MTT assay was performed to detect the percentage of surviving cells after infection with LIVP 1.1.1 (MOI 1.0). g BV-2 and GL261 cells were cultured as direct co-cultures for 24 h in various concentrations. For standard plaque assay cells were infected at an MOI of 0.1 in triplicate. h Viral titers were related to the infection medium for a better comparison and illustrated as ratio (total virus titer/infection medium). Experiments were repeated in independent experiments. Two-sided t test with unequal variances was used for statistics (*p < 0.05).
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Fig3: Analysis of VACV infections in murine GL261, microglial BV-2 and astrocytic IMA2.1 cells in cell culture. a–c Viral replication in GL261, BV-2 and IMA2.1 cells infected with LIVP 1.1.1 at an MOI of 0.1 was analyzed by standard plaque assay. The red line separates active replication from no replication. d–f MTT assay was performed to detect the percentage of surviving cells after infection with LIVP 1.1.1 (MOI 1.0). g BV-2 and GL261 cells were cultured as direct co-cultures for 24 h in various concentrations. For standard plaque assay cells were infected at an MOI of 0.1 in triplicate. h Viral titers were related to the infection medium for a better comparison and illustrated as ratio (total virus titer/infection medium). Experiments were repeated in independent experiments. Two-sided t test with unequal variances was used for statistics (*p < 0.05).
Mentions: One explanation of reduced VACV replication in the mouse brain could be the preferential uptake of viral particles into microglia or astrocytes. We tested this hypothesis in cell culture, using two specific cell lines, BV-2 and IMA2.1, with similar growth characteristics as primary cells. To determine LIVP 1.1.1 virus progeny in these cell lines in vitro, cells were infected at an MOI of 0.1 followed by standard plaque assay performed at different time points. Data was compared with infection of GL261 glioma cells under the same culture conditions (Figure 3a–c). We confirmed that LIVP 1.1.1 replicated efficiently in GL261 cells, determined by increasing cell-associated virus titers from 2 to 48 h post infection (hpi) and a plateau phase reached at 48 hpi at a virus dose of 1.5 × 106 ± 2.1 × 105 pfu/ml. Already, 24 hpi virus titers were higher than the infection medium (1.6 × 104 ± 2.3 × 103 pfu/ml). Virus titers in the supernatant steadily increased over time, reaching a maximum at 96 hpi (2.9 × 105 ± 5.1 × 104 pfu/ml) (Figure 3a). In contrast, the virus titer in BV-2 cells remained slightly below the infection medium at all times that were analyzed except for the 48 hpi time point (Figure 3b). Surprisingly, in IMA2.1 cells, the virus titer was far below the infection medium during the whole time course of the experiment (Figure 3c). No virus spreading occurred as indicated by the complete lack of viral particles in the supernatant of BV-2 and IMA2.1 infected cells. Cell viability after virus infection was examined by MTT assay at an MOI of 1.0. The analysis showed that GL261 and BV-2 cells were lysed upon infection with LIVP 1.1.1, whereas IMA2.1 cells were intact with a slight decrease in the amount of living cells (Figure 3d–f).Figure 3

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