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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

FACS analyses of GL261 and BV-2 cells. a Cells that have lost part of their DNA due to DNA fragmentation in apoptosis are represented in sub-G1 peaks on DNA histograms. The percentage of BV-2 or GL261 cells in sub-G1 phase 24, 48 or 72 h after viral infection (MOI 0.5) are shown in the bar chart. b The percentages of PI-dim (apoptotic) and PI-bright (necrotic) cells in controls and LIVP 1.1.1-infected samples (MOI 1.0) are shown for BV-2 and GL261 cells. The experiment was performed in triplicate and repeated in an independent experiment.
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Fig4: FACS analyses of GL261 and BV-2 cells. a Cells that have lost part of their DNA due to DNA fragmentation in apoptosis are represented in sub-G1 peaks on DNA histograms. The percentage of BV-2 or GL261 cells in sub-G1 phase 24, 48 or 72 h after viral infection (MOI 0.5) are shown in the bar chart. b The percentages of PI-dim (apoptotic) and PI-bright (necrotic) cells in controls and LIVP 1.1.1-infected samples (MOI 1.0) are shown for BV-2 and GL261 cells. The experiment was performed in triplicate and repeated in an independent experiment.

Mentions: To quantify late apoptosis, the percentage of cells in sub-G1 phase was analyzed by flow cytometry. The percentages of mock-infected and VACV-infected cells in sub-G1 phase were compared at 24, 48, and 72 hpi (Figure 4a). We found that the percentage of cells in sub-G1 phase was higher in BV-2 cells than in GL261 cells at all times. 40% of BV-2 cells were in sub-G1 phase after 24 h and this amount of cells increased over time to 70% after 48 h and to 80% after 72 h. On the other hand, at 24 hpi the amount of GL261 cells in sub-G1 phase was around 10% and increased to 45% after 72 h (Figure 4a). Additionally, distribution of PI-positive cells in PI-dim (apoptotic) and PI-bright (necrotic) cells [43, 44] revealed higher numbers of apoptotic cells in the VACV-infected BV-2 sample compared to infected GL261 cells at all time points (Figure 4b). Our data indicates that VACV infection activates different cell death pathways and mechanisms in microglial BV-2 cells in contrast to GL261 tumor cells.Figure 4


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)

FACS analyses of GL261 and BV-2 cells. a Cells that have lost part of their DNA due to DNA fragmentation in apoptosis are represented in sub-G1 peaks on DNA histograms. The percentage of BV-2 or GL261 cells in sub-G1 phase 24, 48 or 72 h after viral infection (MOI 0.5) are shown in the bar chart. b The percentages of PI-dim (apoptotic) and PI-bright (necrotic) cells in controls and LIVP 1.1.1-infected samples (MOI 1.0) are shown for BV-2 and GL261 cells. The experiment was performed in triplicate and repeated in an independent experiment.
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Related In: Results  -  Collection

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Fig4: FACS analyses of GL261 and BV-2 cells. a Cells that have lost part of their DNA due to DNA fragmentation in apoptosis are represented in sub-G1 peaks on DNA histograms. The percentage of BV-2 or GL261 cells in sub-G1 phase 24, 48 or 72 h after viral infection (MOI 0.5) are shown in the bar chart. b The percentages of PI-dim (apoptotic) and PI-bright (necrotic) cells in controls and LIVP 1.1.1-infected samples (MOI 1.0) are shown for BV-2 and GL261 cells. The experiment was performed in triplicate and repeated in an independent experiment.
Mentions: To quantify late apoptosis, the percentage of cells in sub-G1 phase was analyzed by flow cytometry. The percentages of mock-infected and VACV-infected cells in sub-G1 phase were compared at 24, 48, and 72 hpi (Figure 4a). We found that the percentage of cells in sub-G1 phase was higher in BV-2 cells than in GL261 cells at all times. 40% of BV-2 cells were in sub-G1 phase after 24 h and this amount of cells increased over time to 70% after 48 h and to 80% after 72 h. On the other hand, at 24 hpi the amount of GL261 cells in sub-G1 phase was around 10% and increased to 45% after 72 h (Figure 4a). Additionally, distribution of PI-positive cells in PI-dim (apoptotic) and PI-bright (necrotic) cells [43, 44] revealed higher numbers of apoptotic cells in the VACV-infected BV-2 sample compared to infected GL261 cells at all time points (Figure 4b). Our data indicates that VACV infection activates different cell death pathways and mechanisms in microglial BV-2 cells in contrast to GL261 tumor cells.Figure 4

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