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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 infection in organotypic slice cultures with tumor cell implants. a Organotypic slice cultures (OSCs) from adult mice were established and implanted with 1 × 104 Gl261-RFP or U87-MG-RFP cells. 7 days after implantation, OSCs were infected with 5 × 106 pfu LIVP 1.1.1/well. b Standard plaque assay was performed 24 and 72 h after infection. Two-sided t test with unequal variances was used for statistics *p < 0.05. 72 h after infection OSCs implanted with GL261-RFP (c) and U87-MG-RFP (d) cells were stained with Hoechst and anti-VACV antibody to visualize infected tumor spheres. e, f IFN-γ-stimulated or unstimulated BV-2 cells were applied directly onto the OSCs implanted with GL261 cells. 24 h later OSCs were infected with LIVP 1.1.1 and stained after additional 24 h with FITC-IB4, Hoechst and anti-VACV antibody. Yellow arrows indicate exemplarily infected or non-infected BV-2 microglial cells.
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Fig7: Analysis of VACV infection in organotypic slice cultures with tumor cell implants. a Organotypic slice cultures (OSCs) from adult mice were established and implanted with 1 × 104 Gl261-RFP or U87-MG-RFP cells. 7 days after implantation, OSCs were infected with 5 × 106 pfu LIVP 1.1.1/well. b Standard plaque assay was performed 24 and 72 h after infection. Two-sided t test with unequal variances was used for statistics *p < 0.05. 72 h after infection OSCs implanted with GL261-RFP (c) and U87-MG-RFP (d) cells were stained with Hoechst and anti-VACV antibody to visualize infected tumor spheres. e, f IFN-γ-stimulated or unstimulated BV-2 cells were applied directly onto the OSCs implanted with GL261 cells. 24 h later OSCs were infected with LIVP 1.1.1 and stained after additional 24 h with FITC-IB4, Hoechst and anti-VACV antibody. Yellow arrows indicate exemplarily infected or non-infected BV-2 microglial cells.

Mentions: We established organotypic slice cultures (OSCs) from 2 to 3 month old C57BL/6 mice, the same age as the mice that received intracranial injections. The murine GL261 glioma cell line can be viewed as syngeneic non-responder tumor model [52] and the human U87-MG glioblastoma cell line, in contrast, as the responder model [53]. OSCs were implanted with either GL261-RFP or U87-MG-RFP cells and the tumors were allowed to establish for 7 days. The OSCs containing the tumors were infected with 5x106 pfu LIVP 1.1.1 by adding VACV directly onto the tumor spheres (Figure 7a). Viral plaque assay of OSCs with GL261-RFP tumors revealed no difference of the virus titer between 24 and 72 h. In contrast, U87-MG-RFP tumors showed a tenfold increase of VACV LIVP 1.1.1 progeny after 72 h (Figure 7b). Immunohistochemical staining of tumor spheres (Figure 7c) confirmed that VACV is able to infect GL261 tumors expressing RFP but viral particles were only detectable at the periphery of the tumor. In contrast, OSCs with U87-MG tumors expressing RFP were completely infected 72 h after addition of the virus onto the slices (Figure 7d). The same results were observed in OSCs derived from an immunodeficient mouse brain (data not shown). Here the results obtained from intracranial implantation and infection experiments were also confirmed in OSC tumors ex vivo.Figure 7


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 infection in organotypic slice cultures with tumor cell implants. a Organotypic slice cultures (OSCs) from adult mice were established and implanted with 1 × 104 Gl261-RFP or U87-MG-RFP cells. 7 days after implantation, OSCs were infected with 5 × 106 pfu LIVP 1.1.1/well. b Standard plaque assay was performed 24 and 72 h after infection. Two-sided t test with unequal variances was used for statistics *p < 0.05. 72 h after infection OSCs implanted with GL261-RFP (c) and U87-MG-RFP (d) cells were stained with Hoechst and anti-VACV antibody to visualize infected tumor spheres. e, f IFN-γ-stimulated or unstimulated BV-2 cells were applied directly onto the OSCs implanted with GL261 cells. 24 h later OSCs were infected with LIVP 1.1.1 and stained after additional 24 h with FITC-IB4, Hoechst and anti-VACV antibody. Yellow arrows indicate exemplarily infected or non-infected BV-2 microglial cells.
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Fig7: Analysis of VACV infection in organotypic slice cultures with tumor cell implants. a Organotypic slice cultures (OSCs) from adult mice were established and implanted with 1 × 104 Gl261-RFP or U87-MG-RFP cells. 7 days after implantation, OSCs were infected with 5 × 106 pfu LIVP 1.1.1/well. b Standard plaque assay was performed 24 and 72 h after infection. Two-sided t test with unequal variances was used for statistics *p < 0.05. 72 h after infection OSCs implanted with GL261-RFP (c) and U87-MG-RFP (d) cells were stained with Hoechst and anti-VACV antibody to visualize infected tumor spheres. e, f IFN-γ-stimulated or unstimulated BV-2 cells were applied directly onto the OSCs implanted with GL261 cells. 24 h later OSCs were infected with LIVP 1.1.1 and stained after additional 24 h with FITC-IB4, Hoechst and anti-VACV antibody. Yellow arrows indicate exemplarily infected or non-infected BV-2 microglial cells.
Mentions: We established organotypic slice cultures (OSCs) from 2 to 3 month old C57BL/6 mice, the same age as the mice that received intracranial injections. The murine GL261 glioma cell line can be viewed as syngeneic non-responder tumor model [52] and the human U87-MG glioblastoma cell line, in contrast, as the responder model [53]. OSCs were implanted with either GL261-RFP or U87-MG-RFP cells and the tumors were allowed to establish for 7 days. The OSCs containing the tumors were infected with 5x106 pfu LIVP 1.1.1 by adding VACV directly onto the tumor spheres (Figure 7a). Viral plaque assay of OSCs with GL261-RFP tumors revealed no difference of the virus titer between 24 and 72 h. In contrast, U87-MG-RFP tumors showed a tenfold increase of VACV LIVP 1.1.1 progeny after 72 h (Figure 7b). Immunohistochemical staining of tumor spheres (Figure 7c) confirmed that VACV is able to infect GL261 tumors expressing RFP but viral particles were only detectable at the periphery of the tumor. In contrast, OSCs with U87-MG tumors expressing RFP were completely infected 72 h after addition of the virus onto the slices (Figure 7d). The same results were observed in OSCs derived from an immunodeficient mouse brain (data not shown). Here the results obtained from intracranial implantation and infection experiments were also confirmed in OSC tumors ex vivo.Figure 7

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