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Rat astrocytic tumour cells are associated with an anti-inflammatory microglial phenotype in an organotypic model

Rat astrocytic tumour cells are associated with an anti-inflammatory microglial phenotype in an organotypic model
Rat astrocytic tumour cells are associated with an anti-inflammatory microglial phenotype in an organotypic model
Aim: Microglia form a high proportion of cells in glial tumours but their role in supporting or inhibiting tumour growth is unclear. Here we describe the establishment of an in vitro model to investigate their role in astrocytomas.

Methods: Rat hippocampal slices were prepared and, after 7 days to allow microglia to become quiescent, rat C6 astrocytic tumour cells were added. Over the following 7 days, infiltration and cell death were studied using fluorescent C6 tumour cells and confocal microscopy; immunophenotyping of microglia was performed using CD68 (phagocytosis), MHCII (antigen-presentation) and Iba1 (microglial marker regardless of functional state). Cell proliferation was assessed using Ki67 and qPCR to detect cytokine expression. Sham and control groups were included.

Results: Microscopy showed proliferation of C6 tumour cells with both infiltration of tumour cells into the hippocampal tissue and of microglia amongst the tumour cells. Confocal experiments confirmed increasing tumour cell infiltration into the hippocampal slice with time (P<0.001), associated with cell death (?=0.313, P=0.022). Ki67 showed increased proliferation (P<0.001), of both tumour cells and Iba1+ microglia and increased microglial phagocytosis (CD68: P<0.001). Expression of proinflammatory cytokines IL1, IL6 and TNF? were downregulated with expression of the anti-inflammatory cytokine TGF?1 maintained.

Conclusion: This model allows study of the proliferation and infiltration of astrocytic tumour cells in CNS tissue and their interaction with microglia. Our data suggest that microglial function is altered in the presence of tumour cells, putatively facilitating tumour progression. Manipulation of the microglial functional state may have therapeutic value for astrocytic tumours.
astrocytoma, microglia, organotypic slice, cytokines
0305-1846
Billingham, C.
9f598865-18f3-453f-ad4e-6e8f7b923bf9
Powell, M.R.
2b42fb3d-98cf-4306-82ad-6ea1e72164d3
Jenner, K.A.
9f068e80-4743-4a1a-990a-f2c2a611e278
Johnston, D.A.
b41163c9-b9d2-425c-af99-2a357204014e
Gatherer, M.
b0aae216-21c4-4737-b042-865a65658f06
Nicoll, J.A.R.
88c0685f-000e-4eb7-8f72-f36b4985e8ed
Boche, D.
bdcca10e-6302-4dd0-919f-67218f7e0d61
Billingham, C.
9f598865-18f3-453f-ad4e-6e8f7b923bf9
Powell, M.R.
2b42fb3d-98cf-4306-82ad-6ea1e72164d3
Jenner, K.A.
9f068e80-4743-4a1a-990a-f2c2a611e278
Johnston, D.A.
b41163c9-b9d2-425c-af99-2a357204014e
Gatherer, M.
b0aae216-21c4-4737-b042-865a65658f06
Nicoll, J.A.R.
88c0685f-000e-4eb7-8f72-f36b4985e8ed
Boche, D.
bdcca10e-6302-4dd0-919f-67218f7e0d61

Billingham, C., Powell, M.R., Jenner, K.A., Johnston, D.A., Gatherer, M., Nicoll, J.A.R. and Boche, D. (2012) Rat astrocytic tumour cells are associated with an anti-inflammatory microglial phenotype in an organotypic model. Neuropathology and Applied Neurobiology. (doi:10.1111/j.1365-2990.2012.01283.x). (PMID:22631872)

Record type: Article

Abstract

Aim: Microglia form a high proportion of cells in glial tumours but their role in supporting or inhibiting tumour growth is unclear. Here we describe the establishment of an in vitro model to investigate their role in astrocytomas.

Methods: Rat hippocampal slices were prepared and, after 7 days to allow microglia to become quiescent, rat C6 astrocytic tumour cells were added. Over the following 7 days, infiltration and cell death were studied using fluorescent C6 tumour cells and confocal microscopy; immunophenotyping of microglia was performed using CD68 (phagocytosis), MHCII (antigen-presentation) and Iba1 (microglial marker regardless of functional state). Cell proliferation was assessed using Ki67 and qPCR to detect cytokine expression. Sham and control groups were included.

Results: Microscopy showed proliferation of C6 tumour cells with both infiltration of tumour cells into the hippocampal tissue and of microglia amongst the tumour cells. Confocal experiments confirmed increasing tumour cell infiltration into the hippocampal slice with time (P<0.001), associated with cell death (?=0.313, P=0.022). Ki67 showed increased proliferation (P<0.001), of both tumour cells and Iba1+ microglia and increased microglial phagocytosis (CD68: P<0.001). Expression of proinflammatory cytokines IL1, IL6 and TNF? were downregulated with expression of the anti-inflammatory cytokine TGF?1 maintained.

Conclusion: This model allows study of the proliferation and infiltration of astrocytic tumour cells in CNS tissue and their interaction with microglia. Our data suggest that microglial function is altered in the presence of tumour cells, putatively facilitating tumour progression. Manipulation of the microglial functional state may have therapeutic value for astrocytic tumours.

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e-pub ahead of print date: 27 May 2012
Keywords: astrocytoma, microglia, organotypic slice, cytokines
Organisations: Faculty of Medicine

Identifiers

Local EPrints ID: 341418
URI: https://eprints.soton.ac.uk/id/eprint/341418
ISSN: 0305-1846
PURE UUID: 7cfa6f7a-c3e4-4ba9-a440-315ddc813e44
ORCID for D.A. Johnston: ORCID iD orcid.org/0000-0001-6703-6014
ORCID for J.A.R. Nicoll: ORCID iD orcid.org/0000-0002-9444-7246
ORCID for D. Boche: ORCID iD orcid.org/0000-0002-5884-130X

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Date deposited: 25 Jul 2012 10:56
Last modified: 06 Jun 2018 12:52

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Contributors

Author: C. Billingham
Author: M.R. Powell
Author: K.A. Jenner
Author: D.A. Johnston ORCID iD
Author: M. Gatherer
Author: J.A.R. Nicoll ORCID iD
Author: D. Boche ORCID iD

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