Table of Contents

Research-Current work

Microglia in brain tumour biology

The actual role of microglia, the brain resident macrophages, in glioma biology remains largely unknown and controversial. In vitro and in vivo observations strongly suggest that these phagocytes are endowed with tumour-promoting as well as with tumour-destructive capacities. In vivo, the ability of glioma to grow despite of the presence of numerous infiltrating microglia indicates an efficient “silencing” of these cells and goes in line with the known immunosuppressive capacity of glioma. A better understanding of the interactions taking place between tumour cells and microglia would definitely help in designing fully efficient strategies that would be appropriate to the unique environment displayed by the brain. In that regard, the use of viral vectors for local gene therapy directed at glioma cells may help not only to kill tumour cells but also to induce or enhance anti-tumour activities of microglia. Owing to their oncotropic and oncolytic properties, parvovirus-based vectors represent interesting candidates for anti-tumour therapy in the brain. Viral vectors must however fulfil an important criterion for a safe therapeutic use, i.e. their specific targeting at neoplastic cells, hence their innocuousness for normal cells and, if possible, their lack of interference with the host anti-tumour defenses. We focus our investigations on the two following questions, in the mouse and in the human systems:

• Characterisation of microglia pro- and anti-tumour activities
• Parvovirus and microglia-mediated anti-tumour activities

Characterisation of microglia pro- and anti-tumour activities

Whereas the pro-tumour activities of murine microglia are well documented, those of human cells are still scarce. We modified available protocols to obtain human microglia, isolated from brain tumour specimen, and analysed them for their pro- and anti-tumor activities (collaboration with Dr. Christel Herold-Mende, Neurosurgery, Heidelberg). We observed that untreated, i.e. “resting” microglia secrete factors that support tumour growth and migration in a 3D collagen invasion assay. Stimulation of human microglia with a TLR-3 agonist however led to an anti-tumour phenotype characterised by reduction of tumour cell growth and migration and by cytotoxicity. Microglia toxicity was specifically directed at tumor cells, as human astrocytes and neurons were not affected by the supernatant of stimulated microglia. These data indicate that human microglia isolated from tumour tissue have a tumour-supporting phenotype that can be switched to an anti-tumour phenotype by application of (a) proper stimulus/stimuli. However, these anti-tumour activities were not induced when microglia activation was performed in the presence of human glioma cells, indicating a strong control exerted by glioma cells on the regulation of microglia activation state (Kees et al, 2012). We are currently investigating the transcriptome of human microglia treated with the supernatant of glioma cells in order to identify the molecular mediators of this inhibitory effect. We are also setting an experimental model to validate the cytotoxic potential of pre-activated microglia (via TLR-3 or via phagocytosis of dying glioma cells) in vivo.

Very little progress has been made in understanding the molecular basis of microglia cytotoxicity since its first characterisation towards a non-brain tumour cell line. We recently reported the first detailed characterisation of the lipopolysaccharide plus interferon-gamma-induced cytotoxic activity of primary microglia towards glioma cell lines in the mouse system (Mora et al, 2009). Stimulated microglia secreted proteic factors that efficiently killed TNF-a and TRAIL-resistance glioma cell lines without affecting the survival of primary cultures of astrocytes or neurons. Cell death was autophagy-dependent and resulted from a blockade of the basal autophagic flux present in tumour cells. These observations demonstrate that glioma cells resistant to apoptotic death ligands could be successfully and specifically killed through autophagy-dependent death induced by appropriately activated microglia. We could mimic part of these effects through the chemical inhibition of sphingosine kinase in murine and human glioma cells, supporting the promising use of sphingosine kinase inhibitors in glioma therapy (Mora et al., 2010). We are currently investigating the effects of combining sphingosine kinase inhibitors with temozolomide to improve the efficacy and efficiency of these drugs towards tumor cells, in vitro and in vivo (Noack and Régnier-Vigouroux).

In the course of these studies, we also observed that normal astrocytes and glioblastoma cells kept in culture differed in their response to oxidative stress, suggesting differences in the detoxification potential of these cells. We indeed identified the glutathione peroxidase 1 enzyme to be a crucial element over other antioxidant enzymes for oxidative stress regulation in murine and human glioblastoma cells. Our data suggest that mapping the antioxidant enzyme status of glioblastoma may prove to be a useful tool for personalized ROS/RNS inducing therapies (Dokic et al, unpublished data).

Altogether our present observations emphasize the potential of activated microglia as tools for anti-tumor therapies. On the other side, they stress the fact that tumor cells strongly regulate these immune cells. Our objectives in the next future are: (i) to validate the use of human microglia for therapy using mouse glioma models (Cisneros and Régnier-Vigouroux), and (ii) to unravel the nature of the communication signals between glioma and microglia that underlies the inhibition of this microglial cytotoxic activity, in the mouse and in the human system (Choi and Régnier-Vigouroux).

Parvovirus and microglia-mediated anti-tumour activities

Specificity and safety of use of the parvovirus as a vector for brain gene therapy

The safe use of parvovirus for glioma therapy was analysed in a proof-of-the principle study in the mouse. Our data (Abschuetz et al, 2006; Nickles and Régnier-Vigouroux, unpublished data) indicate that, in the natural host, a majority of normal glial cells are not competent for parvovirus replication and that the abortive infection taking place in a minor fraction of these cells fails to impede their survival and immunocompetence. Similar experiments conducted with human microglia demonstrated a lack of parvovirus toxicity towards these cells (Kees and Régnier-Vigouroux, unpublished data). These features argue for the safety of parvovirus-based therapies of brain tumours that may harbour reactive and proliferative glial cells.

Anti-tumour response and parvovirus

As already mentioned, the brain parenchyma is characterised by a tolerogenic status. This, combined with the immunosuppressive features of tumour cells, contributes to decrease the anti-tumour potential of microglia. We would like to explore the possibility to induce a parvovirus-mediated activation of the microglial anti-tumour activities. Infection and killing of tumour cells by wild type parvovirus may indeed provide activation of microglia through their phagocytosis of infected dying cells. Transduction of immunomodulators by recombinant parvovirus-infected tumour cells may lead to the re-activation of anti-tumour activities of microglia. We therefore wish to address the role of parvovirus in boosting microglia immune potential in vitro, using mixed (tumour cells, microglia) spheroid cultures and in vivo, using the mouse model for analysis of intra-cerebral immune responses (collaboration with the laboratory of Dr. Christiane Dinsart).

In conclusion, we have shown that the anti-tumour potential of microglia can be switched on again using appropriate stimuli, in the mouse and in the human models. Identification of microglial toxic factors and of molecules involved in these anti-glioma cell-specific activities is underway and can be expected to provide keys to new targets for glioma therapy. The use of recombinant parvoviral vectors as a delivery system and as a microglia activation tool will be further explored.

References

MORA, R., ABSCHUETZ, A., KEES, T., DOKIC, I., JOSCHKO, N., KLEBER, S., GEIBIG, R., MOSCONI, E., ZENTGRAF, H., MARTIN-VILLALBA, A. and RÉGNIER-VIGOUROUX, A. (2009)
TNF-alpha- and TRAIL-resistant glioma cells undergo autophagy-dependent cell death induced by activated microglia
GLIA, 57, 561-581

MORA, R. and RÉGNIER-VIGOUROUX, A. (2009)
Autophagy-driven cell fate decision maker. Activated microglia induce specific death of glioma cells by a blockade of basal autophagic flux and secondary apoptosis/necrosis
Autophagy, 5, 1-3

MORA, R., DOKIC, I., KEES, T., HÜBER, C.M., KEITEL, D., GEIBIG, R., BRÜGGER, B., ZENTGRAF, H., BRADY, N.R. and RÉGNIER-VIGOUROUX, A. (2010)
Sphingolipid rheostat alterations related to transformation can be exploited for specific induction of lysosomal cell death in murine and human glioma.
GLIA, 58, 1364-1383

KEES, T., LOHR, J., NOACK, J., MORA, R., GDYNIA, G., TÖEDT, G., ERNST, A., RADLWIMMER, B., FALK, C.S., HEROLD-MENDE, C. and RÉGNIER-VIGOUROUX, A. (2012)
Microglia isolated from patients with glioma gain anti-tumor activities on poly (I:C) stimulation.
Neuro-Oncology, 14, 64-78

Project partners

Microglia-glioma cells interactions, human model: collaboration with Christel Herold-Mende, Heidelberg University Hospital, Heidelberg
Microglia-glioma cells interactions, mouse model: collaboration with Harald Neumann, Institute of Reconstructive Neurobiology, Bonn
Parvovirus, microglia and glioma: collaboration with Christiane Dinsart, DKFZ, Heidelberg and Jean Rommelaere, DKFZ (Parvovirus clinical trial Parvoryx).
Modelling of microglia-glioma cells interactions: collaboration with Thorsten Buzug and Alina Toma, Institute of Medical Engineering, Lübeck
Modelling of sphingolipid metabolism in glioma cells: collaboration with Rodrigo Mora, Faculty of Microbiology, San José, Costa Rica.

Contact

Dr. Anne Régnier-Vigouroux
Function: Group leader
Topic: Microglia in brain tumor biology

Our team belongs to the Faculty of Biology, Johannes Gutenberg University Mainz (JGU) and is currently hosted at the DKFZ in the frame of a collaboration with the JGU.

E-Mail: regnier@dkfz.de
Tel.: +49 6221 42 49 71
Fax: +49 6221 42 49 62
Address: DKFZ, ATV, INF242, 69120 Heidelberg