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Increase in tumor size following intratumoral injection of immunostimulatory CpG-containing oligonucleotides in a rat glioma model

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Abstract

The immunosuppressive environment of malignant gliomas is likely to suppress the anti-tumor activity of infiltrating microglial cells and lymphocytes. Macrophages and microglial cells may be activated by oligonucleotides containing unmethylated CpG-motifs, although their value in cancer immunotherapy has remained controversial. Following injection of CpG-containing oligonucleotides (ODN) into normal rat brain, we observed a local inflammatory response with CD8+ T cell infiltration, upregulation of MHC 2, and ED1 expression proving the immunogenic capacity of the CpG-ODN used. This was not observed with a control ODN mutated in the immunostimulatory sequence (m-CpG). To study their effect in a syngeneic tumor model, we implanted rat 9L gliosarcoma cells into the striatum of Fisher 344 rats. After 3 days, immunostimulatory CpG-ODN, control m-CpG-ODN, or saline was injected stereotactically into the tumors (day 3 group). In another group of animals (day 0 group), CpG-ODN were mixed with 9L cells prior to implantation without further treatment on day 3. After 3 weeks, the animals were killed and the brains and spleens were removed. Rather unexpectedly, the tumors in several of the animals treated with CpG-ODN (both day 0 and day 3 group) were larger than in saline or m-CpG-ODN treated control animals. The tumor size in CpG-ODN-treated animals was more variable than in both control groups. This was associated with inflammatory responses and necrosis which was observed in most tumors following CpG treatment. This, however, did not prevent excessive growth of solid tumor masses in the CpG-treated animals similar to the control-treated animals. Dense infiltration with microglial cells resembling ramified microglia was observed within the solid tumor masses of control- and CpG-treated animals. In necrotic areas (phagocytic), activation of microglial cells was suggested by ED1 expression and a more macrophage-like morphology. Dense lymphocytic infiltrates consisting predominantly of CD8+ T cells and fewer NK cells were detected in all tumors including the control-treated animals. Expression of perforin serving as a marker for T cell or NK cell activation was detected only on isolated cells in all treatment groups. Tumors of all treatment groups revealed CD25 expression indicating T cells presumed to maintain peripheral tolerance to self-antigens. Cytotoxic T cell assays with in vitro restimulated lymphocytes (51chromium release assay) as well as interferon-gamma production by fresh splenocytes (Elispot assay) revealed specific responses to 9L cells but not another syngeneic cell line (MADB 106 adenocarcinoma). Surprisingly, the lysis rates with lymphocytes from CpG-ODN-treated animals were lower compared to control-treated animals. The tumor size of individual animals did not correlate with the response in both immune assays. Taken together, our data support the immunostimulatory capacity of CpG-ODN in normal brain. However, intratumoral application proved ineffective in a rat glioma model. CpG-ODN treatment may not yield beneficial effects in glioma patients.

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Acknowledgments

We are indebted to Dr. Axel Heiser for his support in performing the Elispot assays. We thank Dr. T. von Hörsten (Medizinische Hochschule Hannover, Germany) for supplying us with the MADB 106 adenocarcinoma cell line. The Iba1 antibody was a kind gift from Dr. Y. Imai (Department of Neurochemistry, National Institute of Neuroscience, Tokyo). The technical support by Mrs. Bärbel Hufnagel is appreciated. This work has been supported by the ‘Interdisziplinäres Zentrum für Krebsforschung der Christian-Albrechts Universität zu Kiel’ and by the ‘Hensel Stiftung’.

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Authors and Affiliations

  1. Klinik für Neurochirurgie, Universitätsklinikum Schleswig-Holstein, Campus Kiel, Germany

    Christian Ginzkey, Sven O. Eicker , Joerg Krause, Hans H. Hugo, H. M. Mehdorn & Wolfgang Hamel

  2. Institut für Immunologie, Universitätsklinikum Schleswig-Holstein, Campus Kiel, Germany

    Matthias Marget & Joerg Steinmann

  3. Institut für Pharmakologie, Universitätsklinikum Schleswig-Holstein, Campus Kiel, Germany

    Stephan Brecht

  4. Klinik für Hals-, Nasen- und Ohrenkrankheiten der Universität Würzburg, University of Würzburg, Würzburg, Germany

    Christian Ginzkey

  5. Neurochirurgische Klinik, Universitätsklinikum Hamburg-Eppendorf, Martinistr. 52, 20246, Hamburg, Germany

    Manfred Westphal & Wolfgang Hamel

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  1. Christian Ginzkey
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  2. Sven O. Eicker
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Correspondence to Wolfgang Hamel.

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Ginzkey, C., Eicker , S.O., Marget, M. et al. Increase in tumor size following intratumoral injection of immunostimulatory CpG-containing oligonucleotides in a rat glioma model. Cancer Immunol Immunother 59, 541–551 (2010). https://doi.org/10.1007/s00262-009-0771-y

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  • DOI: https://doi.org/10.1007/s00262-009-0771-y

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