Article
NKG2D ligands in glioma stem-like cells: Expression in situ and in vitro
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Authors
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Charlotte Flüh
- Department of Neurosurgery, University Medical Center Schleswig-Holstein, Campus Kiel, Kiel, Deutschland
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Vivian Adamski
- Department of Neurosurgery, University Medical Center Schleswig-Holstein, Campus Kiel, Kiel, Deutschland
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Kirsten Hattermann-Koch
- Institute of Anatomy, Christian-Albrechts-University Kiel, Kiel, Deutschland
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Guranda Chitadze
- Institute of Immunology, University Medical Center Schleswig-Holstein, Campus Kiel, Kiel, Deutschland
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Michael Synowitz
- Department of Neurosurgery, University Medical Center Schleswig-Holstein, Campus Kiel, Kiel, Deutschland
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Dieter Kabelitz
- Institute of Immunology, University Medical Center Schleswig-Holstein, Campus Kiel, Kiel, Deutschland
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Janka Held-Feindt
- Department of Neurosurgery, University Medical Center Schleswig-Holstein, Campus Kiel, Kiel, Deutschland
| Published: | June 9, 2017 |
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Outline
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Objective: Glioblastoma multiforme (GBM) is a highly malignant brain tumor. Tumor stem cells have a major influence on tumor malignancy and progression, and immunological escape mechanisms, involving the Natural Killer Group 2, member D (NKG2D) receptor-ligand system, are key elements in tumor progression. Cell-bound NKG2D-ligands (NKG2DL) such as MHC class I related molecule A and B (MICA and MICB), and the UL-16 binding protein family (ULBP1-6) are recognized by the NKG2D-receptor and trigger cytotoxic effector activity in NK- and T-cells. By releasing soluble NKG2DL, tumor cells inhibit the killing potential of effector cells. Numerous studies documented the importance of the NKG2D-system in in vitro GBM-model systems, but the role for glioma stem cells has not been described yet.
Methods: We analyzed the expression profile and localization of NKG2DL (MICA, MICB, ULBP1 and 2) and embryonic and neural stem cell markers (Klf-4, Oct-4, Sox-2, Nanog, Musashi-1) in solid human GBM and stem-like cells isolated from glioma cell lines using quantitative RT-PCR and two color immuno-staining. We also evaluated the effect of temozolomide (TMZ) on NKG2DL and stem cell marker expression in stem-like cells derived from glioma cell lines by qRT-PCR.
Results: Whereas Musashi-1 and Oct-4 were rarely costained with NKG2DL, Sox-2 and Nanog showed partial costaining. For Klf-4, we observed a complete costaining with MICA, MICB, ULBP1 and ULBP2. NKG2DL were found in a distinct tumor cell subpopulation and were broadly costained with each other. qRT-PCR of stem-like cells derived from glioma cell lines T98G and U251MG in comparison to differentiated cells revealed that T98G stem-like cells were predominantly positive for MICB and ULBP1, whereas MICA, ULBP2, Sox-2, Nanog and Musashi-1 were more pronounced in U251MG stem-like cells. Upon differentiation, T98G displayed significantly less MICA, MICB and ULBP2, whereas in U251MG expression of NKG2LD was mostly unaltered, and expression of most stem cell markers decreased significantly, as expected. This was consistent with findings on protein level, which were obtained by immunostaining. Stimulation with TMZ led to a significant upregulation of NKG2DL in stem-like cells derived from T98G and U251MG.
Conclusion: The role of the NKG2D system concerning glioma stem cells is complex: in solid glioblastomas NKG2DL are found in a subset of tumor cells that coexpress some but not all investigated embryonic and neural stem cell markers. Stem-like cells derived from glioma cell lines show a heterogeneous expression pattern of NKG2DL and stem cell marker expression. TMZ leads to an upregulation of NKG2DL, with some variation between cell lines. As stem-like cells from GBM cell lines in vitro show a higher expression of NKG2DL than more differentiated tumor cells, the NKG2D system might play an important role in regulation of tumor stem cell survival.
