Previously we defined a pathway of transforming growth factor beta (TGF-beta) and stromal cell-derived factor-1/CXC chemokine ligand 12 (SDF-1alpha/CXCL12) dependent migration of adult haematopoietic stem and progenitor cells (HPC) towards glioma cells in vitro and their homing to experimental gliomas in vivo. Hypoxia is a critical aspect of the microenvironment of gliomas and irradiation is an essential part of the standard therapy. To evaluate the therapeutic potential of HPC as vectors for a cell-based therapy of gliomas, we investigated the impact of hypoxia and irradiation on the attraction of HPC by glioma cells. Temozolomide (TMZ) treatment and hyperthermia served as controls. Supernatants of irradiated or hypoxic LNT-229 glioma cells promote HPC migration in vitro. Reporter assays reveal that the CXCL12 promoter activity is enhanced in LNT-229 cells at 24 h after irradiation at 8 Gy or after exposure to 1% oxygen for 12 h. The irradiation- and hypoxia-induced release of CXCL12 depends on hypoxia inducible factor-1 alpha (HIF-1alpha), but not on p53. Induction of transcriptional activity of HIF-1alpha by hypoxia or irradiation requires an intact TGF-beta signalling cascade. This delineates a novel stress signalling cascade in glioma cells involving TGF-beta, HIF-1alpha and CXCL12. Stress stimuli can be irradiation, hypoxia or TMZ, but not hyperthermia. Cerebral irradiation of nude mice at 21 days after intracerebral implantation of LNT-229 glioma induces tumour satellite formation and enhances the glioma tropism of HPC to the tumour bulk and even to these satellites in vivo. These data suggest that the use of HPC as cellular vectors in the treatment of glioblastoma may well be combined with irradiation or other anti-angiogenic therapies that induce tumour hypoxia.

Macrophage migration inhibitory factor (MIF), which inhibits apoptosis and promotes angiogenesis, is expressed in cancers suppressing immune surveillance. Its biological role in human glioblastoma is, however, only poorly understood. We examined in-vivo expression of MIF in 166 gliomas and 23 normal control brains by immunohistochemistry. MIF immunoreactivity was enhanced in neoplastic astrocytes in WHO grade II glioma and increased significantly in higher tumour grades (III-IV). MIF expression was further assessed in 12 glioma cell lines in vitro. Quantitative RT-PCR showed that MIF mRNA expression was elevated up to 800-fold in malignant glioma cells compared with normal brain. This translated into high protein levels as assessed by immunoblotting of total cell lysates and by ELISA-based measurement of secreted MIF. Wild-type p53-retaining glioma cell lines expressed higher levels of MIF, which may be connected with the previously described role of MIF as a negative regulator of wild-type p53 signalling in tumour cells. Stable knockdown of MIF by shRNA in glioma cells significantly increased tumour cell susceptibility towards NK cell-mediated cytotoxicity. Furthermore, supernatant from mock-transfected cells, but not from MIF knockdown cells, induced downregulation of the activating immune receptor NKG2D on NK and CD8+ T cells. We thus propose that human glioma cell-derived MIF contributes to the immune escape of malignant gliomas by counteracting NK and cytotoxic T-cell-mediated tumour immune surveillance. Considering its further cell-intrinsic and extrinsic tumour-promoting effects and the availability of small molecule inhibitors, MIF seems to be a promising candidate for future glioma therapy.

Long-term administration of ENZA after radiotherapy is feasible and leads to long-term survival without neurotoxicity.

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