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Abstract

The aim of this project was to develop a mouse allograft model based on neural stem cells displaying the key properties of human glioma on the molecular level. Therefore, neural stem cells were prepared from mice harboring different genetic features of astrocytoma or oligodendroglioma. Common to both are mutations of isocitrate dehydrogenase. A specific type of mutation leads to production of D-2-hydroxyglutarate, a known oncometabolite, which is thought to be essential for tumor initiation. The accumulation of D-2-hydroxyglutarate leads to epigenetic reprogramming and thereby to tumor formation.

Neural stem cells with inducible isocitrate dehydrogenase R132H knock-in and inducible Tp53 knock-out were analyzed for their tumorigenic potential with different assays. None of the manipulations or combinations thereof showed an enhanced tumorigenic potential. While in cells with isocitrate dehydrogenase mutation R132H cell growth was compromised by D-2-hydroxyglutarate production, this was not observed in combination with the loss of p53. To analyze if these cells show a different tumorigenic potential in vivo, they were injected intracranially into immunodeficient mice. Potential tumor outgrowth was monitored with regular MRI screens and was evident after eight months. It was reasonable to keep the cells in culture for a longer time, because epigenetic reprogramming can take several passages to manifest. Those “long-term” cultures grew more aggressively compared to their recently induced (“short-term”) counterparts. This could mainly be explained by the loss of the isocitrate dehydrogenase mutation, which is a negative selection marker under in vitro conditions. Such a phenomenon is already described in human cell lines. In contrast to the in vitro situation, the isocitrate dehydrogenase mutation was kept in vivo, although the tumors disappeared within twelve months.

In conclusion, the project could show that the combination of isocitrate dehydrogenase mutation R132H together with the loss of p53 is beneficial for the cells and sufficient to initiate tumor formation. The model nicely pictures the human system, in which cells in culture lose the mutation, but tumors only rarely do. It is perfectly suited for the investigation of the essential function of isocitrate dehydrogenase mutation in glioma. By this it will help to understand this function, uncover weaknesses of these tumors and help to predict resistance mechanisms against isocitrate dehydrogenase mutation inhibitors. Further development of the system by addition of other genetic alterations will potentially lead to more durable tumors in vivo.