gms | German Medical Science

Objective: The glioblastoma multiforme (GBM) and the gliosarcoma (GSarc) are primary brain tumors with a high morbidity and mortality, offering only palliation. Stem-like glioma cells (SLGCs) are considered as one major cause of treatment failure and relapse. It has been shown that alterations in receptor tyrosine kinase PDGFRa (platelet-derived growth factor receptor), a member of the type III receptor tyrosine kinase (RTK) family, may contribute to GBM initiation and progression. The potential roles of the close relative PDGFRb or the type III RTK cKit remain elusive.

Methods: We have isolated and characterized SLGCs from patients with WHO grade II, III and IV neuroepithelial primary brain tumors. Nine GBM- and two GSarc-derived SLGCs lines underwent a detailed cellular and molecular characterization and were treated with increasing doses of the type III RTK inhibitor Imatinib (Ib; Glivec^©). In order to elucidate whether the two PDGF receptors and/or cKit would be responsive for the Ib-mediated effects, we generated functional knock-outs of the PDGFRa- and b-genes, as well as of the cKit-gene by CRISPR-Cas9-mediated genome editing.

Results: We observed that increasing doses of Ib affected proliferation, viability and migration of SLGCs. Responsiveness was distinct for SLGCs from different patients and additionally varied between clones derived from the same SLGC mother cell lines, supporting previous suggestions that SLGCs display a high degree of cellular and molecular heterogeneity. We edited the genes coding for the PDGFRa or b, or cKit in six distinct SLGC-lines, which differed with respect to their Tp53- and PTEN-status. It turned out that bi-allelic editing of both PDGF receptor genes in the same cell was possible, but with very low efficacy. Notably, the respective clones did not display full inactivation of all pdgfr alleles, but small in frame deletions in at least one of the four alleles. This indicated the requirement of functional PDGF-signaling and suggested that PDGFRa might replace PDGFRb and vice versa in vitro. It appeared that Ib-responsiveness of the edited clones was distinct.

Conclusion: The edited SLGC clones will serve asvery precious tools for future in vitro and in vivo experiments, addressing the function of the type III RTKs in glioma progression, metabolism and invasiveness.