gms | German Medical Science

Objective: About 95 % of GBM patients show tumor relapse leaving them with limited therapeutic options as recurrent tumors are often resistant to temozolomide (TMZ). GBM-like stem cells (GSCs) are considered to be the major obstacle in therapy resistance, so that characterization of their response to TMZ is helpful in identifying genes associated with TMZ resistance.

Methods: GSCs were isolated from GBM patients (n=5) and characterized by IF-staining for GFAP and Nestin. GSCs from 3 different GBM patients were continuously exposed to TMZ treatment similar to patient treatment intervals (10 mM, 5 out of 7 days) over a period of 20 weeks. Dose-response curves were generated to determine IC50 values for TMZ. RNAs from resistant cell clones were subjected to RNA sequencing. Expression changes of candidate genes were analyzed by qPCR, western blotting, and immunohistochemistry in resistant GSCs and in matched patient samples (GBM vs. recurrent GBM).

Results: RNA sequencing analysis revealed 49 genes dysregulated in TMZ resistant cell clones. By qPCR analyses of the most prominent 10 genes (up- or downregulated), we found that only the gene encoding for Carbonic Anhydrase 2 (CA-2) was consistently upregulated in all GSCs cell clones and in recurrent GBM vs. GBM. Further analyses confirmed a strong expression of CA-2 in GBM tissue and in GSCs. No expression of CA-2 was observed in differentiated patient-derived GBM cell lines. This also applies for other CAs identified in GBM such as CA-9 and CA-12. Furthermore, we used an inhibitor for Carbonic Anhydrases, Acetazolamide (ACZ) in conjunction with TMZ and observed a significant sensitization of GSCs (p<0.01) for TMZ in all GSCs cell lines tested.

Conclusion: To minimize the occurrence of GBM recurrence, it is essential to eliminate GSCs, which are particularly resistant to standard therapy. Here we provide a rationale for a specific and potent inhibition of Carbonic Anhydrase 2 as a means to optimize the existing chemotherapy of GBM.