gms | German Medical Science

Objective: Aldehyde dehydrogenase 1 (ALDH1) is a detoxifying enzyme involved in the oxidation of intracellular aldehydes. In glioblastomas (GB), isoforms A1 and A3 have been implicated in the regulation of tumor growth and resistance to cytotoxic therapy. Recent evidence suggests that A1 or A3 may have non-redundant functions in newly diagnosed and recurrent GB (ndGB and recGB, respectively). Up to date, investigations on ALDH1 isoforms have been confined to analyses of GB tissues or long-term cell lines lacking the properties of cancer stem cells. This study aims to characterize ALDH1 isoforms A1 and A3 in glioblastoma stem cells (GSC) representing a therapeutically relevant target in GB.

Methods: ALDH1 expression in FFPE tissue or cultured GSC was analyzed. FFPE tissue and GSC were obtained from freshly resected ndGB oder recGB. GSC were isolated using the Neural Tissue Dissociation Kit (Miltenyi Biotec) and maintained in NeuroBasal medium supplemented with B27 component and self-renewal factors bFGF and EGF. GSCs differentiation was induced by bFGF/EGF withdrawal. ALDH1 expression was analyzed by immunostaining using antibodies recognizing A1 or A3 isoforms. Antibody epitope mapping was performed by western blot using a panel of antibodies, binding to different regions of the ALDH1(A3) protein.

Results: Immunohistochemistry showed a high degree of inter- and intra-tumoral heterogeneity of A1 expression in either ndGBs (n=62) or recGBs (n=62). A1 expression was found to be primarily associated with tumor-infiltrating macrophages (CD68 co-staining). In contrast, GSC cultures showed a consistent pattern characterized by homogeneous expression of A3 and a lack of A1 expression. A3 expression in tumor cells could be confirmed in selected cases (n=6) in FFPE material. GSC-associated A3 was found to have an abnormal localization in the nuclear matrix. Biochemical analyses revealed the expression of a 33 kDa protein that is recognizable by different ALDH1(A3) antibodies and abundantly expressed in a panel of 8 ndGB- and 4 recGB-derived GSCs. Antibody epitope mapping suggests the A3 identity of a 33 kDa peptide lacking the A3 catalytic domain.

Conclusion: Our results indicate that ALDH1(A3) but not ALDH1(A1) is a marker associated with GSCs. Our findings urge to unequivocally determine the identity and functions of a putative A3 peptide expressed in GSCs and establish the role of the nuclear A3. Given the diagnostic merits of ALDH1, clarification of these aspects has considerable clinical importance.