gms | German Medical Science

Objective: A transmembrane protein CD133 has been implicated as marker of stem-like glioma cells (GSCs) and as a predictor of therapeutic response in malignant brain tumors. CD133 expression is commonly evaluated by using antibodies specific for AC133 epitope located in the extracellular domain of the CD133 protein. There is conflicting evidence regarding the significance of the AC133 epitope as a marker for GSCs and predictor of clinical outcome in glioma patients. The aim of the study is to evaluate CD133 estimates of AC133-based and AC133-independent assessments of CD133 expression in GSCs.

Method: Six lines of GSCs isolated from fresh GBM specimens were propagated in Neurobasal medium (NB) with bFGF (10 ng/ml) + EGF (20 ng/ml). CD133 expression was assessed by flow cytometry using CD133 specific antibodies binding either to AC133 epitope (Miltenyi Biotech) or to C-terminal epitope (CD133CT) distinct from AC1433 (Abcam). Flow cytometry results were validated by immunofluorescence staining and western blot analysis of unfractionated cell lysates or purified plasma membranes. To assess CD133 expression at different stages of the cell cycle, GSCs were synchronized by using a double-thymidine block and analyzed for surface expression of CD133 by flow cytometry.

Results: Assessments of six GSC lines using anti-AC133 antibody showed considerable heterogeneity in the CD133/AC133 estimates varying from 0.1-28.3% of positive cells in different lines. Strikingly, comparative analyses of CD133 expression by anti-AC133 and anti-CD133CT antibodies revealed more than a 10-fold difference (p<0.001) between AC133 and CD133CT estimates in all GSC lines examined. Furthermore, surface expression of CD133 is not constant but fluctuates throughout the cell cycle in GSCs. Cell cycle arrest induced by a double thymidine block leads to a nearly 2-fold decrease (11.1-6.9%, p<0.05, GSC line No.1051) in the expression of surface CD133, which can be re-gained upon re-entry into the cell cycle (6.9-21.5%, p<0.05, GSC line No. 1051).

Conclusions: Immunoreactivity for the AC133 epitope, which is considered a phenotypic marker of GSCs and a prognostic indicator for patients with GBM, does not adequately reflect the level of CD133 protein. Our data offer a unifying explanation for existing controversies regarding the roles of CD133 in human gliomas and necessitate re-consideration of previous interpretations based on the assumption that AC133 immunoreactivity is synonymous to CD133 abundance in GSCs.