gms | German Medical Science

Objective: The biological significance of CD133 in glioblastoma has been the subject of controversial scientific debate. Above all, there is disagreement concerning the proper approach, the appropriate (cell) model and the suitable microenvironment to study this molecule, which often led to inconsistent experimental results among studies.

Methods: In consideration of a primary need to dissect and to understand the CD133 phenotype in glioblastoma, we performed a comprehensive analysis of CD133 expression and its regulation in a large set of different glioblastoma cell lines (n=20) by qPCR, FACS analysis and immunocytochemistry. Our analysis considered alternatively spliced mRNA transcripts, different protein epitopes as well as varying sub-cellular localizations of CD133 and explored its regulation under pertinent microenvironmental conditions such as normoxia and hypoxia.

Results: In our study sample, CD133 mRNA and the CD133 protein could be detected in all relevant types of cell lines, regardless of the experimental context and without qualitative differences. Corresponding to the prevalence of CD133 mRNA we detected intracellular CD133 protein accumulations, which were either located to the ER, the golgi apparatus or both but were unrelated to particular CD133 mRNA splice variants or individual protein epitopes. In contrast, extracellular membrane-bound expression of CD133 was restricted to tumor cells bearing the extracellular CD133 epitope AC133. In the latter, differentiation-permissive culture conditions and physiological oxygen levels clearly influenced AC133 expression, CD133 mRNA levels and CD133 protein expression. In addition, we demonstrate that modulation of AC133 expression levels in AC133-positive tumor cells occurs independent of changes in CD133 mRNA transcription, CD133 protein translation, protein retention or protein shedding. We propose that this might be due to discrete changes in the extracellular CD133 protein resulting in either exposure or masking of the AC133 epitope.

Conclusions: Our results corroborate an important role for the AC133 epitope because neither CD133 mRNA nor protein content but only AC133 levels mirror cellular changes that occur under malignancy-modulating conditions such as differentiation and reduced oxygen levels.