gms | German Medical Science

Objective: Stem cell associated cells with CD133 as the most popular marker are considered an important factor for the treatment failure in high grade gliomas. But during the last years, the relevance of the surface protein CD133 has become more and more an issue of discussion. In this study we define possible further marker genes describing stem cell and proliferation properties in a tumor sphere model.

Methods: We defined 4 groups of glioblastoma cells being cultivated under different conditions. Group A (n=5): Cells isolated according to an established tumor stem cell isolation protocol, Group B (n=4): Cells from a commercial cell line, Group C (n=6): Cells from adherent primary glioblastoma cell cultures. Group A-C were cultured in serum-free sphere medium to induce a spherical growth pattern, while adherent primary cultures were cultured as Group D (n=6) in serum medium. The three sphere groups were compared according certain features associated to stem cells, like the time to create spheres, metabolic activity (via MTT-assay) and migration ability. Differentiated or sphere stages were control via immunofluorescence staining. Presuming the maximum fraction of potential stem cell like cells in group A, we analyzed the following markers via RT-qPCR: CD133, MUSAHSI-1, SOX2, OCT4, NANOG, NESTIN, GFAP and NOTCH1 with group A as reference.

Results: The stem cell isolated spheres of group A needed in mean 12d more (p=0,012) to create greater spheres and migrated more strongly (p=0,001) with also a ten times lower metabolic activity (p= 0,004 till 0,016) compared to the sphere induced groups (B, C). All spheres showed mostly CD133 on immunofluorescence staining. On mRNA-level, there was a downregulation of MUSASHI-1** (17-680 fold), NESTIN*** (10-130), NOTCH1 (5-15 fold), GFAP** (250-1900 fold) and Sox2* (4-12 fold) and an increased gene expression of the cell surface marker CD133 (12-24 fold) in groups B-D compared to group A. OCT4 (0,5-1,4 fold) and SOX2 (in Gr. C: 1,1 fold) showed no regulation (*significant). NANOG was not representatively detected.

Conclusion: Concerning our data, Musashi-1 is a more valid marker, supporting through the data of physiological level, to define tumor stem cell subpopulation than CD133. Additionally, the significantly increased NESTIN- and NOTCH1-expression correlated with MUSASHI-1 and seems to be associated with stem cell like properties