gms | German Medical Science

Objective: CD133 is a five transmembrane domain glycoprotein, which is widely used for selection of brain tumor stem cells, although the biological function of CD133 is not well understood. CD133 expression in brain tumor tissue is correlated with poor prognosis. Furthermore, the higher expression of CD133 mRNA indicates the presence of CD133 protein, despite the absence of glycosylated epitopes upon cell differentiation. Here we investigated CD133 antibodies for their applicability in the reliable detection of CD133 as a prognostic factor in brain tumors.

Method: Six different CD133 antibodies raised against epitopes mapping within an extracellular domain and intracellular domain were used for immunohistochemical staining and Western Blot analysis in different human brain tumors (meningiomas and gliomas). The positive control consisted of a Y79 cell line and a human retinoblastoma cell line positive for several stem cell markers including CD133. For the negative control, HeLa, which is described as lacking CD133 expression in the literature, was used.

Results: Immunohistochemical staining of CD133 displayed a wide range of results from strongly positive stainings to no stainings in the same tissue using serial paraffin sections. On antibody mapping, the C-terminus of CD133 showed no staining in 11 of 36 meningiomas, in 9 tumors sporadic cells were stained, in 8 meningiomas up to 10% of the cells were stained and in 8 meningiomas more than 10% of the cells were stained. In contrast one antibody mapping within the extracellular domain displayed no staining in 20 of 36 (56%) meningiomas and in 15 (42%) meningiomas only sporadic cells were stained. Only one meningioma showed staining in up to 10% of the cells. In Western Blot analysis, five of six antibodies displayed bands at 97 kDa for positive control Y79 cell lysate and kidney. One widely-used CD133 antibody showed false positive staining for the HeLa cell line. Five of six antibodies showed no stainings for a malignant meningioma cell line, only the CD133 antibody, that showed false positive staining for HeLa cell line also displayed a specific band for CD133.

Conclusions: All six analysed antibodies detected CD133 highly inconsistently, independent of their extra- or intracellular epitope localisation. Antibodies displayed both false positive and false negative CD133 expression in distinct controls in both immunohistochemical stainings and Western Blot. Therefore conclusions based on only one CD133 antibody should be examined carefully and verified with standard negative and positive controls.