Glioblastoma cells negative for the anti-CD133 antibody AC133 express a truncated variant of the CD133 protein

Abstract

Abstract. The transmembrane glycoprotein CD133 is a marker commonly used for isolation and analysis of putative cancer stem-like cells. However, analysis of CD133 expression is potentially confounded by the fact that two of the commonly used anti-CD133 antibodies, AC133 and 293C, only recognize CD133 that has undergone glycosylation. Therefore, our aim was to thoroughly examine antibody recognition and mRNA expression of CD133 in glioblastoma multiforme. Glioblastoma cell lines and primary cultures obtained from resected tumor tissue were analyzed by real-time PCR, Western blot analysis, and flow cytometry for CD133, and immunofluorescence was used to determine cellular localization. The AC133 and 293C antibodies did not detect any CD133 on the surface of the glioblastoma cells despite the fact that a protein was detected using C24B9, an anti-CD133 antibody that recognizes an unglycosylated epitope. This CD133 variant was truncated (~16 kDa) and, unlike typical expression of full-length CD133 protein, was found throughout the cytoplasm instead of localized to the plasma membrane. Levels of mRNA and protein for the variant increased with stress, indicating potential for it to be a functional molecule. Because AC133 and 293C antibodies do not detect all CD133 variants in glioblastoma cells, alternate detection methods need to be utilized for complete analysis of CD133 expression and for accurately determining the relationship between CD133 and cancer stem-like cells.

Emerging roles of prominin-1 (CD133) in the dynamics of plasma membrane architecture and cell signaling pathways in health and disease

“…1 b) [ 10 ]. A 16-kDa truncated variant of the CD133 protein has also been reported in glioblastoma cell lines [ 109 ]. Yet, the precise nature of this potential short form would require further investigation.…”

The Impact of Glycosylation on the Functional Activity of CD133 and the Accuracy of Its Immunodetection

“…The use of different clones of antibodies against CD133 can lead to differences in tissue staining patterns. Partly, this may be due to the detection of alternative splice variants, either tissue-specific or appearing in tumor cells [ 137 ]. However, the absence of binding to extracellular loops is more likely to indicate a change in glycosylation.…”

Engineering Three-Dimensional Tumor Models to Study Glioma Cancer Stem Cells and Tumor Microenvironment

“…Given its superficial location, detection of CD133 may vary depending on several factors such as cell-microenvironment interactions and epigenetic influences. Careful analysis of its informational value is recommended as immediate cell-extracellular matrix (ECM) disassociation, extended in vitro culture, and/or equivocal epitope recognition may give rise to false-negative results (Clément et al, 2009;Osmond et al, 2010;Campos et al, 2011).…”