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Flow cytometry assessment of anti-CD133 antibodies in human glioblastoma multiforme cells

Sunguroglu A, Department of Medical Biology, Faculty of Medicine, Ankara University, Ankara, TURKEY
Akcora Yildiz D, Department of Biology, Art&Science Faculty, Mehmet Akif Ersoy University, Burdur, TURKEY
Yukselten Y, Department of Medical Biology, Faculty of Medicine, Ankara University, Ankara, TURKEY
Bal M.G, Department of Medical Biology, Faculty of Medicine, Ankara University, Ankara, TURKEY
Bunsuz M, Department of Medical Biology, Faculty of Medicine, Ankara University, Ankara, TURKEY
Demircan K, Department of Medical Biology, Faculty of Medicine, Turgut Özal University, Ankara, TURKEY
Ugur H.C, Department of Neurosurgery, Faculty of Medicine, Ankara University, Ankara, TURKEY


Cancer stem cells (CSCs), commonly recognized by the expression of cell surface markers, are thought to be responsible for initiation of the primary disease, recurrence and chemo- / radio-resistance in a growing number of hematological and solid tissue malignancies including glioblastome multiforme (GBM). GBM is the most prevalent and aggressive type of brain tumors in adults. In GBM, the neural stem cell marker CD133, known as prominin-1, has been widely used as a CSCs marker. Although, CD133+ GBM cells were shown to have strong tumorigenicity and be responsible for initiation and maintenance of tumor, CD133- GBM cells were also reported to exhibit similar properties. Antibodies recognizing AC133 (CD133/1), a glycosylated CD133 epitope, have been frequently used to distinguish CD133 positive and negative cell populations. In addition to AC133, 293C/AC141(CD133/2), a second glycosylated CD133 epitope has also been used for the same purpose. Recent studies indicated that post-translational modifications like glycosylation or change in folding of the CD133 protein may lead to obtain higly variable CD133 staining results. Therefore, we have investigated the staining of two PE (phyco-erythrin) conjugated anti-CD133 monoclonal antibodies, recognizing AC133 and 293C, in flow cytometry analysis (BD Biosciences) by comparing their capacities to detect human CD133+ GBM cells derived from both cell lines (U87, U118, U138G and T98G) and newly diagnosed GBM patients (n=3). We found that U138G, T98G, U118 and U87MG cells stained with AC133 antibody have 5,83%, 32,3%, 12,29 and 9,79% CD133 expression, respectively. Whereas these cells in the same order stained with 293C antibody had 8,5%, 2,5% and 4,55% CD133 expression. Such an increased expression of CD133 was observed with the use of AC133 rather than 293C antibody in T98G cells. Furthermore, two of three patient-derived GBM tumor cells stained with both AC133 and anti-293C antibodies had similar amount of CD133 positive cells. However, GBM tumor cells derived from one patient’s specimen stained with AC133 and 293C antibodies had 9,39% and 19,71% CD133 staining, respectively. In conclusion, we revealed a staining variation in GBM cells derived from T98G cell line or one patient’s specimen by using two different antibodies recognizing two distinct glycosylated epitopes. Therefore, our results suggest that the detection of CD133 positivity with the use of different antibodies recognizing different glycosylated epitopes in GBM patients and the use of antibodies detecting the high rate expression of CD133 could be important for studying the stem cell biology of GBM. This research has been supported by The Scientific and Technological Research Council of Turkey (No:114S189).

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