university of groningen role of differentiation in

University of Groningen

Role of differentiation in glioblastoma invasionVareecal Joseph, Justin

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CHAPTER8Similar neurosphere formation, multilineage differentiation

and DNA damage repair potential in CD133+ and CD133- glioblastoma cells

Justin V. Joseph1, Saravanan Yuvaraj1, Tomar T2, Veerakumar Balasubramaniyan3, Michiel Wagemakers4, Frank A.E. Kruyt1

1Department of Medical Oncology, 2Department of Gynecological Oncology, 3Department of Neuroscience, 4Department of Neuro-surgery University of Groningen, University Medical Center

Groningen, Hanzeplein 1, 9713, Groningen, The Netherlands.

Manuscript in progress

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CD133+ and CD133- GBM cells exhibit similar stem cell properties Chapter 8

ABSTRACT

Glioblastoma (GBM) is an extremely aggressive and clinically unresponsive form of cancer. Increasing evidence supports the cancer stem cell hypothesis in GBM, which postulates that the GBM stem cells (GSCs) are responsible for tumor initiation, progression and resistance to treatment. Hence targeting the stem cell fraction in GBM could be of potential benefit in improving the poor prognosis associated with this disease. Over the recent years CD133 (Prominin1) has been used extensively as a marker for the identification of stem cell fraction in GBM. Here, we examined the potential of CD133 to enrich for GSCs in several newly generated GBM cell lines. We focused our study in testing three characteristics predominantly associated with the GSCs namely- neurosphere formation, multilineage differentiation and DNA damage repair potential. Our results indicate that both the CD133+ and CD133- population share all the above mentioned characteristics of GSCs. Further, upon differentiation of neurospheres a drastic decrease in CD133 expression was detected and preliminary experiments showed an increased sensitivity of the cells to radiation-induced apoptosis. However CD133+ and CD133- cells did not show any difference in response to radiation-induced DNA damage repair. Hence our findings add up to other studies that question the credibility of CD133 as a universal marker of GSCs, and show that stem cell properties are not restricted to the CD133+ fraction.

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INTRODUCTION

Glioblastomas (GBM) are extremely lethal primary brain tumors. Despite advances made in the therapeutic regime of other solid cancers, the treatment of GBM remains essentially palliative.1 The median survival of GBM patients treated with multimodal therapies including surgical resection, radiation and chemotherapy is less than 16 months.2, 3 This poor prognosis of GBM patients has not improved significantly over decades, underscoring the difficulties and challenges in effectively detecting and treating these lethal cancers. The fundamental problem associated with these malignancies is their highly infiltrative nature and extreme resistance to conventional treatments.1 One of the rapidly progressing yet controversial areas in glioma research is that of cancer stem cells (CSCs). The American Association for Cancer Research (AACR) in its workshop on CSCs defined these cells as a subpopulation of cells in the tumor that have self-renewal capacity and can give rise to heterogeneous cancer cells that comprise the original tumor.4 GSCs were one of the first CSCs isolated from solid tumors.5 In the original report, as few as 100 GSCs could give rise to tumors that recapitulate the parental tumors when implanted in immunodeficient mice, whereas as many as 1,000,000 non-GSCs could not.5 The cell of origin of GBM is still a matter of discussion and debate. There are studies advocating that normal neuronal stem cells could potentially serve as the cell of origin of GBM6, 7 while at the same time the credibility of such a hypothesis is questioned in studies from others, who demonstrated that even differentiated cells when transduced with short hairpin RNAs (shRNAs) targeting the gene encoding neurofibromatosis type 1 (NF1) and p53 can produce GBM in mice.8

The identification of GSCs usually involves molecular markers-based sorting of a potential stem cell population from freshly isolated tumor tissue or from in vitro cultured cells and culturing in media with defined growth factors in the absence of serum, and comparing the tumorigenic properties of this sorted population to unsorted and/ or the remaining cells in neurosphere formation assays in vitro or in an in vivo orthotopic transplantation assay.9 In this way, multiple markers like SSEA1/CD15, ALDH1, CD44 and CD133 have been proposed to enrich for GSCs.10, 13 Of the multiple markers being used for tracing stem cell fractions in GBM, CD133 remains the most widely used biomarker for the separation of CD133+ GSCs from the CD133- non-GSCs. CD133+ cells have been associated with the key features of GSCs, such as enhanced tumorigenic potential, drug/radio-resistance and neurosphere formation potential in comparison to the CD133- cells, which were largely devoid of these features.5, 14

CD133 (also known as AC133 and Prominin-1) is an 865 amino acid protein, which contains 5 transmembrane domains and 2 large glycosylated extracellular loops and has an actual molecular weight of 120 kDa.15, 16 CD133 gene (PROM1) is conserved throughout the animal

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kingdom and human PROM1 is mapped on chromosome 4p15.16, 17 CD133 expression in humans can be detected in multiple adult epithelial cells18, 20 and in non-epithelial cells like the hematopoietic stem/progenitor cells.15, 16 Other than in GBM, CD133+ stem cell have also been found in colorectal cancer, breast cancer, hepatocarcinomas, melanomas, head and neck cancers and osteosarcomas.5, 21-27 The exact physiological function of CD133 is unknown, however, there is a study linking CD133 overexpression to drug resistance in C6 glioma cells, where it was observed that the CD133 overexpressing cells showed an upregulation of P-glycoprotein mRNA transcription and elevated ABC transporter activity, which contributed to the protection from cytotoxic reagents.28 Some studies on GBM have also linked CD133 to bioenergetics stress as an elevated expression of CD133 was observed as a consequence of exposure of cells to hypoxia.29, 30 On the other hand there are reports suggesting that CD133 may not be an absolute indicator of GSCs.31, 32 However, CD133 remains a popular marker to identify GSCs.33, 34 In the present study, we sought to identify the credibility of CD133 as a potential GSC marker in some of our newly generated GBM cell lines that were propagated as neurospheres in serum free medium. Our results showed that many of the GSC associated features like neurosphere formation, multilineage differentiation and DNA damage repair potential are equally efficient in the CD133+ and CD133- cells. Hence our data further question the credibility of CD133 as a potential GSC marker.

MATERIALS AND METHODS

Cell culture and irradiation

The cell line GSC23 was a kind gift from dr. Kenneth Aldape and dr. Howard Colman, Department of Pathology, Houston, TX. The other cell lines namely GG1, GG6 and GG9 were newly generated from GBM surgical samples as described in chapter 2 and 4 of this thesis. GSC23, GG1, GG6 and GG9 were cultured as neurospheres in neural stem cell medium (NSM), which is composed of Neurobasal A-Medium (Gibco Life Technologies) supplemented with 2% B27 supplement (Gibco Life Technologies), 20ng/ml EGF (R&D systems, Abingdon, UK) , 20 ng/ml bFGF (Merck-Millipore, Billerica, MA, USA), 1% pen/strep and 1% L-glutamine (Gibco Life Technologies). Cells were passaged following dissociation with accutase (life technologies) treatment. For differentiation, neurospheres were dissociated into single cells and resuspended in media containing 10% FCS and seeded over poly-l-lysin-coated cover slips placed in 6 well plates. Once in 2-3 days fresh medium was added and the cover slips where processed after 10 days for further experiments.

Cells cultured in 6 well plates were irradiated with a 137Cs irradiator (Shepherd Mark-I, model 68, SN 643) at a dose rate of 1 Gy per minute. Cell lines were maintained at 37°C in a humidified atmosphere with 5% CO2.

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FACS analysis and FACS sorting

Briefly, neurospheres were dissociated in serum-free medium and the single cell suspension was transferred to FACS tubes and, subsequently centrifuged at 1000 RPM for 5 min, washed with FACS buffer (PBS +1% BSA), pelleted again at 1000 RPM for 5 min and resuspended in 100µl of FACS buffer. 10µl of CD133 epitope 1 targeted mAb, PE(phycoerythrin)-conjugated CD133/1, 1:10; clone AC133; [Meltenyi Biotec] was added and incubated in the dark at 4 0C for 30 min. Cells were incubated with IgG isotypes (Meilenyi Biotec) matched antibodies as a negative control. After incubation the cells were washed 3 times with the FACS buffer prior to analyses. The threshold was adjusted in forward and side scatter dot plot to exclude cellular debris and a total of 10000 events were analyzed on a FACSCaliburTM flow cytometer running CellQuest software (BD Biosciences). For FACS sorting of the CD133+/- fractions ~ 5 x 10 6 cells were labeled as described above. After sorting, aliquots of CD133+ and CD133- sorted cells were evaluated for purity by using a mAb targeting epitope 2 of CD133 [PE(phycoerythrin)-conjugated CD133/2, 1:10; clone AC133; Miltenyi Biotec] and subsequent flow cytometry with a FACSCalibur (BD Biosciences). Sorted cell populations were resuspended in serum-free media or media containing 10% FCS when appropriate for further analyses.

Limiting dilution assay

Limiting dilution assay was performed as previously described.39, 40 In brief, neurospheres were dissociated and resuspended in PBS at a density of ~ 2x106 cells/ml. Using FACS (BD Biosciences) 10, 20 and 40 cells were seeded into 96 well plates containing 100 µl of NSM; a total of three 96 well plates were used for each cell number. An extra 75 µl of NSM was added to the plates every week for replenishing the growth factors. After 21 days the formed neurospheres were counted using an inverted microscope at 5x magnification. The average number of neurospheres formed per condition was calculated and graphically depicted.

Western blotting

Briefly, cells were harvested, washed with cold PBS and lysed with M-per mammalian protein extraction agent (Thermo Scientific) supplemented with 1% protease inhibitor (Thermo Scientific) and 1% phosphatase inhibitor (Thermo Scientific) for 1.5 hours on ice. Next, the suspension was centrifuged for 10 minutes at 14000 RPM at 4˚C and the supernatant was taken for determining protein concentrations using a Bradford assay (Bio-Rad). 25µg of proteins per sample per lane was loaded for SDS-PAGE electrophoresis. Proteins were then transferred to PVDF membrane (Millipore IPVH00010 0,45 µm). The membrane was blocked for 1 h at room temperature (RT) with 5% milk in TBST (20 mmol/1 Tris-HCL (pH 8.0), 137 mmol/l NaCl and 0.1 % Tween-20). Primary antibodies were incubated overnight at 4 0C. Primary antibodies used were: Nestin [1:500, Santa Cruz Biotechnology INC (sc-23927)], Vimentin

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[1:500, Santa Cruz Biotechnology INC (sc-373717)], Oct4 [1:1000, abcam, (ab-19857)], Anti-phospho-Histone H2A.X [1:200, Millipore, (05-636)]. After incubation membranes were washed with TBST, and incubated with appropriate HRP-conjugated secondary antibodies, either anti-mouse immunoglobulin G (IgG), anti-rabbit IgG or anti-goat IgG) (Dako) for 1 h at RT. Proteins were visualized using Amersham Biosciences enhanced chemiluminescence (ECL) detection system (GEHealthcare, UK).

Immunofluorescence microscopy

Cells cultured on poly L lysine (Sigma-Aldrich)-coated cover slips were fixed for 10 min using 4% formaldehyde or 100% methanol. After 3 times washing with ice cold PBS, cells were permeabilized with 0.1% Triton (Sigma-Aldrich) in PBS, washed again with PBS followed by a blocking step for 1 hr with PBS + 0.1% Tween-20 (Sigma-Aldrich), 2% BSA (PAA Laboratories GmbH, Germany) and 1:50 dilution of normal goat serum (Dako Denmark A/S, Denmark). Subsequently, cells were incubated with the indicated primary antibodies at room temperature for 1.5 hrs. Primary antibodies used: Polyclonal Rabbit Anti-Glial Fibrially Acidic Protein (GFAP) [1:400, Dako (N1506)], Anti-Olig2 antibody [1:100, abcam (ab81093)], Anti-beta ІІІ Tubulin antibody [1:200, Abcam, (ab76287)], Anti-phospho-Histone H2A.X [1:100, Millipore, (05-636)]. After 3-times washing with PBS, slides were incubated for 1 hr with the appropriate secondary antibodies: goat anti-Mouse Alexa488 (1:200, Life Technologies), or Goat anti-Rabbit IgG Antibody, Cy3 conjugate (1:400, Millipore (AP132 C)). Hoechst (Sigma H6024) staining was performed for 5 minutes followed by mounting the coverslips with Kaisers glycerin (Merck, Germany). Cells were examined by fluorescent microscopy (Leica DM6000, Leica Microsystems GmbH, Mannheim, Germany) and images were captured using Leica DFC360 FX camera.

Statistical analysis

In-vitro data are presented as the mean ± standard error of the mean (SEM) using the GraphPad Prism version 5.01 (GraphPad for Science, San Diego, CA). Statistical significance was calculated by two way student’s t-test and multiple comparisons between different groups were performed by one-way ANOVA with Bonferroni post-test unless otherwise mentioned in the figure legends. p values < 0.05 were assumed as statistically significant for all the tests.

RESULTS

CD133 expression in GBM cell lines

Earlier established GSC23 cells35 and newly generated GBM cell lines, GG1, GG6 and GG9, were propagated and maintained as neurospheres in NSM (Figure 1 a). FACS analysis of these GBM cell lines showed differential expression of CD133; GSC23 had approximately 24,5(±15) % of CD133+ cells, GG1 0,4(±0.11) % , GG6 3(±0.5) % and GG9 17(±1.3) %

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8Figure 1. Variable levels of CD133 expression in newly generated GBM cell lines. (a) Phase contrast microscopic pictures (x10) of GSC23 and newly generated GBM cell lines GG1, GG6 and GG9 being propagated as neurospheres in serum free medium. (b) GSC23, GG1, GG6 and GG9 cells were stained with anti-CD133 (blue histograms) or isotype-matched control antibodies (black or red histograms) and analyzed by flow cytometry. A representative experiment is shown.

(Figure 1 b). It should be noted that the percentage of CD133 expressing cells in these GBM lines also varied; in particular GSC23 showed strong fluctuation in CD133 expression levels.

CD133+ and CD133- cells have a similar neurosphere forming potential

The CD133+ and CD133- cell fractions were FACS sorted from GSC23 and GG9 cells (Figure 2a). The sorted CD133+ and CD133- cells had a purity of ~ 95% and 98%, respectively

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(Figure 2 b). Next, the neurosphere forming potential of CD133+ and CD133- cells derived from GSC23 and GG9 was compared and showed no significant differences between the number and size of the neurospheres. On an average around 30% and 20% of both CD133+

and CD133- populations of GSC23 and GG9, respectively, produced neurospheres in serum free medium (Figure 2 c-f). Following sorting of the CD133+ and CD133- fractions from GSC23 cells and prolonged culturing for a month CD133 expression levels remained mostly constant, unlike the unsorted parental GSC23 cells that as earlier mentioned showed significant variation in the CD133 expression levels in different passages (Supplementary Figure 1a). In the CD133+ and CD133- fractions of GSC23 we also tested the expression levels of other stem cell-associated factors, such as Vimentin, Nestin and OCT4,6, 36, 37 and no significant difference was observed in the expression pattern of these proteins (Supplementary Figure 1b).

CD133+ and CD133- populations can be differentiated by serum

One of the key features associated with stem cells is their ability to differentiate into one or more different lineages of the tissue in which the stem cells reside.38 In order to test the differentiation potential of the CD133+ and CD133- cells these cells were exposed to media containing 10% FCS for 10 days. Serum-induced differentiation was observed that was accompanied by the adherence of cells with astrocytic morphology (Figure 3 c). Differentiation was confirmed by immunofluorescent staining of cells with astrocytic (GFAP), neuronal (βІІІ tubulin) and oligodendroglial (OLIG2) markers. Both the CD133+ and CD133- fraction of GSC23 and GG9 cells showed similar differentiation potential and with particularly high expression of GFAP(Figure 3 a, b). Activation of the differentiation program also reduced the expression of the stem cell marker SOX2 (not shown) and interestingly, differentiation also reduced the CD133 expression levels by four fold in comparison to the undifferentiated GSC23 cells (Figure 3 d).

Radiation-induced DNA damage repair is equally efficient in both CD133+ and CD133- GSC23 cells

Previously it was reported that the CD133+ GSCs are more resistant to radiation-induced DNA damage in comparison to the CD133- non-GSCs.14 Here we also tested this finding by irradiating (5 Gy) the FACS-sorted GSC23 CD133+ and CD133- cells and after 2 and 24 hrs recovery cells were stained with gH2AX in order to determine the level and repair of double strand(ds) DNA breaks. The non-irradiated CD133+ and CD133- cells showed little or no dsDNA breaks (Figure 4a). At 2 hrs post-irradiation both the CD133+ and CD133- fraction showed high numbers and equal amounts of foci representing dsDNA breaks (Figure 4b). At 24 hrs post-irradiation a significant decrease of breaks was detected in both cell fractions (Figure 4c) indicating more or less equal DNA damage repair activities. The quantification of

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8Figure 2. CD133+ and CD133- GBM cells have similar neurosphere forming potential. (a) Representative dot plots of FACS sorting of CD133+ and CD133- cell fractions from GSC23 and GG9 using CD133-PE antibody. The red boxes indicate the sorted cell fractions. (b) Sorted CD133+ and CD133- cells from GSC23 were re-stained with CD133/2-PE antibody (blue histogram) or isotype-matched control antibodies (black histogram) and analyzed by FACS to evaluate the purity of the sorted population. (c-d) CD133+ and CD133- cells were sorted from GSC23 or GG9 cells and seeded in 96 well plates in serum free medium and the average numbers (no.) of neurospheres counted at day 21 post seeding are indicated and quantified (e-f) Phase contrast microscopic pictures (x10) of neurospheres generated from CD133+ and CD133- GSC23 and GG9 cells.

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the amount of DNA damage sustained and repaired by these cells did not show a significant difference between the CD133+ and CD133- cells (Fig 4d). These results indicate that the CD133+ and CD133- GSC23 cells have similar abilities to repair radiation-induced dsDNA breaks.

Figure 3. Multilineage differentiation potential is equally induced in CD133+ and CD133- cells. (a-b) Immunofluorescence staining of serum differentiated CD133+/- cells from GSC23 and GG9. Cell are stained for the GFAP-astrocyte marker, OLIG2-oligodendrocyte marker and βІІІ tubulin-neuronal marker (all in red) and DAPI (blue) to label all nuclei. The images were acquired at x20 magnification using a fluorescence microscope. (c) Phase contrast microscopic pictures (x10) of GSC23 neurospheres and serum differentiated monolayer cells (GSC23D). (d) CD133 expression levels were assayed by FACS in GSC23 and GSC23D cells showing ~5 fold decline following differentiation.

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Figure 4. CD133+ and CD133- cells display similar DNA damage repair potential post irradiation. (a-c) Immunofluorescence microscopy of unirradiated CD133+/- GSC23 cells and irradiated (5 Gy) cells at 2 hrs and 24 hrs post irradiation being stained for dsDNA breaks using γH2AX (green) or with DAPI (blue) to label nuclei. The images were captured at x40 magnification. Quantification shown in (d).

Radiation-induced DNA damage repair is equally efficient in neurospheres and differentiated GSC23 cells

Finally, we examined whether serum-induced differentiation of GSC23 cells may affect the response to irradiation-induced DNA damage. In addition also apoptosis activation was studied. Both the differentiated and undifferentiated GSC23 cells were exposed to 5Gy radiation and analyzed for γH2AX foci and caspase-3 cleavage as a read-out for apoptosis. Both differentiated and undifferentiated cells had similar levels of foci after 2 and 24 hrs post-irradiation, however, differentiated cells appeared to have higher levels of cleaved caspase-3 (Supplementary Figure 2 a, b). Although preliminary, these findings suggest that the differentiation state of the GSC23 cells does not influence the ability to repair dsDNA breaks, but may affect the induction of apoptosis.

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DISCUSSION

In order to evaluate the use and credibility of CD133 as a potential stem cell marker in several of our newly generated GBM cell lines we sorted the CD133+/- fractions and compared these fractions in terms of their neurosphere formation potential, multi-lineage differentiation potential and DNA damage repair capacity, as these three features have been traditionally linked to stem cells. We found that both the CD133+ and CD133- cells showed striking similarities in there neurosphere formation potential both in terms of the number of neurospheres produced and the size/morphology of the neurospheres. We also observed that the CD133- and CD133+ cells maintained their CD133 negativity and positivity over an extended time period, which might indicate that the CD133+ cells largely gives rise to CD133+ cells and similarly the CD133- cells might end up producing largely CD133- cells. The finding that in unsorted GSC23 cells CD133 levels are more variable suggests the possible involvement of yet unknown interactions between CD133-/+ cells. Neurospheres derived from both the CD133- and CD133+ cells both showed high expression of other stem cell markers like Vimentin, Nestin and OCT4 and could be differentiated by exposure to serum containing medium in to adherent astrocytic cells characterized mainly by GFAP expression. Of note, serum-induced differentiation of CD133+ cells resulted in a 5-fold decrease in CD133 expression, and was the only finding suggestive of CD133 being a potential stem cell marker. Taken together, our findings strongly suggest that CD133- cells possess similar stem cell properties as the CD133+ cells.

There are also studies advocating for enhanced DNA damage repair potential in CD133+ cell in comparison to the CD133- cells.14 We also examined this by looking at the repair of dsDNA breaks induced by radiation and in contrast found that the DNA damage repair potential was equally effective in the CD133+ and CD133- cell fractions as well as upon serum-induced differentiation. Difference between our and the previous reported findings may involve cell-dependent differences. Additional experiments in a broader panel of GBM cell lines are required to further examine a possible correlation between CD133 expression and differentiation state and DNA damage responses. Interestingly, our preliminary results showed that differentiation of neurospheres sensitized the cells to radiation-induced apoptosis as determined by cleaved caspase-3 (CC-3) staining. This difference in apoptosis activation could not be related to differences in DNA repair, which was independent of differentiation as mentioned above. Additional work is required, including clonogenic survival assays and detection of additional apoptotic markers, to further establish a possible relationship between the differentiation state and radiation-induced apoptosis in GBM.

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Several early reports proposed that the expression of CD133 was synonymous with, or at least a prerequisite for GSCs.5, 14, 41-43 However, in agreement with our current findings there are also multiple reports suggesting that CD133 may not be an absolute indicator of stem cell properties.29, 31, 32, 44, 45 The clear discrepancies in the literature on CD133 being a GSC marker could be explained in part by poor reliability of antibodies detecting CD133+ expression32,

46 and/or observations showing that the expression of CD133 in itself may be modulated by environmental factors such as hypoxia and cellular stress.29, 47 A recent study showed that exposure of patient derived as well as established glioma cell lines to therapeutic doses of temozolomide (TMZ), which is the most commonly used chemotherapeutic drug against gliomas, consistently increased the GSC pool, and was also associated with an enhanced expression of pluripotency and stemness associated genes, such as SOX2, OCT4, and Nestin and also CD133.34 Furthermore, the level of CD133 expression was previously found to highly vary between individual cases of GBM, ranging from 0.5% to 82%.48 Thus, CD133 appears not a universal marker for GSCs, and in line with this also other GSC markers have been reported (CD15, ALDHA1, CD44).10-13

GBM has been classified into four transcriptional subtypes, mesenchymal, classical, neural and proneural.49 Recently, Zarkoob et al.50 made a comparison between a genetic signature selective for CD133 positive GBM cells with each of the molecular GBM subtypes and found a significant positive correlation of the CD133 signature with the mesenchymal and neural subtypes, compared to the classical and proneural subtype. On the other hand the CD133 gene itself was found to be less expressed in the mesenchymal subtype when compared to the other subtypes. This observation is in agreement with earlier findings of Chen et al.51 who showed that the mesenchymal subtype of GBMs mostly comprised CD133- cells. Zarkoob et al. explain this discrepancy by assuming that there is a difference between a gene signature that reflects cellular processes and expression of the gene itself. Of note, the GBM cells we used in our current study represented 2 subgroups with GSC23 and GG9 belonging to the proneural and GG1 and GG6 to the mesenchymal subtype, respectively.

It has been proposed that CD133 may be a good marker for detecting GSCs from freshly isolated tumor material, and that this property is lost during in vitro culturing.52 Culturing on plastic inevitably gives rise to clonal selection and depletion of the supporting microenvironment and this might all together change the marker expression pattern of these cells. It is possible that passaging freshly isolated tumor cells in mouse instead of plastic flasks might help retain the credibility of CD133 as a potential stem cells marker.

Despite multiple markers that are being used in tracking the stem cell fraction in gliomas as well as normal NSCs, so far there is no one universal marker or a combination of markers

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which could identify and define these cells in pure form. Taking in to account the extremely complex genetic and epigenetic heterogeneity of GBMs, it is highly unlikely that the expression of a single marker such as CD133 will detect GSCs in all tumors.

In conclusion, our findings further support the view that CD133 has its clear limitations in tracing/sorting GSCs in GBM.

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SUPPLEMENTARY DATA

Supplementary Figure 1. CD133 expression is maintained over extended periods in sorted cells. (a) CD133 expression was monitored in CD133+ and CD133- GSC23 cells at day 1, 14 and 31 post sorting. (b) Western blot analysis comparing the expression of NSC markers Nestin, Vimentin and OCT4 in CD133+/- sorted and unsorted GSC23 cells.

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Supplementary Figure 2. Radiation-induced DNA repair and apoptosis in neurosphere-cultured and differentiated GSC23 cells. (a) Immunofluorescence microscopy of GSC23 neurosphere and differentiated derivatives irradiated and stained for γH2AX (green), cleaved caspase-3 (red) and with DAPI (blue) at 2 hrs and 24 hrs post irradiation. The images were obtained at x40 magnification. (b) Western blot analysis comparing the expression of γH2AX

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