Surface Protein Dynamics in Glioma Stem Cells

Research Article

Austin Neurosurg Open Access. 2014;1(3): 1015.

Surface Protein Dynamics in Glioma Stem Cells

Soeda A1*, Ohe N1, Lee D2, Iwama T1 and Park DM2

1Department of Neurosurgery, Gifu University School of Medicine, Japan

2Department of Neurological Surgery, University of Virginia, USA

*Corresponding author: Soeda A, Department of Neurosurgery, Gifu University School of Medicine, 1-1 Yanagido, Gifu City, Gifu 501-1194, Japan

Received: May 14, 2014; Accepted: June 05, 2014; Published: June 05, 2014


Introduction: Flow cytometry is used for isolating Cancer Stem Cells (CSCs) by recognizing their surface markers such as CD133, CD44, CD24, and CD15. Although several CSC markers are helpful to identify CSCs, it remains unknown whether these markers can be used only when freshly derived from surgical tissue specimens or even after long–term exposure to the in vitro environment.

Material and Methods: We established and evaluated 8 glioma cell lines from glioblastoma multiforme tissue specimens. Flow cytometry was used to analyze the stem cells, glia, and neuronal– and cell–adhesion molecules at the following time periods: (1) within 3 months of tumorsphere culturing (primary analysis), (2) between the stage of dissociated single cells and subsequently developed spheres at the same passage period (passage analysis), and (3) 3–36 months after primary analysis (mid⁄long–term analysis). We also evaluated the differences in the surface markers after differentiation following serum addition.

Results: The primary analysis of the fresh surgical tissue specimens revealed different expression patterns, especially for CD24 (5–99%) and CD184 (3–40%), with higher expression levels for CD44 and CD146. After sphere development from the dissociated single cells, the CD54 expression was elevated with epidermal growth factor receptor (EGFR) degradation during cell passages. After serum addition, the CD133, A2B5, CD24, CD56, and CD184 expression decreased, suggesting the potential of these proteins as stemness markers. At long–term analysis, most surface markers were found to be stable; however, the expression profiles of several markers differed among the CSC lines.

Conclusion: Glioma CSCs maintain stable expression of stem cell markers even under long–term in vitro propagation. Our results may facilitate identification of novel cell markers for application in the diagnosis and treatment of gliomas.

Keywords: Glioma; Glioma stem cell; Flow cytometry; CD133; Cell adhesion molecule


Cancer Stem Cells (CSCs) have been identified in several malignancies, and a variety of experimental approaches have been initiated to analyze their properties [1]. Comprehending the unique properties of CSCs is a high priority for researches aimed at elucidating the molecular mechanisms driving tumor initiation and for the development of therapeutic strategies specifically targeting CSC population [2]. Recent advances in stem cell biology, cell signaling, computational technology, and genetic model systems have revolutionized our understanding of the mechanisms underlying the genetics, biology, and clinical behavior of cancer [3]. Among these, flow cytometry technique can be applied for the isolation of CSCs by recognizing the CSC surface markers such as CD133, CD44, CD24, and CD15 [4–6].

CD133 is the most commonly used glioma CSC marker for studying aspects such as in vivo–tumor formation ability [4]. Although the function of CD133 remains unknown, it has proven useful in several other solid cancers such as colorectal cancers [7]. However, several reports have suggested a less clear distinction between the abilities of CD133+ and CD133 cells to form tumors [8,9]. Several strategies have been used for the identification of CSCs of gliomas and other cancers such as colon, prostate, and lung cancers. For example, CD44 has been identified as a potential breast and prostate CSC marker [5,10]. Moreover, the expression profile of CD44 also identifies the astrocytic progenitors, and most gliomas express this marker [11,12]. High CD24 expression level helps identify transitamplifying cells as well as differentiated neurons, and CD24 is also required for the terminal differentiation of neuronal progenitors [13]. Furthermore, A2B5–expressing glial–restricted precursor is capable of generating oligodendrocytes astrocytes and gliomas [9]. CD184 is a chemokine receptor involved in Neural Stem Cell (NSC) migration; it has been implicated in the invasion of gliomas and metastasis of pancreatic CSCs [14]. Moreover, several CAMs are also involved in gliomagenesis [15,16]. However, it remains unknown whether thesemarkers are useful only when used immediately after derivation from surgical specimens or even after long–term exposure to the in vitro environment.

Patient–specific CSC lines are a powerful tool in the study of CSC biology that can be exploited for the development of therapies targeted at specific patients. However, CSCs may adapt differently to the prevailing culture conditions or may reflect the intrinsic genetic fluctuations typical of tumor cells [17–19]. The isolation and manipulation of CSCs may introduce artifacts under in vitro conditions at primary analysis, long–term analysis, and under in vivo microenvironment [20]. Alternatively, CSCs may be reprogrammed in long–term in vitro culture settings or may dedifferentiate in vitro from a more differentiated cell type in response to certain signal transduction cascades [21]. Therefore, to address these issues, it is important to characterize CSC lines from different patients at different time periods.

In this study, we evaluated several surface markers obtained from surgical tissue specimens and glioma CSCs derived from patients by the neurosphere assay [19]. We compared the surface–marker dynamics at the following time periods: (1) within 3 months of tumorsphere culturing (primary analysis), (2) between the stages of dissociated single cells and subsequently developed spheres (during the same passage), and (3) 6–36 months after primary analysis (middle to long–term analysis). To minimize the influence of artificial in vitro effects, long–term analysis was performed at least twice and their results were averaged. Because CSCs can differentiate in the presence of ideal compounds such as retinoic acid, bone morphogenetic protein, and serum, a better understanding of the makers expressed in differentiated CSCs will be useful in CSC biology [18,22]. With this perspective, we evaluated the surface–marker differences after differentiation following serum addition.

Material and Methods

Cell culture

Tumorsphere culturing was performed as described previously with some modifications in the medium. We used Dulbecco’s modified Eagle’s medium⁄nutrient mixture F–12 (DMEM–F12; GIBCO–Invitrogen, La Jolla, CA) supplemented with penicillin G, streptomycin sulfate, B–27 (GIBCO–Invitrogen), recombinant human FGF–2 (20 ng⁄mL; R&D Systems, Minneapolis, MN), and recombinant human epidermal growth factor (EGF; 20 ng⁄mL; R&D Systems) [19]. The cells were cultured in HERA cell incubators (Thermo Electronic Corporation, Asheville, NC) at 37°C, ≥95% relative humidity, and 5% CO2 with 20% O2 conditions. Prior informed consent was obtained from the donor patients. Our study was approved by the Medical Review Boards of University of Pittsburgh, University of Virginia, and Gifu University School of Medicine.

Flow cytometry

For flow cytometry of the surgical specimens within 2 h of tumor removal, the tumor tissues were minced by a surgical scalpel, incubated in Accutase (Sigma–Aldrich, St. Louis, MO) for 20 min under 37°C and washed; the cells were then dissociated in phosphatebuffered saline (PBS; 3X) to remove the cell debris, and titrated in PBS. The cells were then passed through a 40–μm strainer (Falcon, Oxnard, CA) and resuspended in flow cytometry buffer consisting of PBS with 0.1% fraction V of bovine serum albumin (Sigma–Aldrich). The sphere cells were mechanically dissociated by a 5–mL pipette (Corning, NY) and then passed through a 40–μm strainer (Falcon). For long–term analysis, we used clonally expanded frozen–cultured X01, X02, and X03 sphere cells at different time points [18,19]. Briefly, the cells were diluted to 1 × 105 concentration with 50–μL aliquots for each analysis. For surface–marker analysis, we used antibodies against anti–human phycoerythrin (PE)–conjugated anti–human CD184, CD44, CD24, CD15, PDGFRa, CD54 (or intracellular adhesion molecule–1, ICAM–1), CD56 (or neural CAM, NCAM), CD146 (or melanoma CAM, MCAM), CD166 (or activated–leukocyte CAM, ALCAM), EGFR (BD Biosciences, San Jose, CA), PE–conjugated CD133⁄1 (AC133) (Miltenyi Biotec, Auburn, CA), and purified anti–human A2B5 (Miltenyi Biotec). Antibodies were titrated using appropriate dilutions and incubated on an ice bath for 60 min. The cells were then washed with the flow cytometry buffer, and the secondary fluorescent–conjugated antibody for A2B5 was added at appropriate dilutions and incubated on an ice bath for 60 min. For intracellular staining, the cell pellets were incubated with 0.1% Triton X–100 in the flow cytometry buffer on an ice bath for 10 min and then washed with the flow cytometry buffer. Sox2 (R&D Systems) and bmi– 1 (R&D Systems) were used for intracellular staining. The stained cells were washed once with the flow cytometry buffer, resuspended in 500 μL of the same buffer, and evaluated by the Coulter EPICS Cytometer (Beckman Coulter, Fullerton, CA). Appropriate compensation and isotype controls were used in the experiment.

Immunofluorescent staining

Immunocytochemistry of CSCs was performed as described in a previous study [23]. The following antibodies were used: antinestin (rabbit pAb, 1:200; Chemicon, Temecula, CA), anti–CD133⁄1 (1:1; Miltenyi Biotec), anti–ß–III–tubulin (Tuj1; mouse mAb, 1:200; Chemicon) for neurons, and anti–glial fibrillary acidic protein (GFAP; rabbit pAb, 1:500; DAKO, Glostrup, Denmark) for astrocytes. Visualizations were performed with Alexa fluorophore–conjugated secondary antibodies (1:1,000; Molecular Probes, Eugene, OR).

Statistical analysis

The differences among the various surface maker expression patterns were evaluated by Student’s t–test. p < 0.05 was considered statistically significant.


Glioma CSCs and glial⁄neural linage markers of freshly dissected brain tumors and primary–established glioma CSCs

All experiments were performed with malignant–glioma derived CSC lines from freshly resected surgical specimens [19,23]. Our culture system allowed the isolation of clonogenic cells from the human brain tumors and that these tumors contained multipotent, long–term self–renewing, population–expanding cells that satisfy the defining criteria of CSCs [1,4,18]. We analyzed the expression patterns of surface markers CD44, A2B5, and PDGFR–a for glia, CD24 for neural cells, and CD133 and CD15 for stem cells on fresh glioblastoma multiforme (GBM) specimens by flow cytometry. The CD184 expression was also analyzed because it is extremely important for tumor invasion. High CD44 expression was observed in all 8 cases and high CD24 expression was observed in 3 cases (1203, 0320, and 0408) (Table 1). The cells from all 8 GBM samples formed neuronal sphere–like aggregates within 2 to 7 days of culturing. The spherelike aggregation of cells from cell lines 1203 (X04), 0320 (X06), and 0408 (X07) increased continuously, while the cells from the other 5 samples became adherent and lost their proliferative capacity within 3 months of culturing. X04, X06, and X07 could be passaged and amplified by resupplementing with fresh medium twice weekly. Interestingly, in these 3 cases, the CD24 expression was nearly 100%, while the CD184 expression was higher than that in other cases (p < 0.01). No correlation was noted between the CD133 expression and cell amplification.