expression of stem cell markers in human astrocytomas of ... · expression of stem cell markers in...

LAB INVESTIGATION-HUMAN/ANIMAL TISSUE

Expression of stem cell markers in human astrocytomasof different WHO grades

Yue-Hui Ma Æ Rolf Mentlein Æ Friederike Knerlich ÆMarie-Luise Kruse Æ H. Maximilian Mehdorn ÆJanka Held-Feindt

Received: 11 May 2007 / Accepted: 11 June 2007

� Springer Science+Business Media B.V. 2007

Abstract According to new hypotheses astrocytomas/

gliomas either arise from or attract neural stem cells.

Biological markers, particularly antigenic markers, have

played a significant role for the characterization of these

tumour stem cells (TSCc). Because these studies have been

performed with single experimental samples mostly from

gliomas, we investigated the expression of the stem cell

markers CD133/Prominin, Nestin, Sox-2, Musashi-1,

CXCR4, Flt-4/VEGFR-3 and CD105/Endoglin in 72

astrocytomas of different WHO-grades and compared it to

normal adult human brain. Expression of their mRNA was

quantified by quantitative RT-PCR, of their protein by

counting immunopositive cells. In contrast to normal brain,

tumour samples showed a high variability for the expres-

sion of all markers. However, their mean expression was

significantly increased in astrocytomas, but this depended

on the WHO grade only for CD133, Nestin, Sox-2 and

Musashi-1. Confocal microscopy revealed that these

markers mostly could be co-stained with glial fibrillary

acidic protein, a marker for astoglial cells, but less fre-

quently with the proliferation marker Ki-67/MIB-1. These

markers sometimes, but not necessarily could be co-stained

with each other in complex patterns. Our results show that

most astrocytomas contain considerable portions of cells

expressing stem cell markers. It appears that some of these

cells originate from tumour genesis (supporting the stem

cell hypothesis) while others are attracted by the tumours.

Further functional markers are required to differentiate

these TSC-types.

Keywords Astrocytomas � CD133 � CXCR4 � Endoglin �Flt-4 � Musashi-1 � Nestin � SOX-2 � Stem cell markers �Tumour stem cells

Abbreviations

NSC Neural stem cell

TSC Tumour stem cell

HSC Hematopoietic stem cell

NBT Normal brain tissue

IHC Immunohistochemistry

RE Real-time reverse-transcription polymerase

chain reaction

RT-PCR Reverse-transcription polymerase chain

reaction

GFAP Glial fibrillary acidic protein

WHO World Health Organization

Introduction

Astrocytomas are the most common types of primary brain

tumours, accounting for 33.3% of all newly diagnosed

brain tumours [1]. These neuroepthelial tumours can occur

Y.-H. Ma � F. Knerlich � H. M. Mehdorn � J. Held-Feindt (&)

Department of Neurosurgery, University Medical Center

Schleswig-Holstein, Campus Kiel, Schittenhelmstr. 10, 24105

Kiel, Germany

e-mail: [email protected]

Y.-H. Ma

Department of Neurosurgery, the First Affiliated Hospital,

School of Medicine, Zhejiang University, Hangzhou, People’s

Republic of China

R. Mentlein

Department of Anatomy, University of Kiel, Kiel, Germany

M.-L. Kruse

Department of General Internal Medicine, University Medical

Center Schleswig-Holstein, Campus Kiel, Kiel, Germany

123

J Neurooncol

DOI 10.1007/s11060-007-9439-7

at any age, especially at young and middle ages, and are

more common in men. Based on specific histopathological

characteristics such as cellularity, nuclear atypia, mitotic

activity, microvascular proliferation and necrosis, astrocy-

tomas are subdivided into four grades of malignancy

[World Health Organization (WHO) grades I–IV] in order

of increasing malignancy [2]. By this, astrocytomas of

WHO grade IV = glioblastomas are the most malignant

forms.

It has traditionally been thought that astrocytomas

originate from astrocytes or their precursor cells. The

genesis of astrocytomas involves a number of character-

istic genetic alterations including activation of oncogenes

and inactivation of tumour suppressor genes [3, 4], which

lead to a progressive disorder of normal cellular growth,

apoptotic, cell motility and invasion pathways. However,

with discovery of neural stem cells (NSCs) in the central

nervous system and advances on molecular biology of

NSCs since early 1990s, there has been a wide speculation

that normal NSCs or progenitor cells might be principal

targets of mutations that lead to a brain tumour, probably

through dysregulation of self-renewal pathways. Tumour

cells with NSC properties are called tumour stem cells

(TSCs) [5–8].

The TSC hypothesis dictates that tumours arise from a

single, self-renewing cell type, which then gives rise to the

rest of the tumour cells, including a variety of ‘‘more dif-

ferentiated’’ cell types. It has been found that astrocytoma

cells share many characteristics with NSCs such as the

capacity of self-renewal and multi-lineage differentiation

[9–11], and similarities in phenotype and signalling [11,

12]. Imaging studies demonstrate that astrocytomas origi-

nate in two areas, the subventricular zone of the lateral

ventricle and the hippocampus, which have been previ-

ously shown to be reservoirs of normal NSCs [13]. Besides,

subependymal giant cell astrocytomas coexpressed several

neuronal markers (neuronal nuclear antigen, neuron spe-

cific enolase) and markers of NSCs (nestin) and glial cells

(glial fibrillary acidic protein) [14]. These studies suggest

that astrocytomas may result from transformation of NSCs.

The direct evidence supporting this notion comes from

transgenic mice models, which demonstrate that nestin-

expressing progenitors, excessively expressing oncogenes

such as c-Myc, can give rise to gliomas and medulloblas-

tomas [15–17].

Biological markers, particularly antigenic markers,

have played a significant role for the characterization of

various stem cells. Reported markers for detecting TSC

populations are Nestin (glioblastomas) [8], CD133 (glio-

blastomas; medulloblastomas, ependymomas and prostate

cancer) [5, 8, 18, 19], Musashi-1 (glioblastomas) [6],

SOX-2 (glioblastomas) [6], Bmi-1 (glioblastomas) [6],

CD44 (breast cancer; prostate cancer) [18, 20], CD20

(melanomas) [21], CD34 (leukaemia) [22], ABCG2 (ret-

inoblastoma, neuroblastoma) [23, 24] and stem cell anti-

gen-1 (Sca-1) (lung cancer) [25]. Moreover, it could be

shown that CXCR4 is expressed by NSCs [26], Flt-4/

VEGR-3 on neural progenitor cells [27], and CD105/

Endoglin, a specific marker for hematopoetic stem cells,

was found in neoplastic cells of sarcomas and ovary

carcinomas [28].

Tumour stem cells can be isolated and characterized in

many solid tumours, such as brain tumours [5–8, 19, 29–

31], breast cancers [20], melanomas [21], prostate cancers

[18] and lung cancers [25]. Nevertheless, TSCs were

primarily only identified in glioblastomas. However, most

studies were done with single samples and nothing is

known about the expression of these putative TCS

markers in a broader variety of gliomas and especially in

astrocytomas of lower WHO grades. Therefore, we anal-

ysed for the first time the expression of seven different

stem cell markers (CD133, Nestin, SOX-2, Musashi-1,

CXCR4, Flt-4 and Endoglin) in astrocytomas of all WHO

grades with different quantitative methods on the mRNA

and protein level. Moreover, we investigated the co-

expression of theses genes with markers for glial and

proliferating cells, and among themselves, and correlated

the results with the different malignant grades of the used

tumours.

Materials and methods

Patients and tumour specimens

Astrocytoma tissues of different WHO grades were col-

lected from 72 patients who were operated on between

January 1998 and August 2006 at the Department of

Neurosurgery, Kiel, Germany (see Table 1). Four normal

brain tissue (NBT) samples served as controls. The

patients included 28 women and 44 men. The mean age at

diagnosis was 37.6 ± 23.2 and 39.9 ± 21.5 years, respec-

tively. All samples were obtained in accordance with

approved ethical standards of the responsible committee

of the University of Kiel and with the Helsinki Declara-

tion of 1975, as revised in 1983. Tumours were classified

according to the WHO criteria into the various subtypes

of astrocytomas. Eighteen tumours corresponded to

astrocytomas WHO grade I, 12 tumours were astrocyto-

mas WHO grade II, 17 were astrocytomas WHO grade

III, and 25 tumours corresponded to astrocytomas WHO

grade IV (glioblastomas). If possible (enough material

available), for different experiments matched probes of

individual tumour samples were used (for details see

Table 1).

J Neurooncol

123

Real-time RT-PCR

For real-time RT-PCR solid astrocytoma tissues of different

WHO grades (13 astrocytoma I samples, 12 astrocytoma II

samples, 11 astrocytoma III samples and 18 astrocytoma IV

samples), and four NBT samples were chosen (see Table 1).

Total RNAs from different samples were purified with

TRIzol Reagent (Invitrogen, Life Technologies, Karlsruhe,

Germany), treated with RNase free DNase (1 U/ll, Pro-

mega, Madison, WI, USA), and reverse transcribed by

RevertAidTM H Minus M-MuLV Reverse Transcriptase

(200 U/ll, Fermentas, Vilinius, Lithuania) according to the

manufacturer’s instruction.

Real-time RT-PCR was performed in three replicates of

each sample using a total reactive volume of 20 ll, which

contained 1 ll of 20 · Assays-on-DemandTM Gene

Expression Assay Mix (Hs00195682_m1 for CD133,

Hs00707120_s1 for Nestin, Hs00602736_s1 for SOX-2,

Hs00159291_m1 for Musashi-1, Hs00237052_m1 for

CXCR4, Hs00176607_m1 for Flt-4, Hs00164438_m1 for

Table 1 Tumour samples

Sample

number

Gender Age Histology Detection

1 Male 23 Astrocytoma I RE, IHC

2 Female 14 Astrocytoma I RE

3 Male 42 Astrocytoma I RE

4 Female 28 Astrocytoma I RE, IHC


6 Female 7 Astrocytoma I RE








14 Female 11 Astrocytoma I IHC



17 Male 48 Astrocytoma I IHC


19 Male 34 Astrocytoma II RE, IHC


21 Female 40 Astrocytoma II RE, IHC




25 Female 25 Astrocytoma II RE

26 Male 58 Astrocytoma II RE

27 Female 42 Astrocytoma II RE




31 Female 41 Astrocytoma III RE, IHC


33 Male 36 Astrocytoma III RE





38 Male 57 Astrocytoma III RE, IHC

39 Female 51 Astrocytoma III RE


41 Male 32 Astrocytoma III RE, IHC

42 Female 31 Astrocytoma III IHC

43 Female 59 Astrocytoma III IHC

44 Male 43 Astrocytoma III IHC




Table 1 continued

Sample

number

Gender Age Histology Detection

48 Female 63 Astrocytoma IV RE, IHC

49 Male 60 Astrocytoma IV RE

50 Male 71 Astrocytoma IV RE, IHC, Confocal

51 Male 49 Astrocytoma IV RE, IHC

52 Male 69 Astrocytoma IV RE, IHC


54 Female 78 Astrocytoma IV RE












66 Male 65 Astrocytoma IV IHC, Confocal

67 Male 73 Astrocytoma IV IHC

68 Male 75 Astrocytoma IV IHC

69 Female 25 Astrocytoma IV IHC, Confocal

70 Female 57 Astrocytoma IV IHC

71 Female 31 Astrocytoma IV IHC, Confocal

72 Male 68 Astrocytoma IV Confocal

RE real-time reverse transcription polymerase chain reaction, IHCimmunohistochemistry

J Neurooncol

123

Endoglin; Applied Biosystems, Foster City, CA, USA),

10 ll of 2 · TaqMan Universal PCR Master Mix and 100

or 10 ng of cDNA template (diluted in RNase-free water to

9 ll). After 2 min at 50�C and 10 min at 95�C, 40 cycles of

15 s at 95�C and 1 min at 60�C were run. Glyceraldehyde-

3-phosphate dehydrogenase (GAPDH; Hs99999905_m1,

Applied Biosystems) in each sample was tested as intrinsic

positive control. Each plate included at least three ‘‘No

Template Controls (NTC)’’. The reaction was carried out

with the MyiQTM Single Color Real-time PCR Detection

System (Bio-Rad, Munchen, Germany) and fluorescent

data were converted into cycle threshold (CT) measure-

ments. DCT-values of each sample were calculated as

CTgene of interest–CT GAPDH.

Immunohistochemistry

For immunohistochemistry (IHC) examination, fresh-fro-

zen astrocytoma tissues of different WHO grades (11

astrocytoma I samples, 9 astrocytoma II samples, 11

astrocytoma III samples and 10 astrocytoma IV samples)

and two NBT samples were cut in a cryostat into 6 lm

sections (see Table 1). The sections were air dried and

stored in –20�C freezer until used. IHC was performed

using the avidin-biotin-peroxidase complex (ABC) meth-

od. The sections were fixed with 4% para-formaldehyde in

phosphate buffered saline (PBS) for 30 min at room tem-

perature (RT). To block endogenous peroxidase and non-

specific binding, 3% H2O2 in 0.3% Triton·-100/PBS

(30 min at RT) and appropriate 10% normal blocking

serum in PBS (60 min at RT; normal horse serum for

Nestin and Musashi-1, normal rabbit serum for CD133, and

normal donkey serum for SOX-2, CXCR4, Flt-4 and En-

doglin; Jackson Immuno Research Laboratories, Mulkiteo,

WA, USA) were used, respectively. Sections were incu-

bated with appropriate dilutions of primary antibodies over

night at 4�C (anti-CD133, goat polyclonal, 1:100, Santa

Cruz, CA, USA; anti-Nestin, mouse monoclonal, 1:200,

R&D Systems, Wiesbaden, Germany; anti-Musashi-1,

mouse monoclonal, 1:200, R&D Systems; anti-SOX-2,

rabbit polyclonal, 1:100, Santa Cruz; anti-CXCR4, rabbit

polyclonal, 1:100, IMGENEX, San Diego, CA, USA; anti-

Flt-4, rabbit polyclonal, 1:200, Santa Cruz; anti-Endoglin,

rabbit polyclonal, 1:50; Santa Cruz). The antibodies were

diluted in 0.3% Triton·-100 and appropriate 2% normal

blocking serum in PBS. Primary antibodies were omitted

for negative controls. After washing steps with PBS

(10 min for two times), the sections were incubated with

corresponding second antibodies diluted 1:200 in 1.5%

blocking serum/PBS for 60 min at RT (biotinylated horse

anti-mouse IgG for Nestin and Musashi-1, Vector Labo-

ratories, Burlingame, CA, USA; biotinylated rabbit anti-

goat IgG for CD133, Vector Laboratories; biotinylated

donkey anti-rabbit for SOX-2, CXCR4, Flt-4 and Endoglin,

Jackson Immuno Research). Amplification of the signal

was carried out by ABC method with ABC Vectastain� kit

(Vector Laboratories, Burlingame, CA, USA). The signal

was visualized by incubation with 0.06% 3,3¢-diam-

inobenzidine-tetrahydrochloride (Sigma, Munchen, Ger-

many) and 0.003% H2O2 in 0.1 M Tris–HCl (pH 7.6) for

40–55 s. Finally, the sections were counterstained with

hematoxylin for 40 s, mounted with Eukitt� quick-harden

mounting medium (Fluka, Munchen, Germany) and cov-

erslipped for investigation. Brightfield microscopy with

digital photography was performed using a Zeiss micro-

scope (Zeiss, Oberkochen, Germany).

Confocal microscopy

Cryostat sections of five different astrocytoma tissues

WHO grade IV (see Table 1) were fixed in acetone/meth-

anol (1:1; 10 min) at -20�C, washed with Tris-buffered

saline plus 0.1% Tween 20 (TBS-T, 3·, RT), washed with

20%, then 70% ethanol (each 2 min), blocked with Sudan

black (1% in 70% ethanol) for 10 min, rinsed with 70%

ethanol until dye free, then for 2 min with 20% ethanol,

washed with TBS-T (3·), blocked with 0.1% bovine serum

albumin and 0.2% glycine in TBS (1 h), then without

washing incubated in with primary antibodies in TBS-T at

4�C overnight. Primary antibodies were omitted for nega-

tive controls. After a washing step (3 · TBS-T, 10 min)

the first secondary antibody was incubated for 1 h at 37�C

in darkness. The sections were washed with TBS-T

(3 · 10 min). When both primary antibodies were obtained

from mouse, an additional blocking step with goat anti-

mouse FAB-fragments (1:1,000 1 h, RT, from Dianova,

Hamburg, Germany) was necessary. The second primary

antibody was applied after another washing step (3 · TBS-

T) and incubated over night at 4�C. Second primary anti-

bodies were omitted for negative controls. The slides were

washed again (3 · TBS-T) and incubated with the second

secondary antibody for 1 h at 37�C. After washing with

TBS-T (1 · 10 min), TBS (2 · 10 min), nuclei were

stained with diamidino-2-phenylindole (DAPI; Molecular

Probes/Invitrogen, Life Technologies, Karlsruhe, Ger-

many; 1:30,000, 30 min RT), washed with TBS (3·) and

finally distilled water. After embedding in Immu-Mount

(Shandon, Pittsburgh, PA, USA) data were observed with a

confocal microscope (Zeiss).

In combination with anti-GFAP (rabbit polyclonal,

1:100, Santa Cruz), anti-MIB-1/Ki-67 (rabbit polyclonal,

1:2,000, Santa Cruz), and anti-SOX-2 (1:500, rabbit poly-

clonal, Santa Cruz) anti-Nestin (mouse monoclonal, 1:750,

R&D Systems) and anti-Musashi-1 (mouse monoclonal,

1:250, R&D Systems) were always stained first with

anti-mouse Alexa Fluor 555 (goat polyclonal, 1:1,000,

J Neurooncol

123

Invitrogen) as secondary antibody, the second secondary

antibody was anti-rabbit Alexa Fluor 488 (goat polyclonal,

1:1,000, Invitrogen). In combination with anti-Musashi-1

(1:250) anti-Nestin (1:750) was stained first with anti-

mouse Alexa Fluor 555 (goat polyclonal, 1:1,000) as

secondary antibody, the second secondary antibody was

anti-mouse Alexa Fluor 488 (goat polyclonal, 1:1,000). In

combination with anti-GFAP (mouse monoclonal, 1:500;

DAKO, Glostrup, Denmark), and anti-MIB-1/Ki-67

(mouse monoclonal, 1:500, DAKO) anti-SOX-2 (1:500)

was always stained first with anti-rabbit Alexa Fluor 555

(goat polyclonal, 1:1,000) as secondary antibody, the sec-

ond secondary antibody was anti-mouse Alexa Fluor 488

(goat polyclonal, 1:1,000). In combination with anti-GFAP

(mouse monoclonal, 1:500), anti-Nestin (1:750), and anti-

Musashi-1 (1:250) anti-CD133 (goat polyclonal, 1:100,

Santa Cruz) was always stained first with anti-goat Alexa

Fluor 488 (donkey polyclonal, 1:1,000, Invitrogen) as

secondary antibody, the second secondary antibody was

anti-mouse Alexa Fluor 555 (goat polyclonal, 1:1,000). In

combination with anti-SOX-2 (1:500) and anti-MIB-1/Ki-

67 (rabbit polyclonal, 1:2,000) anti-CD133 (1:100) was

first stained with anti-goat Alexa Fluor 488 (donkey

polyclonal, 1:1,000) as secondary antibody, the second

secondary antibody was anti-rabbit Alexa Fluor 488

(donkey polyclonal, 1:1,000, Invitrogen).

Statistical analysis

Statistical methods of Student’s t-test with independent

samples and bivariate correlation analysis (Pearson corre-

lation coefficients) were used. Significance levels ranged

between P < 0.05 (indicated by *), P < 0.01 (indicated by

**) and P < 0.005 (indicated by ***).

Results

Expression of CD133, Nestin, SOX-2 and Musashi-1,

but not of CXCR4, Flt-4 and Endoglin correlated with

malignant grades of astrocytomas

To evaluate mRNA and protein expression levels of the

investigated stem cell markers in astrocytomas of different

WHO grades real-time RT-PCR and IHC of 72 different

tumour samples (see Table 1) and four different NBT

samples were performed. Results are shown in Figs. 1–4.

Although a wide range between single samples oc-

curred, in relation to NBT in astrocytomas of WHO grades

II–IV a clear mRNA over-expression of CD133, Nestin,

SOX-2 and Musashi-1 could be detected (Figs. 1a–4a). The

normalized averaged DCT-values were 9.8, 10.6, 8.5 for

CD133 (NBT 12.5); 1.6, 2.3, 0.6 (NBT 4.6) for Nestin; 6.4,

5.9, 7.1 (NBT 9.5) for SOX-2 and 4.2, 2.3, 4.7 (NBT 6.4)

for Musashi-1 in astrocytomas of WHO grades II, II and

IV, respectively. The statistical differences ranged between

P < 0.05 and 0.005 (see Figs. 1a–4a, indicated by stars). In

contrast to this, with exception of CD133 in astrocytomas

of WHO grade I mean mRNA expression levels of Nestin,

SOX-2 and Musashi-1 were comparable to those of NBT.

These results could be confirmed by IHC. Even if in

relation to NBT not in all cases statistically significant

differences were evaluated and results differed from sam-

ple to sample, in astrocytomas of WHO grade IV the

highest amounts of positive stained cells of CD133, Nestin,

SOX-2 and Musashi-1 were found (Figs. 1b–4b, 1c–4c).

Amounts of positive stained cells were 15.6% for CD133

(NBT 2.3%), 25.3% for Nestin (NBT 4.2%), 9.7% for

SOX-2 (NBT 2.3%) and 9.8% for Musashi-1 (NBT 2.1%),

respectively.

Moreover, a comparison between the averaged DCT-

values and percentages of positive stained cells of CD133,

Nestin, SOX-2 and Musashi-1, especially in astrocytomas

of WHO grade IV, gave interesting results (Figs. 1–4).

While the mean DCT-value of Nestin was the lowest (0.6;

means highest expression) among these four markers, and

similarly, the percentage of nestin positive cells was the

highest (25.3%), the DCT-value of CD133 was the highest

(8.5; means lowest expression) among all markers ob-

served. In contrast to this, the percentage of CD133 posi-

tive cells was higher than that of SOX-2 and Musashi-1

(15.6% for CD133; 9.7% for SOX-2; 9.8% for Musashi-1).

DCT-values of SOX-2 and Musashi-1 were higher than that

of Nestin but lower than that of CD133 (7.1 for SOX-2; 4.7

for Musashi-1).

To evaluate whether a correlation between the expres-

sion of CD133, Nestin, SOX-2 and Musashi-1 and the

pathological grades of astrocytomas could be observed a

bivariate correlation analysis (Pearson correlation coeffi-

cients) was done. Statistically significant positive correla-

tions between the percentages of immunostained cells and

pathological grades of astrocytomas were found for CD133

(cp = 0.318, P < 0.05), Nestin (cp = 0.599, P < 0.001),

SOX-2 (cp = 0.331, P < 0.05) and Musashi-1 (cp = 0.327,

P < 0.05). DCT-values of these four stem cell markers had

a negative correlation with the pathological grades of

astrocytomas, which means that with higher malignant

grades of astrocytomas, higher mRNA amount (low-DCT-

values) could be found. Statistically significant correlations

appeared for Nestin (cp = -0.465, P < 0.005), and SOX-2

(cp = -0.324, P < 0.05).

In relation to NBT mRNA and protein expression levels

of CXCR4, Flt-4 and Endoglin did not correlate signifi-

cantly with malignant grades of astrocytomas (data not

shown). In detail, averaged DCT-values of CXCR4 were

6.82, 6.84, 7.28 and 6.23 in astrocytomas of WHO grades

J Neurooncol

123

I–IV, respectively, and 7.46 in NBT. The amounts of

positive stained CXCR4 cells were 6.17, 5.48, 6.67 and

11.06% in grades I–IV, and 0.97% in NBT. By this,

whereas in astrocytomas of WHO grade IV a notable in-

crease of positive stained CXCR4 cells was detected, and

this difference was statistical significant (P < 0.01), no

correlation between the expression of CXCR4 and the

pathological grades of astrocytomas was found. Same

results were observed for Flt-4/VEGFR-3 and Endoglin/

CD105 mRNA and protein expression levels. Averaged

DCT-values were 8.22, 7.15, 6.96 and 6.77 for Flt-4 (NBT

8.33), and 5.29, 5.15, 5.18 and 3.77 for Endoglin (NBT

5.02) in astrocytomas WHO grades I–IV, respectively. The

amounts of positive stained cells ranged between 1.33, 6.9,

9.71 and 7.91% for Flt-4 (NBT 5.17%) and 3.38, 4.89,

8.85 and 5.71% for Endoglin (NBT 2.27%). Although

Flt-4 and Endoglin mRNA and protein expression levels

increased from low-grade to high-grade astrocytomas, no

statistically significant differences between expression

levels of NBT and tumour tissue samples and no correla-

tion with the pathological grades of astrocytomas was

found.

Fig. 1 Expression of CD133 in

astrocytomas (AC) of different

WHO grades and normal brain

tissue (NBT) evaluated by areal-time RT-PCR (logarithmic

scale, DCT = 3.3 corresponds to

a tenfold difference) and (b, c)

immunohistochemistry (IHC); bgraphical representation of

percentages of CD133 positive

stained cells, c examples of

immunohistochemical staining

of CD133 (original

magnification · 200); inset:

negative control (AC IV)

without primary antibody.

Although a wide range between

single samples occurred,

compared with NBT in high-

grade astrocytomas elevated

mRNA expression levels and

amounts of CD133 positive

cells were found. Normalized

averaged DCT-values were 9.7,

9.8, 10.6 and 8.5 (NBT 12.5),

averaged percentages were 6.9,

6.5, 15 and 15.6% (NBT 2.3%)

in astrocytomas of WHO grades

I–IV, respectively. Results

correlated with malignant

grades of astrocytomas and

were statistical significant

(*P < 0.05; **P < 0.01); UNundetectable

J Neurooncol

123

In summary, the mRNA and protein expression of the

stem cell markers CD133, Nestin, SOX-2 and Musashi-1

correlated with malignant grades of human astrocytomas,

whereas those of CXCR4, Flt-4 and Endoglin did not.

CD133, Nestin, SOX-2 and Musashi-1 could

be co-stained with GFAP and among themselves

in a complex pattern

To analyse whether CD133, Nestin, SOX-2 and Musashi-1

could be co-stained with GFAP (glial fibrillary acidic

protein), Ki-67/MIB-1 (a marker of proliferating cells), or

among themselves confocal microscopy was performed.

Astrocytomas of WHO grade IV were used for investiga-

tions. Results are summarized in Table 2 and exemplary

data are shown in Figs. 5 and 6.

Initially it should be mentioned that both staining

intensities and amounts of positive regions differed among

single samples and within the same sample. Although

especially in tumour regions with high amounts of positive

stained cells results were difficult to interpret, for all

investigated stem cell markers no clearly co-staining with

Fig. 2 Expression of Nestin in







percentages of Nestin positive



of Nestin (original




While mean normalized DCT-

values were 3.9, 1.6, 2.3 and 0.6

(NBT 4.6), and averaged

percentages were 7.9, 7.7, 9.6

and 25.3% (NBT 4.2%) in

astrocytomas of WHO grades

I–IV, respectively, an increase

of Nestin expression could be

observed. Results correlated

with malignant grades of

astrocytomas and were

statistical significant

(*P < 0.05; ***P < 0.005)

J Neurooncol

123

Ki-67/MIB-1 could be observed. While the nuclear antigen

Ki-67/MIB-1 is present during all active phases of the cell

cycle (G1, S, G2 and M-phases), but absent in resting cells

(G0-phases), it seems that cells which were going through

the active cell cycle phases did not express CD133, Nestin,

SOX-2 and Musashi-1 in detectable amounts in parallel

(for example see Figs. 5b, 6b).

In contrast to this, we could show that all investigated

stem cell markers could be co-stained with GFAP. Whilst

the intermediate filament protein GFAP is an immunohis-

tochemical marker for localizing benign astrocyte and

neoplastic cells of glial origin in the central nervous sys-

tem, this means that astroglia also expressed CD133,

Nestin, SOX-2 and Musashi-1 (examples for merged

regions are shown in Figs. 5a, 6d). Additionally, cells

which were solely positive for GFAP or the individual stem

cell marker (or negative for both) existed within the tumour

sections (see Figs. 5a, 6d; Table 2).

Expression of both CD133 and Nestin was found in

combination with SOX-2, Musashi-1 and between each

other, respectively (for example see Fig. 5c, d, 6a, c;

Table 2; since different markers are not localized within

the same structures in the cells, signals did not merged in

all cases, but were found in the same regions). Thereby, it

seems that amounts of CD133 and Nestin positive stained

cells were higher than that of SOX-2 and Musashi-1 (see

Fig. 5d, 6a). This was in accordance with the results

obtained by IHC (in astrocytomas of grade IV percentages

of CD133 and Nestin positive stained cells were clearly

higher that that of SOX-2 and Musashi-1, Figs. 1–4).

Fig. 3 Expression of SOX-2 in







percentages of SOX-2 positive



of SOX-2 (original




Especially on mRNA level a

clear SOX-2 over-expression

was detected. Mean normalized

DCT-values were 8.8, 6.4, 5.9

and 7.1 (NBT 9.5) in

astrocytomas of WHO grades I-

IV, respectively (*P < 0.05).

Although, the amounts of SOX-

2 positive cells showed no

statistically significant

differences in relation to NBT,

the percentages of SOX-2

labelled cells correlated with

grades of malignancy (1.9, 6.6,

6.4 and 9.7% in astrocytomas

grades I–IV and 2.3% in NBT);

UN undetectable

J Neurooncol

123

Fig. 4 Expression of Musashi-1

in astrocytomas (AC) of

different WHO grades and

normal brain tissue (NBT)

evaluated by a real-time RT-

PCR (logarithmic scale,

DCT = 3.3 corresponds to a

tenfold difference) and (b, c)


percentages of Musashi-1

positive stained cells, cexamples of


of Musashi-1 (original



without primary antibody. High

mRNA and protein expression

levels of Musashi-1 were found

in astrocytomas of different

WHO grades, especially in

astrocytomas of WHO grade IV.

In detail, CT-values were 5.6,

4.2, 2.3 and 4.7 in astrocytomas

of WHO grades I–IV,

respectively, and 6.4 in NBT

(*P < 0.05, **P < 0.01;

***P < 0.005). Although, the

amounts of Musashi-1 positive

cells showed no statistically

significant differences in

relation to NBT, the percentages

of Musashi-1 labelled cells

correlated with grades of

malignancy (3.4, 2.9, 6.3 and

9.8% in astrocytomas grades

I–IV and 2.1% in NBT); UNundetectable

Table 2 Summarized results of confocal microscopy

CD133 Nestin SOX-2 Musashi-1

Positive Negative Positive Negative Positive Negative Positive Negative

Nestin Positive · ·Negative · ·

SOX-2 Positive · · · ·Negative · · · ·

Musashi-1 Positive · (–) · (–) · (–)

Negative · · · · (·) ·GFAP Positive · · · · · · · ·

Negative · · · · · · · ·MIB-1 Positive – · – · – · – ·

Negative · · · · · · · ·

J Neurooncol

123

Consequently, CD133 and Nestin positive cells existed not

only in combination with SOX-2 or Musashi-1, but also

without any co-staining (see Table 2). Moreover, as ini-

tially mentioned in different tumour sections regions with

high and low amounts of positive stained cells existed in

parallel (for CD133 see for example Figs. 5b, 6c).

SOX-2 positive signals were found alone or in combi-

nation with CD133 and Nestin (for example see Fig. 6a;

Fig. 5 Examples of co-staining of different stem cell markers in

astrocytomas grade IV determined by confocal microscopy. Upper

panel: CD133 (green) stained in combination with a GFAP (red) or bKi-67/MIB-1 (red). Beside cells which were solely positive for

CD133 or GFAP also clearly CD133/GFAP double positive cells

were observed (merged regions, yellow). No co-expression of CD133

and Ki-67/MIB-1 could be detected. Lower panel: Nestin (red)

stained in combination with c CD133 (green) and d Musashi-1

(green). Since different markers for Nestin/CD133 or Nestin/

Musashi-1 are not localized within the same structures in the cells,

signals did not merged in all cases, but were found in the same

regions. Whereas, Nestin and CD133 solely stained cells were found,

Musashi-1 seemed to be expressed only in combination with Nestin

Fig. 6 Examples of co-staining of different stem cell markers in

astrocytomas grade IV determined by confocal microscopy. Upper

panel: SOX-2 (red) stained in combination with a CD133 (green) or bKi-67/MIB-1 (green). Beside many CD133 and single SOX-2 positive

cells also some cells which could be co-stained with CD133 and

SOX-2 exited within the tumour section. By this, signals did not

merge, but were found in the same regions. No co-expression of SOX-

2 and Ki-67/MIB-1 could be detected. Lower panel: Musashi-1 (red)

stained in combination with c CD133 (green) and d GFAP (green).

Whereas, Musashi-1 seemed to be expressed only in combination

with CD133, it could be found alone or in combination with GFAP

(merged regions, yellow). Moreover, with this example it should be

shown that in astrocytomas of WHO grade IV regions with high

amounts of CD133 positive cells (c) and those with low amounts (d)

exited

J Neurooncol

123

Table 2). By this, since different markers are not localized

within the same structures in the cells, fluorescence signals

did not merged in all cases, but were found in the same

regions (e.g. see Fig. 6a). If a co-staining of SOX-2 and

Musashi-1 was performed, it seems that SOX-2 positive

cells were found with and without Musashi-1 expression in

parallel. Since in astrocytomas WHO grade IV averaged

percentages of SOX-2 and Musashi-1 positive cells were

only 9.7 and 9.8%, respectively, and sometimes single

structures were hard to investigate, it was very difficult to

evaluate whether SOX-2 positive/Musashi-1 negative cells

exist. Thereby, in Table 2 this result was demonstrated

with brackets. Moreover, it seems that Musashi-1 positive

could be only evaluated in combination with CD133,

Nestin and SOX-2 (for example see Figs. 5d, 6c, d). Flu-

orescence signals did not merged in all cases, but were

found in the same regions. For same reasons as mentioned

above in Table 2 results were shown with brackets.

In summary, CD133, Nestin, SOX-2 and Musashi-1

could be co-stained with GFAP and among themselves in a

complex pattern. In relation to CD133 and Nestin, SOX-2

and Musashi-1 were found in lower amounts in astrocytoma

grade IV tumour sections, and Musashi-1 seems to be ex-

pressed only in combination with other stem cell markers.

Discussion

Several recent reports suggest that TSCs exist in high-grade

astrocytomas and could be isolated from these tumours.

Nevertheless, little is known about the expression of these

markers in solid astrocytomas, especially in relation with

the malignant grades of these tumours. Herein, the aim of

this study was to investigate the expression of CD133,

Nestin, SOX-2, Musashi-1, CXCR4, Flt-4 and Endoglin on

mRNA and protein level, to analyse their relation with the

malignant grades of different astrocytomas, and to study the

co-expression of theses genes with markers for glial and

proliferating cells, and among themselves.

CD133/Prominin

Which belongs to the pentaspan membrane glycoproteins

family, was originally described to be expressed in neu-

roepithelial and hematopoietic stem cells (HSCs) [32, 33],

CD133 could be used to identify and isolate human stem

cells from various sources including CNS [33, 34]. We

could show that in relation to NBT in astrocytomas

amounts of CD133 positive stained cells were higher

(15.6% in astrocytomas of WHO grade IV; 2.3% in NBT)

and correlated with the malignant grades of these tumours.

Results were confirmed by real-time RT-PCR (DCT-values

were 12.5 and 8.5 in astrocytomas grade IV and NBT,

respectively). The high amount of CD133 positive cells

exceeds our expectation; however it is similar to the result

that 6–29% of putative TSCs (CD133 positive cells) were

sorted from glioblastomas [8]. It was found that more than

10,000 unsorted cancer cells were needed to initiate tu-

mours [19], which implied that the TSCs fraction was less

than 0.01% of the unsorted population. Even if as low as

100 CD133 positive glioblastoma cells were necessary to

form tumours in immunodeficient mice [8], theoretically,

the proportion of TSCs (sorted CD133 positive cell popu-

lation) in solid tumour should be only 1%. But, in contrast

to that and in accordance with our result, 11–35% cells that

expressed the stem cell markers CD44+/CD24– were sorted

from breast cancer in another study [20]. These data sug-

gest that not all of the CD133 positive cells are true TSCs,

even though the CD133 positive cell portion represents

undifferentiated cells. In fact, primary tumour sphere-de-

rived cells (CD133 positive) formed secondary spheres

with a range of 0.53–1.0 secondary spheres/100 primary

sphere cells [19]. So, CD133 positive cells are not always

linked with the ability of self-renewal and tumourigenesis.

Nestin

An intermediate filament protein typical for neural pre-

cursor cells, has been extensively used as a marker for

NSCs [35, 36]. Based on the observations of a correlation

of Nestin expression with NSCs in vivo, now Nestin is also

used as a typical marker for identifying TSCs from CNS

tumours [5–8, 19, 29, 37]. In primary malignant CNS tu-

mours high amounts (more than 50%) of Nestin positive

cells have been reported [38, 39]. Our results are in

agreement with these reports. Astrocytomas were abun-

dantly positive for Nestin, and the amounts of Nestin po-

sitive cells increased with malignant grades of these

tumours. The highest proportion of Nestin positive cells

(average 25.3%) was found in astrocytomas of WHO grade

IV with a statistically significant difference in relation to

NBT (4.2%). Moreover, compared with NBT in astrocy-

tomas of WHO grades II, III and IV a statistically signifi-

cant up-regulated expression of Nestin mRNA was found

(DCT-values were 1.6, 2.3, 0.6, respectively, and 4.6 in

NBT).

Nestin is expressed in primitive neuroepithelial cells of

all regions of the CNS during development [40], whereas in

the adult CNS, Nestin is a marker for NSCs lining in

the ventricular wall and the central canal [41]. However,

other studies showed that Nestin was also detected in

NBT ?tul?> outside of the subependymal zone [42–45],

and could be re-expressed in astrocytes of the adult CNS in

response to cellular stress, including brain trauma/trans-

plantation [46–48], CNS ischemia [48, 49], neurotoxicant

[50], neoplastic transformation [38, 39] and even altered

J Neurooncol

123

trophic stimulation [51]. The relation between the ability of

neurosphere formation and Nestin expression in adult brain

is different from that in embryonic brain [52]. So, Nestin

expression is complex at every stage of stem cell devel-

opment and differentiation.

SOX-2

Encodes a SRY-related high mobility group of box tran-

scription factors, which act as transcriptional activators for

a variety of genes that are developmentally regulated [53,

54]. SOX-2 expression is found in at least three types of

stem cells (NSCs, embryonic and trophoblast stem cells)

[54, 55]. Glioma-derived neurospheres can express SOX-2,

and SOX-2 mRNA expression could be detected in brain

tumours [6]. In this study we could show that SOX-2 was

expressed in astrocytomas, and its expression was up reg-

ulated in relation to NBT. Although no statistically sig-

nificant differences of SOX-2 protein expression could be

detected by IHC between astrocytomas and NBT (9.7% in

astrocytomas grade IV, 2.3% in NBT), SOX-2 mRNA

expression was really significantly up regulated in astro-

cytomas of WHO grades II, III and IV (DCT-values were

6.4, 5.6, 7.1, respectively, and 9.5 in NBT) Moreover,

amounts of SOX-2 positive cells and mRNA expression

levels increased with malignant grades of these tumours.

SOX-2 appears to be a common marker for all neural

cells within heterogeneous neurospheres derived from

different parts of the brain [56]. However, that may be not

right for tumour-derived spheres. Tumour-derived spheres

could acquire SOX-2 mRNA expression that was absent in

their parent tumours or they could lose SOX-2 expression

[6].

Musashi-1

Encodes an evolutionarily conserved group of RNA-bind-

ing proteins, which are selectively expressed in neural

progenitor/stem cells, and play a key role in the mainte-

nance of the stem cell state during embryonic CNS

development. Musashi-1 is also an excellent marker for

neural progenitor/stem cells in adult brains [57], and is

expressed in neuronal precursors of the cerebellum and

subventricular zone [58, 59]. In our study, high mRNA and

protein expression levels of Musashi-1 were found in

astrocytomas of different WHO grades, especially in

astrocytomas of WHO grade IV. In detail, DCT-values

were 4.2, 2.3 and 4.7 in astrocytomas of WHO grades II–

IV, respectively, and 6.4 in NBT. Although, the amounts of

Musashi-1 positive cells showed no statistically significant

differences in relation to NBT, the percentages of Musashi-

1 labelled cells correlated with grades of malignancy (2.9,

6.3 and 9.8% in astrocytomas grade II–IV and 2.1% in

NBT). In accordance with these results, previous studies

showed that the expression of Musashi-1 could be corre-

lated to the proliferating activity of gliomas [57]. No

relationship between the grades of malignancy and the

degree of Musashi-1 expression could be observed in hu-

man hepatoma cells [60]. Tumour-derived spheres (undif-

ferentiated and differentiated) could acquire Musashi-1

mRNA expression which was absent in their parent tu-

mours, or they could loss Musashi-1 expression [6]. So,

mRNA expression of Musashi-1 appears to be dynamic.

CXCR4

A chemokine receptor, belongs to the family of G protein-

coupled receptors and is selective for a single chemokine,

the stromal cell-derived factor 1 (SDF-1, CXCL12).

CXCR4 is expressed by NSCs of the cerebellar external

granular layer [26], and CXCR4 positive NSCs could be

isolated from mouse neurospheres. Moreover, the intrace-

rebral administration of SDF-1 enabled intravenously in-

jected CXCR4 positive NSCs to migrate into the CNS and

differentiate into neurons [61]. Although in our study no

correlation between the expression of CXCR4 and the

pathological grades of astrocytomas was found, in astro-

cytomas of WHO grade IV a notable increase of positive

stained CXCR4 cells was detected, and this difference was

statistical significant (P < 0.01).

Flt-4/VEGFR-3 (fms-like-tyrosine kinase-4, also

named vascular endothelial growth factor receptor-3)

A receptor for VEGF-C and VEGF-D, is a member of the

tyrosine protein kinase family, and was first observed in

endothelial cell precursors of mouse embryos [62]. CD34

positive cells, isolated from human fetal liver, were posi-

tive for Flt-4 [63]. These CD34+/Flt-4+ cells, which also

co-expressed CD133, were functionally endothelial cell

precursors, which demonstrated that Flt-4 might be a po-

tential stem cell marker. Furthermore, murine neural pro-

genitor cells express VEGFR-3, and VEGF-C is a trophic

factor for these cells [27].

Endoglin (CD105)

Is a homodimeric cell surface component of the trans-

forming growth factor-beta (TGF-b) type I receptor com-

plex [64]. Several reports showed that Endoglin was a

highly specific marker for HSCs and could be used to

purify HSCs from bone marrow [65–67]. TGF-b1 induces

Endoglin expression and is a crucial factor for maintaining

immaturity of Endoglin positive HSCs [67].

Although Flt-4 and Endoglin mRNA and protein

expression levels increased from low-grade to high-grade

J Neurooncol

123

astrocytomas, in our study and no correlation with the

pathological grades of astrocytomas was found.

To evaluate whether in astrocytomas of WHO grade IV

CD133, Nestin, SOX-2 and Musashi-1 could be co-stained

with GFAP, Ki-67/MIB-1 or among themselves, confocal

microscopy was performed. Whereas, in our study none of

these stem cell markers were co-expressed with Ki-67/

MIB-1, a co-staining of CD133, Nestin, SOX-2 and Mus-

ashi-1 with GFAP could be observed in some cells. These

results are in accordance with findings that Nestin positive

cells can co-express GFAP or beta III-tubulin [44–47]. In

contrast to this, Kanemura et al. demonstrated that Mus-

ashi-1 was expressed in GFAP-negative tumour cells that

formed foci and were surrounded by GFAP-positive cells

[57]. While the expression of Musashi-1 could be corre-

lated with the proliferating activity of gliomas [68], and

this molecule could be found in proliferating cells of the

neural retina [69], some studies demonstrated that Mus-

ashi-1 positive tumour cells did not co-express PCNA or

Ki-67 [70]. Now we could show that in astrocytomas grade

IV CD133, Nestin, SOX-2 and Musashi-1 could be co-

stained with each other, and Musashi-1 seems to be ex-

pressed only in combination with other stem cell markers.

Our results reveal a high heterogeneity of stem cell

marker expression within astrocytomas. Furthermore, the

expression pattern is rather complex, and the different

markers do not necessarily conincide with each other. Also,

cells positive for stem cell marker are not always highly

proliferative ones. It appears that some of the markers, e.g.

Nestin, result from dedifferentiation or from activated state

of the glial tumour cells. Clearly, there is a need for

defining TSCs by more appropriate functional markers, e.g.

by receptors for growth or chemotactic factors that are

involved in stem cell maintenance or differentiation.

Assuming that the most of the investigated markers

represent more or less NSCs or TSCs, astrocytomas in situ

contain a considerable amount of cells with stem cell

character. With increasing WHO grade some of these

markers, namely, CD133, Nestin, Sox-2 and Musashi-1

increase, while others (CXCR4, Flt-4, Endoglin) are ele-

vated over NBT, but are independent for the tumour grade.

It cannot be judged from the in situ expression data whe-

ther these stem cells are the origin of ‘‘differentiated’’

astrocytoma cells or whether astroglial tumours attract

adult stem cells from other parts of the brain. The latter

hypothesis could be supported by an increase of cells po-

sitive for stem cell markers with the WHO grade, whereas

for the first (TSC) hypothesis this would not be required.

Perhaps, both possibilities exist side by side, and mixed

populations of NSCs and (more differentiated) TSCs exist

together, especially in gliomas. Therefore, further investi-

gations on stem cells in astrocytomas/gliomas should

consider their heterogeneity and focus more on functional

markers and properties.

Acknowledgements We thank Brigitte Rehmke, Ute Malcus-Cos-

kun and Jorg Krause for expert technical assistance and Clemens

Franke for drawing pictures. This work was supported by an intra-

mural grant of the Universitatsklinikum Schleswig-Holstein, Campus

Kiel (JH-F & RM) ‘‘Stem cells in brain tumours’’.

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