Cell Stem Cell
Perivascular Nitric Oxide Activates
Notch Signaling and Promotes Stem-like
Character in PDGF-Induced Glioma Cells
Nikki Charles,1,2Tatsuya Ozawa,1,2Massimo Squatrito,1,2Anne-Marie Bleau,1,2Cameron W. Brennan,2,3
Dolores Hambardzumyan,1,2and Eric C. Holland1,2,3,*
1Department of Cancer Biology and Genetics
2Brain Tumor Center
3Department of Neurosurgery and Surgery
Memorial Sloan Kettering Cancer Center, New York, NY 10021, USA
and correlated with increased tumor growth and
aggressive character. We investigated the potential
role of nitric oxide (NO) activity in the perivascular
niche (PVN) using a genetic engineered mouse
model of PDGF-induced gliomas. eNOS expression
is highly elevated in tumor vascular endothelium
adjacent to perivascular glioma cells expressing
Nestin, Notch, and the NO receptor, sGC. In addition,
the NO/cGMP/PKG pathway drives Notch signaling
in PDGF-induced gliomas in vitro, and induces the
side population phenotype in primary glioma cell
cultures. NO also increases neurosphere forming
capacity of PDGF-driven glioma primary cultures,
and enhances their tumorigenic capacity in vivo.
signaling in vivo and prolongs survival of mice. This
mechanism is conserved in human PDGFR amplified
therapeutic targets for this subset of gliomas.
In the normal brain, capillaries located in the subventricular zone
(SVZ) and hippocampus form the major structural entity of the
neural stem cell niche (Riquelme et al., 2008), and the perivascu-
lar region is the location for neural stem cells (Palmer et al., 2000;
Louissaint et al., 2002). This proximiy between neural stem cells
and the vasculature is believed to facilitate intercellular commu-
nication between neural stem cells and endothelia that release
soluble factors critical for promoting stem cell renewal (Shen
et al., 2004; Ramı ´rez-Castillejo et al., 2006).
Glioblastoma multiforme (GBM) is the most malignant and
aggressive type of central nervous system tumor (Legler et al.,
1999) and is classified into groups based on gene expression
profiles where approximately 30% are designated ‘‘proneural’’
and show evidence of PDGF signaling (Phillips et al., 2006;
Brennan et al., 2009). As gliomas progress to higher grade,
they develop several histologic structures that define malignant
behavior, including microvascular proliferation or hypercellular
vasculature. Microvascular proliferating regions of these tumors
are grossly disorganized angiogenic vessels that are a hallmark
of malignant behavior and are surrounded by a perivascular
niche that is a habitat for brain tumor stem-like cells (Calabrese
et al., 2007). Soluble factors released from endothelial cells
promote the self-renewal and proliferation of brain tumor stem-
like cells (Calabrese et al., 2007; Folkins et al., 2007). In medullo-
blastomas, stem-like cells in the perivascular niche are resistant
to radiation and are believed to give rise to tumor recurrence
(Hambardzumyan et al., 2008b). The underlying mechanism(s)
responsible for the formation of vascular stem cell niches that
maintain tumor cells in a stem-like state are not understood;
moreover, the mechanisms responsible for driving stem-like
character in the perivascular niche and the endothelia-derived
factors that support cancer stem-like cells of the perivascular
niche in brain tumors have not been identified.
tric oxide (NO) from their substrate L-arginine. NO regulates
many physiological processes through the NO/cGMP pathway,
as well as through protein S-nitrosylation (Fukumura et al.,
2006). During NO/cGMP signaling, NO produced from one cell
diffusesto neighboring cellswhereitbinds toitsreceptor soluble
guanylate cyclase (sGC). sGC converts GTP to cGMP to activate
several downstream effectors, including cGMP-dependent
protein kinase (PKG). Many of the activities of NO signaling
thelial isoform of nitric oxide synthases, endothelial nitric oxide
synthase (eNOS), is required for initiation and maintenance of
human pancreatic tumor growth (Lim et al., 2008), and eNOS is
elevated in various cancers (Fukumura et al., 2006), including
human gliomas (Bakshi et al., 1998; Broholm et al., 2003), where
its expression is correlated with glioma grade (Cobbs et al.,
1995). Elevated levels of eNOS expression and activity in
gliomas are often localized to the tumor vascular endothelium
(Iwata etal.,1999).However, the specific roleof eNOS in glioma-
genesis has not been fully established.
We hypothesized that NO produced by eNOS in endothelial
cells functions in a paracrine manner to activate signaling
Cell Stem Cell 6, 141–152, February 5, 2010 ª2010 Elsevier Inc. 141
pathways in glioma cells in the perivascular niche and thereby
promotes or reinforces stem cell character. We used a geneti-
cally engineered mouse model of PDGF-induced gliomas to
investigate the role of NO in gliomas. This model shows eNOS
expression restricted to the tumor vascular endothelium and
that a population of stem-like cells expressing Nestin and
Notch1 are tightly apposed to the tumor endothelium. These
Nestin-expressing stem-like cells also express sGC, the
receptor for NO, and NO activates Notch in glioma stem-like
cells through the NO/cGMP/PKG pathway. This NO-induced
activation of Notch signaling in stem-like cells accelerates
glioma initiation and tumor formation in mice. We further show
that mice lacking eNOS have delayed gliomagenesis and subse-
quent enhanced survivalcorrelating with decreased activation of
is conserved in human PDGFR-amplified gliomas. We also show
that NO activates the Notch pathway to enhance the side popu-
lation (SP) phenotype in cultured human glioma cells.
Nitric Oxide Stimulates Nestin and Hes1 Promoter
Activity in Cultured Human Glioma Cells
Factors derived from the tumor vascular endothelium reinforce
the self-renewal of stem-like cells residing in the brain tumor
perivascular niche (PVN) (Calabrese et al., 2007). Since elevated
eNOS expression and activity is restricted to the tumor vascular
endothelium, we investigated whether NO might be one factor
promoting stem-like activity within the niche. In order to investi-
gate thepotentialrole ofNO onstem-like character, weanalyzed
its effect on pathways known to regulate stem cell character,
namely Notch, Shh, and Wnt (Taipale and Beachy 2001; Radtke
and Raj 2003). Luciferase reporters coupled to the promoters
of Hes1, Gli1, or b-Catenin were transiently transfected into
U251 human glioma cells. As shown in (Figure 1A), S-nitrosoglu-
tathione (GSNO), an NO donor, induced a more than two-fold
increase in luciferase expression in the Hes1-luciferase-
expressing cells relative to untreated controls (80.57 ± 0.55
versus 28.32 ± 0.13; p < 0.0001). This effect was specific to acti-
vation of the Hes1 promoter, as there was no statistically signif-
icant difference in activation of Gli1 or b-Catenin promoters
(41.15 ± 0.81 versus 39.91 ± 0.37 and 5.44 ± 0.23 versus
4.23 ± 0.19, respectively) following GSNO treatment. Nestin,
a well-characterized marker of stem/progenitor cells in brain
tumors, is highly expressed in stem-like cells of the glioma
PVN, and Notch signaling is known to activate the Nestin
promoter in gliomas (Shih and Holland 2006). In addition, loss
of eNOS was demonstrated to decrease Nestin expression in
the brain in vivo (Chen et al., 2005). Therefore, we determined
whether NO affects Nestin expression in human glioma cells.
Transient transfection of the U251 cell line with a Nestin-lucif-
erase reporter indicated that GSNO treatment led to an approx-
imately 2-fold induction of the Nestin reporter relative to controls
(77.95 ± 2.55 versus 38.84 ± 0.66; p < 0.0001) (Figure 1A). We
confirmed activation of the Notch pathway in U251 cells by
western blot for HES1 protein, following GSNO treatment (Fig-
ure S1A available online). In addition, we analyzed the mRNA
transcripts encoding HES1, NESTIN, GLI1, and b-CATENIN in
these cells, following treatment with GSNO. The transcriptional
levels of HES1 and NESTIN were significantly elevated relative
to controls (10.8 ± 2.45 versus 1 ± 0.26 and 5.2 ± 1.36 versus
1 ± 0.29, respectively). Gene expression levels for GLI1 and
b-CATENIN were unchanged (1.4 ± 0.56 versus 1 ± 0.39 and
0.97 ± 0.22 versus 1 ± 0.28, respectively) (Figure S1D). These
data indicate that NO can specifically activate the Notch
pathway in human glioma cells.
eNOS and Active Notch1 Proteins Are Significantly
Elevated and Are Expressed in Cells of the PVN
in PDGF-Induced Mouse Gliomas
To further investigate the connection between NO and the Notch
pathway in gliomas, we employed the RCAS/tv, a method for
creating PDGF-induced gliomas in mice, because the well-char-
logical features of this model closely mimic those observed in
human gliomas (Holland 2004). Western blot analysis demon-
strated that both eNOS and cleaved Notch1 (Notch intracellular
domain-NICD) were highly elevated in PDGF-induced mouse
gliomas with respect to the contralateral side of the brain (p <
0.0001) (Figure 1B). Usingimmunofluorescence, we investigated
their spatial relationship to one another within the glioma PVN.
Immunostaining for total eNOS protein within the PDGF-induced
gliomas indicated that eNOS colocalized with CD31-expressing
endothelial cells (Figure 1C) surrounded by a population of
Nestin-expressing cells that also coexpress Notch1 (Figures
1D and 1E). These Nestin-expressing perivascular cells also
express soluble guanylyl cyclase (sGC, the major receptor for
NO) (Madhusoodanan and Murad 2007), whose staining is
limited almost exclusively to the perivascular niche (Figure 1F)
and which therefore may represent a population of cells within
the niche that can respond to NO signaling.
Nitric Oxide Activates Notch Signaling and the SP
Phenotype in Primary Cultured Mouse Glioma Cells
The data above suggest a regional correlation between eNOS
expression and Notch1 activation in vivo. In order to determine
whether there is a direct link between NO signaling and Notch
signaling within PDGF-induced mouse gliomas, we investigated
whether NO could upregulate the Notch signaling pathway in
culture. Western blot analysis of GSNO-treated PDGF-induced
glioma primary cultures (PIGPCs) revealed a dose-dependent
increase in Notch intracellular domain (NICD) indicating activa-
tion of the Notch pathway (Figure S1C). Cell viability was not
adversely affected after 6 hr of treatment (data not shown). We
then examined the effect of NO on the expression of Notch
ligand proteins and the downstream protein targets of Notch,
Hes1, and Hey1. GSNO treatment of PIGPC indicated specific
activation of the Notch pathway, as evidenced by a substantial
increase in components of the activated Notch pathway
(Figure 2A). The expression of the Notch ligand proteins, Delta-
like1 and 4 (DLL1 and DLL4), increased within 30 min, which
coincided with elevated NICD. Expression of the transcription
factor targets of Notch signaling, Hes1, and Hey1 was subse-
quently elevated at 1 hr.
Activation of the Notch pathway plays a critical role in
promoting stem-like character in brain tumors (Fan et al.,
2006). Therefore, we investigated whether NO was involved
in mediating this effect in PDGF-induced gliomas using side
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population(SP)analysis.SP analysisisusedfor theidentification
of stem cells via fluorescence-activated cell sorting and is based
on the capacity of stem cells to efflux Hoechst fluorescent dyes
by the activity of ATP binding cassette transporters (ABC trans-
porters) (Goodell et al., 1996). The SP cells in human glioma cell
lines as well as other tumors are enriched in tumorigenic cells
with stem cell properties, and the SP cells of PDGF-induced
gliomas are more capable of growing as tumor neurospheres
and are more tumorigenic than non-SP cells when transplanted
in mice (Bleau et al., 2009). PDGF-induced glioma primary
cultures (PIGPCs) were incubated with Hoechst 33342 in the
presence or absence of GSNO and assayed for their SP. These
PIGPCs contained an SP that ranged from ?3% to 20% at base-
line dependingon the tumor. Following 2–2.5 hrof NO treatment,
a significant increase (2- to 5-fold) in the SP was observed
relative to vehicle-treated cells derived from the same tumor
Figure 1. Nitric Oxide Stimulates Nestin and Hes1 Promoter Activity in Human Glioma Cells and Elevated eNOS and Notch1 Protein Expres-
sion Is Localized to Cells of the Glioma Perivascular Niche
(A) Luciferase assay of U251 glioma cells transfected with Hes1-, Nestin-, Gli1-, or b-Catenin-responsive luciferase reporters. Positive controls for Gli and Wnt
reporters were cotransfections of CMV-gli- or CMV-beta-Catenin-expressing vectors, respectively (**p < 0.005 reports difference between Nestin and Hes1
promoter activity in control and GSNO-treated U251 cells). Error bars are the mean ± SEM.
(B) Western blot analysis of PDGF-induced mouse gliomas analyzed for total eNOS and Notch intracellular domain (NICD) proteins (n = 7). N and T represent the
contralateral normal and tumor-containing hemispheres of the brain, respectively. Bottom: quantification of eNOS and NICD proteins. Data are represented as
mean ± SEM (***p < 0.005 reports difference between the contralateral normal and tumor-containing hemispheres). Error bars are the mean ± SEM.
(C–F) Coimmmunofluorescence in mouse PDGF-induced gliomas of eNOS, Notch1, sGC (red), and CD31 and Nestin (green) expression. All nuclei are stained
with DAPI. Scale bars, 75 mm.
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NO in the Glioma Perivascular Niche
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(15.50 ± 3.28 versus 4.25 ± 0.48; p = 0.015) (Figure 2B and
Figure S2A). Both control and GSNO-treated cultures were
judged to be approximately 90% viable, indicating that selec-
tion for viable cells was not occurring during the treatment
We hypothesized that NO might preferentially upregulate
Notch signaling in a subpopulation of PIGPC cells. To address
this possibility, primary cultures were pretreated with vehicle or
GSNO, sorted for side and main populations, and analyzed
by western blot for NICD. The relative increase of NICD seen
above was greater in cells of the SP (Figure 2C), suggesting
that NO activates the Notch pathway in a population of glioma
cells, which may promote their SP phenotype or stem cell-like
ABCG2 is expressed in glioma stem-like cells, and its expres-
sion was correlated with increasing glioma grade (Jin et al.,
2009). Furthermore, abcg2 gene expression is specifically upre-
gulated in the cancer stem-like populations of mouse PDGF-
induced gliomas (Bleau et al., 2009). We investigated whether
NO might drive the expression of Abcg2 protein as an additional
measure of NO activation of the Notch pathway. Therefore, we
analyzed 4 PIGPCs treated with GSNO by western blot for
the expression of Abcg2 relative to vehicle-treated controls. All
four primary glioma cultures examined showed increased
Abcg2 protein expression, following GSNO treatment versus
controls (69.67 ± 15.48 versus 22.72 ± 3.21; p = 0.041)
NitricOxide RequiresNotchSignaling toEnhancetheSP
Phenotype in PDGF-Induced Glioma Primary Cultures
To further investigate whether Notch signaling drives the SP
phenotype in gliomas as it does in medulloblastomas (Fan
et al., 2006), we treated these PIGPCs for 2 hours with the
gamma secretase inhibitor (GSI) MRK-003 (Lewis et al., 2007).
The baseline SP in these primary glioma cultures was reduced
by GSI treatment, suggesting that Notch signaling is critical for
the maintenance of the SP phenotype in PDGF-induced gliomas
(Figure S3A). We investigated whether the increase in the SP
phenotype induced by NO is dependent on Notch activation.
PIGPCs were incubated for 2 hours with GSI in the presence
or absence of GSNO, then analyzed for their SP. Treatment of
these primary glioma cultures with GSI abolished the GSNO-
induced increase of the SP (13.88 ± 1.78 versus 0.33 ± 0.13;
p = 0.003) (Figure 3A and Figure S3B), suggesting that NO
requires activation of the Notch pathway to drive the SP pheno-
type in PDGF-induced gliomas. Control, GSNO-, and GSI-
treated cultures were approximately 90% viable by PI staining,
which confirms that cell viability was not adversely affected by
the treatments (Figure S3C). We confirmed the specificity of
(Notch1-SH) knockdown of Notch1 mRNA in PIGPCs resulted
the SP relative to a nonspecific scramble control (p = 0.0109)
(Figures 3B and 3C). To further verify that Notch signaling medi-
infected with a vector expressing constitutively active Notch
(NICD) and then analyzed for their SP phenotype. NICD overex-
pression induced a more than 2-fold increase in the SP when
compared with the empty vector control (14.3 ± 2.70 versus
6.59 ± 1.41; p = 0.0226) (Figure 3D). These data indicate that
Notch signaling is necessary and sufficient for the NO-induced
elevation in SP phenotype in these glioma cells.
Inhibition of Nitric Oxide Activity in PDGF-Induced
Gliomas In Vivo Diminishes Notch Signaling and the SP
Phenotype and Enhances Survival
We next investigated whether the relationship between NO and
Notch signaling seen in culture was conserved in PDGF-induced
mouse gliomas in vivo. Ten glioma-bearing mice were treated
with the NOS inhibitor L-NG-nitroarginine methyl ester (LNAME)
for 24 hr. Tumor tissue and contralateral normal brain were
analyzed by western blot for Notch cleavage (NICD) and Notch
Figure 2. NO Activates Notch Signaling and the SP Phenotype in PDGF-Induced Glioma Primary Cultures
(A) PDGF-induced glioma primary culture (PIGPC) treated with GSNO (100 mM) for the indicated times (n = 6).
(B) SP analysis of GSNO-treated PIGPC. Inset shows cells treated with FTC (fumitremorgen C, an ABCG2 inhibitor). SP, side population; MP, main population.
Data shows one representative graph of four independent experiments.
(C) GSNO-treated PIGPCs, sorted for their SP and MP, then analyzed by western blot for expression of NICD with respect to vehicle-treated controls.
(D) Left: western blot analysis of whole-cell lysates from PIGPC treated with GSNO or vehicle and probed for NICD, ABCG2, and b-actin proteins. Right: quan-
tificationof ABCG2proteinsusingImageJ software. Dataare represented asmean ± SEMofthree individualexperiments (*p< 0.05compared withcontrol). Error
bars are the mean ± SEM.
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ligands. Nine vehicle-treated PDGF glioma-bearing mice were
elevated relative to the normal brain in all cases (p < 0.0001)
(Figure 4A and Figure S4A). NICD was significantly diminished
in LNAME-treated mice relative to untreated controls (79.35 ±
7.23 versus 130.4 ± 8.99; p < 0.0004), and the expression of
the Notch ligand Jagged 2 was also significantly lower relative
to the untreated controls (64.73 ± 12.57 versus 114.8 ± 3.51;
p < 0.05) (Figure 4A). To determine if suppression of NO activity
by LNAME would affect the SP in these PDGF-induced gliomas,
we analyzed the SP of gliomas in six mice treated for 3 days with
LNAME relative to six vehicle-treated tumor-bearing mice of
the same age and background. We found that the SP for the
treated group was significantly lower than the vehicle-treated
controls (2.8 ± 0.22 versus 4.2 ± 0.43; p < 0.0196) (Figure 4B
and Figure S5).
To assess whether complete loss of eNOS would affect the
development of PDGF-induced gliomas in vivo, we crossed
mice carrying a homozygous disruption of eNOS (eNOS?/?)
into Nestin tv-a (N-tva) mice and infected the progeny with
RCAS-PDGF. We compared the survival of eNOS?/?mice with
their respective wild-type littermates (eNOS+/+). The survival of
eNOS?/?mice (n = 43) was significantly longer than eNOS+/+
mice (n = 42) (p = 0.0042) (Figure 4C). Since NO activates the
Notch pathway in these PDGF gliomas, we tested whether the
increased survival observed with the loss of eNOS correlated
with a decrease in activation of the Notch pathway during tumor
progression or at the time of death. Using western blot analysis,
we compared the levels of protein expression of Notch
pathway components between eNOS+/+mice (approximately
54 days, the median age of death in eNOS+/+mice) with respec-
tive eNOS?/?counterparts of the same age. eNOS+/+mice
expressed significantly higher levels of NICD and the Notch
ligand DLL1 relative to their eNOS?/?counterparts. However,
the expression levels of NICD and Notch ligands Jagged 1 and
DLL 1 and -2 in the tumors at death were similar in both
populations (54 days for eNOS+/+and 84 days for eNOS?/?)
(Figure S4B), implying that the development of Notch signaling
was delayed in the absence of eNOS, but eventually reached
a level similar to eNOS+/+mice by the time the tumors were suffi-
ciently aggressive to be lethal.
NO Activates Notch Signaling to Enhance the SP
Phenotype through cGMP Activation of PKG
Downstream NO signaling can be cGMP dependent or cGMP
independent. However, manyphysiological processes regulated
by NO involve cGMP, which activates cGMP-dependent protein
kinase (PKG) (Blaise et al., 2005). To investigate whether the
Figure 3. Notch Signaling Is Required for Nitric Oxide Enhancement of the SP Phenotype
(A) Left: SP analysis of GSNO (100 mM)- and GSI (3 mM)-treated PIGPCs. Inset shows cells treated with FTC control. Right: bar chart shows quantification of SP
analyzed data on the left. Error bars are the mean ± SEM of three individual experiments (***p < 0.0005 compared with GSNO treatment).
(B) Western blot to confirm knockdown of Notch1 proteins.
(C) Left: SP analysis shows a decrease in the percent of SP cells by Notch1-sh-RNA (Notch1-SH) relative to nonspecific scramble control. Inset shows a negative
control, FTC. Graph represents one of six independent PIGPCs samples. Right: quantification of Notch1-shRNA mediated knockdown of the SP in six indepen-
(D) SP analysis of PIGPCs infected with empty vector or vector expressing constitutively active Notch (NICD). Inset shows cells treated with FTC.
Cell Stem Cell
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Cell Stem Cell 6, 141–152, February 5, 2010 ª2010 Elsevier Inc. 145
increase in the SP phenotype induced by NO involved PKG, we
determined if PKGactivation couldincrease the SPphenotype in
PIGPCs similar to NO. We utilized the nonhydrolyzable cGMP
analog and potent activator of PKG, 8-Br-PET-cGMP (PET-
cGMP), to address this question. PIGPCs were treated with
vehicle or Pet-cGMP for 2 hr and then analyzed for their SP.
When compared with vehicle-treated controls, PET-cGMP
enhanced the percent of SP cells in PIGPCs to levels exceeding
that achieved by GSNO alone (9.00 ± 5.03 versus 48.33 ± 7.67;
9.00 ± 5.03 versus 26.7 ± 7.84, respectively) (Figure 5A). This
induction ranged from a 3- to 11-fold increase in the SP relative
to vehicle treated controls (Figure S6). Furthermore, the increase
in the SP observed with GSNO and Pet-cGMP combined was no
more than that observed by either individual treatment
(Figure 5A), suggesting that GSNO and Pet-cGMP act in the
same pathway.ToconfirmactivationofNotch signaling,PIGPCs
were treated for 2 hr with Pet-cGMP, then analyzed by western
blot for activation of the Notch pathway. Western blot analysis
on these primary cultures revealed increased NICD and
enhanced expression of the Notch downstream target Hes1
(Figure 5B). As an additional measure of the potential effect of
PKG activity on Notch signaling, we used an alternative
approach to enhance the activity of PKG in our glioma primary
cultures. PIGPCs were treated with the phosphodiesterase 5
(PDE5) inhibitor, sildenafil, which enhances steady-state levels
of cGMP by suppressing its degradation by PDE5 (Bender and
Beavo, 2006). As shown in Figure 5C, PIGPCs treated with silde-
nafil for 1 hr show increased expression of NICD protein. The
PKG target vasodilator-stimulated phosphoprotein (VASP) was
phosphorylated at serine 239 (Butt, 2009), indicating that PKG
was activated by Pet-cGMP and sildenafil (Figures 5B and 5C).
Sildenafil is a direct inhibitor of ABC transporters (Oesterheld,
2009); therefore, SP analysis of primary glioma cultures treated
with sildenafil could not be conducted. To confirm that PKG
activity is required for NO-mediated enhancement of the SP
phenotype, PIGPCs were treated with the PKG inhibitor
KT5823 in the absence or presence of GSNO and analyzed for
their SP characteristics. As shown in Figure 5D, in the presence
of KT5823, induction of the SP by GSNO or Pet-cGMP was
diminished relative to their respective individual treatments
(6.11 ± 2.43 versus 17.30 ± 3.6 and 5.36 ± 0.87 versus 24.95 ±
0.55; p = 0.0027). VASP phosphorylation was used to confirm
inhibition of PKG by KT5823 (Figure 5E). Similar results were
obtained using a second inhibitor of PKG, Rp-8-pCPT-cGMP,
which inhibits PKG by a different mechanism than KT5823
(data not shown). Collectively, these data indicate that NO
enhances Notch signaling and the SP phenotype in PDGF-
induced gliomas through activation of PKG. Endothelial cells
are known to express ABC transporters and are a component
of the SP in PDGF-induced gliomas (Bleau et al., 2009). More-
over, they can activate the cGMP/PKG signaling pathway in
response to NO (Fukumura et al., 2006). We verified that the
NO-induced increase in the SP phenotype was not due to the
presence of contaminating endothelial cells within these PIGPC
by analyzing six independent PIGPCs by western blot for the
expression of the endothelial cell markers, eNOS and CD31
Transient Activation of the NO/cGMP Pathway
Enhances the Neural Stem Cell-Forming Capacity
of PDGF Gliomas In Vitro
Thus far, we have shown that in a population of PIGPC cells,
NO activates the SP phenotype and Notch signaling, both
consistent with stem-like characteristics. These characteristics
are observed after transient (2 hr) activation of the NO/cGMP
pathway with GSNO or the cGMP analog, Pet-cGMP. Using
a neurosphere formation assay, we then investigated whether
this transient activation might have long-term effects on the
overall capacity of these PIGPC to behave in a stem-like manner
after withdrawal of treatment. When cultured with the growth
factors EGF and bFGF, in the absence of serum, neural stem
cells form neurospheres. Neurosphere-forming glioma cells
Figure 4. Suppression of NO Activity In Vivo
Decreases Notch Signaling and the SP
Phenotype and Enhances Survival In Vivo
(A) Western blot on whole-cell lysates of PDGF-
induced gliomas derived from vehicle (water)- or
LNAME (45 mg/kg; 24 hr)-treated mice using
NICD, Jagged 1 and 2 and Delta-like 1 antibodies.
N represents the contralateral nontumor hemi-
sphere of mouse brain. T represents the tumor-
bearing portion of mouse brain. Vehicle (n = 3);
LNAME (n = 4). Bar chart represents quantification
of proteins analyzed on the left using ImageJ
software. (*p < 0.05 and ***p < 0.0005 reports the
difference between NICD and Jagged 2 in vehicle
and LNAME-treated PDGF-induced gliomas).
in vivo decreases the SP phenotype in PDGF-
induced gliomas (*p < 0.05 reports the difference
between vehicle [n = 6]- and LNAME [n = 6]-
(C) Kaplan-Meier survival curve shows loss of
eNOS expression inPDGF-induced
prolongs survival of mice (eNOS+/+n = 41;
eNOS?/?n = 42).
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represent the more stem-like cell populations in brain tumors
(Singh et al., 2003; Galli et al., 2004) and are enriched in the SP
(Patrawala et al., 2005; Harris et al., 2008). In addition, SP cells
generated from these PDGF-induced gliomas were previously
shown to be enriched with the self-renewing stem-like cell pop-
ulations, which propagate the neurosphere forming capacity of
these gliomas (Bleau et al., 2009). Therefore, we pretreated
treatment waswithdrawn. Thecellswere resuspended, plated at
clonal density (1 cell/ml) in neurosphere medium, and then sub-
jected to an in vitro tumor neurosphere-forming assay that was
quantified by counting the number of neurospheres formed
2 weeks after treatment. Neurosphere formation in GSNO- or
Pet-cGMP-treated glioma primary cells was approximately
twice as fast and more than double the number observed in
vehicle-treated plates (48.83 ± 8.167 versus 20.33 ± 2.171;
p < 0.05 and 76.17 ± 5.845 versus 20.33 ± 2.171; p < 0.005)
(Figure 6A). In addition, tumor neurospheres generated from
each group could be serially passaged to generate secondary
and tertiary neurospheres, demonstrating their capacity for
self-renewal. These neurospheres also stained with antibodies
against the stem cell markers Nestin, Nanog, Musashi, and
Oct-4 (Figure 6B). These results indicate that short-term activa-
tion of the NO/cGMP pathway in a population of PIGPCs can
to a more stem cell-like phenotype.
Transient Activation of the NO/cGMP Signaling Pathway
in PIGPCs Enhance Their Capacity to Establish Tumors
The SP cells of PDGF-induced gliomas are enriched with the
self-renewing stem cell-like populations and are more tumori-
genic than non-SP cells when injected into neonatal mice (Bleau
et al., 2009). The data observed from the neurosphere formation
assay above suggest that transient activation of the NO/cGMP
pathway in vitro in a population of PIGPCs is sufficient to effect
long-term changes toward a more stem cell-like phenotype
within these gliomas. Therefore, we then determined if the
pro-stem-cell-like characteristics achieved in these glioma cells
by transient activation of the NO/cGMP pathway affected
Figure 5. Nitric Oxide Induces the SP Phenotype through cGMP Activation of PKG
(A) Left: SP analysis of GSNO (100 mM)- and Pet-cGMP (200 mM)-treated PIGPCs. Inset shows cells treated with FTC. Right: quantification of SP analysis on the
left. Error bars are the mean ± SEM of three individual experiments.
(C) Western blot analysis on whole-cell lysates of PIGPC treated with sildenafil (1mM) for the indicated times and probed with pVASP, VASP, NICD, and b-actin
(D) Left: SP analysis on PIGPC treated with GSNO, Pet-cGMP, and the PKG inhibitor KT5823 (3 mM). Right: quantification of SP analysis on the left (**p < 0.005
reports the difference between Pet-cGMP alone and in the presence of KT5823). Error bars are the mean ± SEM.
(E) Western blot analysis on whole-cell lysates of PIGPC analyzed in (D) and probed with pVASP, VASP, NICD, and b-actin antibodies.
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Cell Stem Cell 6, 141–152, February 5, 2010 ª2010 Elsevier Inc. 147
tumorigenesis in vivo. PIGPC cells were treated for 2 hr with Pet-
cGMP or GSNO as above and immediately injected into the
cortex of neonatal mice (8 3 104cells per pup, n = 20 Pet-
cGMP population; n = 15 GSNO population). As a control, the
same number of untreated PIGPC cells were injected into the
cortex of a second population of neonatal mice (n = 31). Mice
were closely monitored and sacrificed if they developed symp-
toms of hydrocephalus, lethargy, or cachexia. Tumor formation
was confirmed by histology, which showed tumors to be diffuse
and some to exhibit histological features of high-grade gliomas
such as the presence of microvascular proliferation and pseudo-
palisading necrosis (Figure 6C). As shown in Figure 6D, mice
injected with PIGPCs pretreated with the cGMP analog Pet-
cGMP developed tumors with both higher incidence and shorter
latency than their control counterparts. Injection of Pet-cGMP-
treated primary glioma cells resulted in tumors at 45% incidence
(9 of 20), compared with a 3% incidence (1 of 31) obtained from
injections of untreated primary glioma cells (p = 0.0004). In
addition, GSNO-treated PIGPCs pretreated for 2 hr and injected
into a similar number of mice of the same background resulted
in a tumor incidence of 27% (4 of 15) compared with the lower
incidence seen in untreated controls (p = 0.033). Moreover, the
20% less than the 45% seen with PET-cGMP and consistent
with the relative effects of these two treatments on the SP
phenotype described above.
Activation of the NO/cGMP Signaling Pathway Also
Enhances Notch Signaling and the SP Phenotype in
Human Glioma Cell Cultures and Diminishes Survival
Thus far, using the RCAS/tv, a mouse model, we demonstrated
that activation of NO/cGMP signaling in PDGF-induced gliomas
promotes stem cell-like characteristics. In light of these data
from this mouse model, along with previous findings showing
Figure 6. Enhanced NO/cGMP Signaling Promotes the Neural Stem Cell-Forming Capacity of PDGF-Induced Gliomas and Enhances Tumor-
**p < 0.01 reports the difference between vehicle-treated controls and GSNO or pet-cGMP treatments, respectively). Error bars are the mean ± SEM.
(B) Oct-4, musashi, Nanog, and Nestin immunofluorescence on NSCs generated in (A). Scale bars, 40 mm.
(C) Representative whole-mount H&E of tumors generated from Pet-cGMP-treated PIGPC. Arrows indicate high-grade features such as pseudopalisades (white
arrow) and microvascular proliferation (black arrow).
(D) A comparison of tumor incidence between vehicle (water)-, Pet-cGMP-, and GSNO-treated primary glioma cells (Fisher’s exact test; *p = 0.033 and
**p = 0.0004 reports the difference between vehicle-treated controls and GSNO and pet-cGMP treatments, respectively).
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148 Cell Stem Cell 6, 141–152, February 5, 2010 ª2010 Elsevier Inc.
elevated expression of eNOS in human gliomas, (Cobbs et al.,
1995; Iwata et al., 1999), we wondered if NO might play a role
in promoting stem cell-like activity within the PVN in the context
of human gliomas. The T98G human glioma cell line was previ-
ously shown to contain a SP (Chua et al., 2008). We, therefore,
treated these cells with GSNO and analyzed them for their SP
phenotype relative to controls. GSNO induced an approximate
5-fold increase in the SP when compared with vehicle treated
controls (13.87 ± 0.22 versus 3.43 ± 0.15; p < 0.0001)
(Figure 7A). The induction of the SP by GSNO was blocked in
the presence of the GSI (13.87 ± 0.22 versus 1.16 ± 0.04;
p < 0.0001), suggesting a role for Notch in mediating this
effect. Confirmation of Notch pathway activation was performed
by western blot analysis (Figure S1B). As the PDGFR amplified
subtype of human gliomas is accurately modeled in the PDGF-
induced mouse model, we treated human primary cultured
neurospheres from a PDGF receptor (PDGFR)-amplified human
glioblastoma sample with GSNO. Following NO treatment,
there was an approximate 3-fold induction of the SP when
compared with vehicle treated controls (18.87 ± 0.79 versus
6.87 ± 0.37; p < 0.005). This GSNO-induced increase in the SP
was blocked in the presence of the GSI (18.87 ± 0.79 versus
2.87 ± 1.14; p < 0.005) (Figure 7B), indicating that NO induction
of the SP phenotype through activation of Notch is conserved in
a subset of human gliomas. When combined, the percent
increase in the SP by GSNO and Pet-cGMP was no more than
with either individual treatment, as was seen in our PDGF-
induced glioma mouse model. In addition, we analyzed five
PDGFR amplified human glioma tissue samples by immunohis-
tochemistry for the expression of ENOS, SGC, and NESTIN.
Figure 7. NO Enhances the SP Phenotype in Human Glioma Cells and Decreases Survival
(A) SP analysis of GSNO- and GSI-treated T98G human glioma cell lines. Inset shows cells treated with FTC.
(B) Above: SP analysis of GSNO (35 mM)- and GSI (3 mM)-treated human tumor neurospheres. Below: SP analysis of GSNO- and Pet-cGMP (80 mM)-treated
human tumor neurospheres. Inset shows cells treated with FTC. Plots represent one of three experiments.
(C) Coimmmunofluorescence on PDGFR-amplified human gliomas to detect eNOS (green), CD31, and Nestin (red) protein expression. All nuclei are stained with
DAPI. All scale bars, 75 mm.
GSNO-treated human primary glioma cultures was 36 and 29 days, respectively (n = 10).
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In all five PDGFR-amplified human glioma samples, ENOS
showed vivid expression in the tumor vasculature, and in three
of five samples, NESTIN expression was enhanced in cells
surrounding the tumor vascular endothelium (Figure 7C), with
the remaining two samples showing diffuse NESTIN staining
throughout the tumor. The SGC staining paralleled the NESTIN
expression in these tumors (data not shown). Identical immuno-
histochemical analysis of six human EGFR amplified human
gliomas did not reveal similar ENOS and NESTIN expression
limited to the PVN. (Figure S7).
To assess the tumorigenic potential of NO-activated human
glioma primary neurospheres, immunocompromised mice
were injected with 2.5 3 105GSNO-treated primary glioma cells
and compared to their age-matched controls injected with an
equalnumber ofthesame vehicle-treated humanprimary glioma
cells (10 mice per group). All mice developed tumors; however,
the survival difference between the two groups was statistically
significant (p < 0.005) with a shorter survival for mice receiving
the NO-activated cells. The median survival of vehicle- and
GSNO-treated human primary glioma cultures was 36 and
29 days, respectively (Figure 7D). These data suggest that within
the PDGFR gene amplified subset of human gliomas, elevated
nitric oxide activity within the tumor vasculature may play
a role in promoting stem cell-like characteristics within some of
Stemcellswithin thebrainarelocalized tospecificmicroenviron-
ments that regulate their maintenance (Doetsch 2003). A peri-
vascular location for stem cells has been described for normal
(Shen et al., 2004; Ramı ´rez-Castillejo et al., 2006) and brain
tumor stem-like cells (Calabrese et al., 2007; Hambardzumyan
cular proliferation represent these vascular stem-like cell niches.
As progression to more aggressive gliomas is associated with
a prominent increase in the density of these vascular structures
(known as microvascular proliferation), their presence in gliomas
is a criteria for malignant histology by the WHO grading system
(Kleihues et al., 1995). This association with increasing grade
has led to proposals that the perivascular niche might directly
contribute to progression of brain tumors. One proposal for the
perivascular location of stem-like cells has been that factors
released from endothelial cells intricately associated with the
niche and activate one or more signaling pathways in adjacent
cells that reinforces their stem-cell like character (Calabrese
et al., 2007). Our finding suggests that one of these mechanisms
is the activation of Notch signaling by NO produced from the
endothelial cells. We propose that NO released from the tumor
endothelium diffuses to neighboring glioma stem-like cells,
tightly associated with the tumor vasculature, and activates the
Notch pathway within these stem-like cells (Figure S8).
The tumor perivascular niche is a complex microenvironment
with multiple cell types and signaling factors involved in the
crosstalk between endothelial cells and stem-like cells residing
within the niche. The contribution of aberrant NO signaling within
the niche is not the only component involved in the crosstalk
between the tumor endothelium and perivascular stem-like
cells. In vivo, glioma stem-like cells are likely influenced by the
cular niche. In addition to glioma stem-like Nestin-expressing
cells, there are other cell types known to reside within the brain
tumor perivascular niche, such as reactive astrocytes that
mediate Shh signaling (Becher et al., 2008) known to promote
stem cell renewal in gliomas (Clement et al., 2007) and pericytes
(Hambardzumyan et al., 2008a) that likely contribute either
through direct cellular interactions or in a paracrine fashion to
regulate the activity of glioma stem-like cells.
It is worth noting that transient activation of the NO/cGMP
pathway by GSNO or Pet-cGMP activates Notch signaling and
increases the percentage of SP cells in PIGPC within 2–2.5 hr.
These cells then show long-lasting fundamental changes in their
behavior, as demonstrated by their increased capacity for neural
stem cell formation 2 weeks after treatment withdrawal and
increased capacity for tumor formation more than 4 weeks
after tumor cell transplantation. This effect might involve the
conversion of a subpopulation of glioma cells within our primary
cultures, with an inherent capacity to be primed by NO/cGMP
signaling to a more stem cell-like state. The data strengthens
the argument that stem cell character can be acquired by
specific signaling from the tumor microenvironment. As the
RCAS/tv-a PDGF mouse model mimics the PDGFR subtype of
human gliomas, this observation may be limited to this subgroup
of human gliomas. We have identified PKG as a critical mediator
of NO-induced Notch pathway activation in PDGF-induced
mouse gliomas; however, the mechanism through which PKG
activates the Notch pathway remains to be determined.
induction of stem-like activity complements its established role
in tumor angiogenesis and underscores the emerging role of
the glioma perivascular niche as a direct participant in the
disease process. This work also highlights several potential
therapeutic targets, such as inhibitors of endothelial nitric
oxide synthase (eNOS), PKG, and Notch. Although these effects
were ascribed to inhibition of eNOS-induced angiogenesis, it is
possible that these therapies additionally modified stem-like
characteristics within these tumors. In addition, the use of anti-
angiogenic agents that disrupt these aberrant glioma vascular
stem cell-like niches could potentially sensitize tumors to
conventional treatments by depriving the stem-like cells of niche
signals, such as NO, required for their maintenance. Such
synergy was previously demonstrated with bevacizumab, which
after treatment of glioma-bearing mice, resulted in depletion of
tumor blood vessels and a significant reduction of tumor stem
cells (Calabrese et al., 2007). Since antiangiogenics, such as
endostatin and thrombospondin, exert their effects through inhi-
bition of eNOS (Fukumura et al., 2006), the effect observed with
bevacizumab, could also involve modifications to stem-like
character within these tumors. Targeting of the Notch pathway
was demonstrated to be an effective anti-tumor strategy in brain
tumor preclinical studies (Fan et al., 2006). Pharmacologic
suppression of the Notch pathway in medulloblastoma cells
significantly decreased their cancer stem-cell populations, as
indicated by SP analysis, CD133, and Nestin expression, and
further diminished their capacity to form tumors following trans-
plantation in mice. These cells were also more sensitized to
apoptosis than more differentiated cells (Fan et al., 2006). This
finding supports the notion that targeting the Notch pathway
Cell Stem Cell
NO in the Glioma Perivascular Niche
150 Cell Stem Cell 6, 141–152, February 5, 2010 ª2010 Elsevier Inc.
in gliomas could potentially sensitize the stem cell-like fractions
to apoptosis. The prediction from the preponderance of the liter-
ature is that these approaches might be more effective when
combined with radiation and chemotherapy where perivascular
resistant cells contribute to tumor recurrence after conventional
Hoechst 33342 Staining and Flow Cytometry
PDGF-induced glioma primary cultures (PIGPC) were suspended at 1 3
106cells/ml in 15 ml falcon tubes in DMEM+10FBS medium, then preincu-
bated at 37?C for 35 min in the presence of vehicle (water), GSNO (100 mM)
(Sigma Aldrich, USA), Pet-Br-cGMP (200 mM) (Axxora Platform, San Diego,
CA), Fumitremorgin C (FTC), and ABCG2 inhibitor (5 mM) (Axxora Platform,
San Diego, CA) or the gamma secretase inhibitor (GSI)-MK-003 (3mM),
a kind donation from Merck, USA. Cells were then incubated with Hoechst
33342 (5 mg/ml) for 90 min at 37?C with periodic shaking. Following Hoechst
staining, cells were incubated on ice for 10 min then washed 23 in ice-cold
PBS. Hoechst dye was excited at 407 nm using a trigon violet laser, and
dual wavelength detection was performed using 450/40 (Hoechst 33342-
Blue) and 695/40 (Hoechst 33342-Red) filters. Dead cell exclusion in control,
GSNO-, pet-cGMP-, and GSI-treated glioma primary cultures occurred by
forward and side scatter gating and the exclusion of PI-positive populations.
Data were analyzed using FlowJo (Ashland, OR).
Generation of PDGF-Induced Primary Glioma Cultures
Neonatal Nestin tv-a (N-tva) INK4A/Arf?/?mice described previously (Uhrbom
et al., 2005) were injected intracranially with DF1 chicken fibroblasts,
producing RCAS-PDGF retroviral particles to generate gliomas as described
previously (Shih et al., 2004). PDGF-induced gliomas were dissected and
enzymatically digested for 15 min in 13 Earles balanced salt solution contain-
ing 12% papain (Worthington, Lakewood, NJ) and 10 mg/ml DNase at 37?C.
The digestion was stopped with 1mg/mlovomucoid(Worthington, Lakewood,
NJ). Cells were washed and resuspended 33 in basal medium. Single-cell
suspensions were plated in DMEM+10% FBS, and cells were grown as
a monolayer. The medium was replaced 24–48 hrs later. Primary culture
experiments were performed using PDGF-induced tumors from different
mice; however, control and treated groups compared in all experiments
were generated from the same tumor. Primary cultures used in all experiments
wereperformed using 10cmculturesdishesat50%–60% confluence. Inaddi-
tion, all PIGPC were used between passage numbers p0–p4.
Intracranial Injection of Mouse Glioma Cells
To assess the tumorigenic capacity of PIGPC following GSNO treatment,
approximately 8 3 104vehicle- or GSNO-treated (2 hr) PIGPC were injected
into the cortex of newborn pups. Tumor development occurred between
Injection of human cells was done by stereotactic injection.NOD-SCID mice
between 5–10 weeks old were anaesthetized by i.p. injection of ketamine
(0.1 mg/g) and xylazine (0.02 mg/g). 1ml of 2.5 3 105cells were injected into
the cortex. Stereotactic coordinates were taken relative to bregma, right hemi-
sphere: AP, 0 mm from bregma; Lat, 3.0 mm (left or right); depth, 1.0 mm from
dural surface. Locations were determined using mouse atlas (Franklin and
Paxinos, 2007). Tumor cells were injected using a Hamilton syringe. Mice
were closely monitored for tumor development, as assessed by the presence
of hydrocephalus, lethargy, or cachexia.
Comparisons between two groups were made using two-sided t tests. Chi
square test was use to compare groups in Kaplan Meier graphs. Two-way
ANOVA was used to analyze data from luciferase assays. Fisher’s Exact
Test was used to compare tumorigenicity between vehicle- and pet-cGMP-
treated primary glioma cells. Data represent the mean of three experiments
unless otherwise noted. p values of <0.05 were considered statistically
The Supplemental Information includes seven figures and Supplemental
Experimental Procedures and can be found with this article online at
We thank J. Finney and Q. Zhang for technical assistance and A. Pedraza-
Fernandez for providing human tumor neurospheres for analyses. We extend
thanks to R. Bish for critical reading and feedback of this manuscript. Sincere
thanks totheflowcytometrycore facility for acquisition offlowcytometrydata.
This work was supported by NIH grant U54 CA126518, NIH grant U54
CA143798, and the Litwin Foundation. D.H. is supported by the Leon Levy
Received: July 16, 2009
Revised: December 2, 2009
Accepted: January 4, 2010
Published: February 4, 2010
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