Review

The brain tumor microenvironment

Nikki A. Charles1,*, Eric C. Holland1, Richard Gilbertson2, Rainer Glass3, Helmut Kettenmann4

Article first published online: 28 MAR 2011 DOI: 10.1002/glia.21136

Abstract

High-grade brain tumors are heterogeneous with respect to the composition of bona fide tumors cells
and with respect to a range of intermingling parenchymal cells. Glioblastomas harbor multiple cell types,
some with increased tumorigenicity and stem cell-like capacity. The stem-like cells may be the cells of
origin for tumor relapse. However, the tumor-associated parenchymal cells—such as vascular cells,
microglia, peripheral immune cells, and neural precursor cells—also play a vital role in controlling the
course of pathology. In this review, we describe the multiple interactions of bulk glioma cells and glioma
stem cells with parenchymal cell populations and highlight the pathological impact and signaling
pathways known for these types of cell–cell communication. The tumor-vasculature not only nourishes
glioblastomas, but also provides a specialized niche for these stem-like cells. In addition, microglial cells,
which can contribute up to 30% of a brain tumor mass, play a role in glioblastoma cell invasion.
Moreover, non-neoplastic astrocytes can be converted into a reactive phenotype by the glioma
microenvironment and can then secrete a number of factors which influences tumor biology. The young
brain may have the capacity to inhibit gliomagenesis by the endogenous neural stem and progenitor
cells, which secrete tumor suppressive factors. The factors, pathways, and interactions described in this
review provide a new prospective on the cell biology of primary brain tumors, which may ultimately
generate new treatment modalities. However, our picture of the multiple interactions between
parenchymal and tumor cells is still incomplete. © 2011 Wiley-Liss, Inc.

MICROGLIA

Microglia/Macrophages Accumulate Within and Around Gliomas

Glioma tissue as obtained by surgical resection does not only contain cancer cells but also a considerable
amount of nontransformed cells. The majority of these cells are tumor-associated macrophages
(Graeber et al.,2002; Watters et al.,2005). Resident macrophages of the brain are termed microglia.
These cells invade the brain early in development and differentiate into so-called resident, ramified
microglia (Hanisch and Kettenmann,2007). The turn-over of this cell population under normal conditions
is very low and as a consequence these cells reside as a permanent population in the brain (Mildner et
al., 2007). The presence of microglia in brain tumors was first detected by Wilder Penfield using silver
carbonate staining of tumor sections in 1925 (Penfield, 1925). Penfield detected microglia throughout
tumor sections, with some showing phagocytic activity. These tumor-associated microglia displayed an
ameboid morphology similar to that described in other pathologies. Since microglia share many
properties with macrophages found in nonneuronal tissues including the blood, it was possible that
glioma-associated microglia could be recruited de novo from the microglial population resident in the
brain, or alternatively, migrate into brain tumors from the periphery. To address this question, Badie
and Schartner (2000) used CD45 labeling of cells in rodent tumor models to distinguish brain microglia
(CD45 low) from peripheral macrophages (CD45 high). They used CD11b/c as a general marker for
macrophages and sorted the CD11b/c −positive cells into two populations showing high and low CD45
expression. From this, they concluded that the macrophages are primarily found within tumors, while
microglia was detected in all brain tissue. A similar distribution was subsequently identified in human
gliomas by Parney et al. (2009). They demonstrated that CD45(+)/CD11b(+) cells are the predominant
inflammatory cells infiltrating gliomas. An important caveat of labeling studies is that they assume that
CD45 expression is rather stable and not affected by the brain or glioma environments. It is possible that
the glioma environment upregulates CD45 in microglia. Thus, the precise origin of tumor microglia
remains to be determined. Regardless of origin, we can define any glioma-associated monocytic cell
with macrophage characteristics as a tumor-associated microglia.

Candidate chemoattractants of tumor-associated microglia include monocyte chemotactic protein-3

(MCP-3), colony-stimulating factor 1 (CSF-1), granulocyte-colony stimulatory factor (G-CSF), and
hepathocyte growth factor/scatter factor: each of these has been shown to be released by gliomas and
microglia are known to express receptors for these chemoattractant growth factors (Alterman and
Stanley,1994; Badie et al., 1999; Okada et al.,2009; Platten et al.,2003; Suzuki etal.,2008). Platten et al.
(2005) identified MCP-1 as a potent chemoattractant for glioma-infiltrating microglia: MCP-1-
transfected rat CNS-1 gliomas were massively infiltrated by microglia. However, Okada et al. (2009),
reported that MCP-3 and not MCP-1 is predominantly expressed in glioma cell lines and that levels of
MCP-3 in human gliomas correlate with microglia content. Thus, MCP-3 might be a more clinically
relevant chemoattractant in the human disease.

The Glioma Associated Microglial Cell Is Distinct from a Proinflammatory Phenotype

Microglia activation is not an all or none phenotype but can vary depending on the type of pathology or
during the pathologic process (Hanisch,2007 p. 182). Evidence suggests that the immune function of
microglia might be suppressed when these cells are located in the glioma environment similar as
described for other tumors (Graeber et al.,2002). Glioma cells produce cytokines with an anti-
inflammatory phenotype, such as IL-10, IL-4, IL-6, TGF beta, and prostaglandin E2. TGF beta in particular
suppresses the activation and proliferation of microglia (Suzumura et al., 1993; Wei, 282; Zou, 1999; p.
281). Glioma associated microglia also appear to express low levels of the MHC class II molecule relative
to microglia in nonmalignant diseased brain e.g., multiple sclerosis or stroke (Badie et al.,2002; Flügel et
al., 1999; Schartner et al., 2005; Tran et al., 1998). MHC class II is important for the interaction of
antigen presenting cells with T lymphocytes. This is also the case for the co-stimulatory molecules B7
where microglial expression of B7 has a suppressive effect (Badie et al.,2002). Glioma-released factors
trigger the production of prostaglandin2 in microglia that leads to a suppression of TNF production. This
goes in hand with the upregulation of microsomal PGE synthase-1 (a terminal enzyme of the
arachidonate cascade) by conditioned glioma medium, while TNF production was not suppressed in
microglia from mPGES-1-deficient mice (Nakano et al., 2008). The proportion of glioma-associated
microglia that resembles the M2 phenotype of (CD163 and CD204-positive) macrophages positively
correlates with the histological malignancy of the tumor. This population shift is thought to be regulated
by M-CSF (Komohara, 2008; p. 277). The M2 macrophage phenotype is represents most likely the
default setting under homeostatic conditions while the M1 phenotype is associated with inflammation.
In general, microglia associated with gliomas has the morphological appearance of the typical activated
microglial cell but has a functional phenotype that does not reflect the one described in inflammation.

Microglia Promote Glioma Migration and Tumor Growth

The first indication that microglia might modulate the biology of glioma cells came from cell culture
studies using Boyden chamber assays. Microglia, but not oligodendrocytes or endothelial cells promoted
the migration of GL261 mouse glioma cells (Bettinger et al., 2001). In a cultured mouse brain slice
model, the invasion and growth of glioma cells was compared between normal and microglia-depleted
slices. Selective microglial depletion within 24 h was accomplished by adding clodronate filled liposomes
to the slice. Glioma cell invasion was significantly reduced in microglia depleted slices relative to control
slices (Markovic et al.,2005). The impact of microglia on glioma migration might relate to the production
of membrane type 1 metalloprotease (MT1-MMP or MMP-14) that are produced by microglia in
response to soluble factors released from glioma cells. Glioma cells also release metalloprotease 2 that
is fully activated by MT1-MMP released from microglia. The consequent degradation of the extracellular
matrix has been postulated to enhance the invasion of glioma cells into the brain parenchyma. Glioma-
released factors trigger the expression and activity of MT1-MMP via microglial toll-like receptors and the
activation of the p38 MAPK pathway. The upregulation of MT1-MMP was mediated by toll-like receptor
signaling since it was abolished in a mouse line with deletion of a molecule essential for most toll-like
receptor signaling. Glioma growth was also strongly attenuated in the MyD88-deficient mouse model
(Markovic etal., 2009). The importance of microglia for glioma growth was further substantiated by
studying animals in which microglia was depleted. The microglia-depletion in vivo was achieved by using
the CD11b-HSVTK mouse model. Seven days after intracerebral glioma inoculation, ganciclovir (a specific
substrate for the viral thymidine kinase HSVTK) was infused via mini-pumps into the tumor area for a
further 7 days. To restrict the effect of ganciclovir on the intrinsic microglial population, the mice were
irradiated before bone marrow transplanted with wild-type monocytes. The ganciclovir treatment led to
a considerable depletion of microglia and to an 80% reduction in glioma volume (Markovic et al., 2009).
Using the similar mouse model, Galarneau et al. (2007) injected ganciclovir i.p. twice daily for 6 days
starting 7 days after tumor implantation. In this model, the macrophage population was reduced and
they observed the opposite effect on glioma growth, namely a 33% increase in tumor volume after
macrophage depletion. This difference would indicate that macrophages from the periphery and
intrinsic microglia have distinct effects on glioma growth.

Matrix metalloprotease activity is also regulated by the CX3CL1/CX3CR1 signaling pathway. For example,
matrix metalloproteases 2, 9, and 14 are upregulated in microglia following activation of
CX3CL1/CX3CR1 signaling. Notably, CX3CR1 is upregulated in glioma associated microglia (Held-Feindt et
al.,2010) and polymorphisms in the chemokine receptor CX3CR1 have been associated with prognosis
among patients with glioma. The common CX3CR1 allele (termed V249I) was a favorable prognostic
factor. Patients who had only this CX3CR1 allele had a more than 1.5 longer mean survival time. This
common allele was associated with reduced microglial cell infiltration in primary tumor biopsies (Rodero
et al., 2008).

The Glioma Induced Phenotype of Microglia Is Important for Their Protumorigenic Activity

Lipopolysacharides can activate a proinflammatory phenotype in microglia. Lipopolysacharide treatment

of mouse primary microglia was shown to endow these cells with antitumor activities against TNF-alpha
and TRAIL resistant glioma cell lines, triggering autophagy-dependent death in these glioma cells (Mora
et al.,2009). The effect might also occur in vivo since intratumoral application of lipopolysaccharide to
mice implanted with intracranial glioblastomas, increased the survival times modestly. This benefit was
abrogated in mice deficient for lipopolysaccharide receptor Tlr-4-indicating that lipopolysaccharide does
not affect the tumor cells, but the intrinsic macrophage/microglia population (Chicoine et al., 2007).
Another indication that the antitumorigenic effect of microglia is due to their specific glioma-associated
phenotype came from experiments interfering with the signaling pathway involving signal transducers
and activators of transcription 3 (Stat3). Inactivation of Stat3 in experimental gliomas by siRNA resulted
in microglial activation and tumor growth inhibition (Gao, #278). Cyclosporin A at clinically relevant
concentrations impairs the protumorigenic phenotype of microglia when studying the glioma migation
in organotypic brain slices. The inhibitory effect on glioblastoma invasion was lost when glioblastoma
cells were injected into microglia-depleted brain slices indicating that cyclosporine A affects microglia
(Sliwa et al., 2007).

All these reports point to a glioma promoting role of microglia, if they are in the glioma-induced
phenotype as opposed to the proinflammatory phenotype.

Interaction of T-lymphocytes with Glioma

Fifty years ago the recognition of lymphocytes infiltrating into human gliomas was reported; however,
its significance was not fully understood (Bertrand and Mannen,1960). On the basis of morphological
criteria after hematoxylin and eosin staining, Ridley and Cavanagh described significant lymphocyte
infiltration in about a third of human postmortem glioma tissues samples. In 28% they described slight
infiltration, while in 42% of tissue samples they found no evidence for lymphocyte invasion. The
lymphocyte infiltration was predominantly in tumors with high malignancy. In surgically resected human
glioma tissue, Farmer et al. (1989) found CD8-positive cells in a higher proportion as in blood by
imunolabeling. They also found that high-grade glioma showed a higher leukocyte density as compared
with low-grade cases. Overall, the expression of CD8 and CD4 showed a considerable variability among
tumor specimen (Kuppner et al., 1988). The correlation between lymphocyte infiltration and clinical
outcome is still controversial (see Dunn et al., 2007 for review). The authors, however, noted that these
clinical studies were conducted prior to the emergence of the concept of regulatory T cells (Tregs).
Tregs, identified by the antigen FoxP3, play an important role in the regulation of the immune response.
El Andaloussi and Lesniak (2007) found a positive correlation between disease progression and FoxP3
expression in different grades of astrocytomas. Hussain et al. (2006) even concluded from analysis of
postoperative tissue specimens that a prominent population of Tregs (positive for the markers CD4,
CD25, and FOXP3) infiltrated the tumor tissue. The heme oxygenase-1 seems to play a role in the
immune suppressive phenotype of Tregs. Tumor infiltrating Tregs express heme oxygenase-1 and
expression increased with tumor progression (El Andaloussi and Lesniak, 2007). The chemokines CCL2
and CCL22 are candidates as chemoattractant factors for Tregs (Jordan et al., 2008). The Treg fraction is
not only increased in human, but also in experimental glioma mouse models (El Andaloussi et al., 2006a,
Grauer etal., 2007). This made it possible to address the question of whether Tregs were a
protumorigenic component of the neoplasm. Using anti-CD25 antibodies in an experimental mouse
model, the number of Tregs decreased and the treated mice lived significantly longer as compared with
control animals (El Andaloussi et al., 2006a). Toll-like receptors seem to play an important role for
controlling Treg activity. Stimulation of TLR9 decreased the number of infiltrating Tregs and prolonged
survival (El Andaloussi etal., 2006b). The infiltration of Tregs is paralled by an increase in TGF-beta1
expression in experimental intracranial gliomas. The brain environment plays an important role since
this correlation was not observed in gliomas injected subcutaneously (Biollaz etal., 2009). Low-dose
application of Temozolomide, the standard chemotherapeutic agent for glioblastoma, reduced the
number of circulating Tregs and induced a slight and nearly significant decrease in the percentage of
Treg within the tumor (Banissi et al., 2009). Thus Tregs associated with glioma represent a novel target
for glioma therapy.

THE INTERACTION OF GLIOBLASTOMAS WITH NEURAL PRECURSOR CELLS

It was discovered only recently, that glioblastomas can interact with neural precursor cells (NPCs). The
attraction of NPCs to experimental glioblastoma was initially observed with exogenously cultivated and
immortalized precursors (Aboody et al.,2000). These immortalized cells show strong, directed migration
towards primary brain tumors over large distances and can even track-down single, scattered
glioblastoma cells. Hence, they are now considered as a cell-based delivery system for glioblastoma
therapeutics (Aboody et al.,2008). However, the attraction of NPCs to CNS neoplasms is a phenomenon
that is not restricted to exogenous NPCs, but also occurs endogenously in the brain. Large numbers of
endogenous NPCs, e.g. from the subventricular zone (SVZ) lining the lateral ventricles or from the corpus
callosum, migrate towards experimental brain tumors (Assanah etal.,2006; Assanah et al.,2009;
Chirasani,2010; p. 243; Glass et al.,2005; Walzlein etal.,2008). NPCs home in to pathologic brain tissue
and possibly also to tumors due to expression of CXCR4 (Kokovay et al. ; Xu et al., 2009). The association
of endogenous NPCs with glioblastomas may have been overlooked for many years, since it is only
abundant in the young brain and since special animal models are necessary to distinguish glioblastoma
cells from NPCs. Throughout the last decades evidence is accumulating that glioblastomas likely arise
from genetically transformed stem and precursor cells in the brain (Sanai et al.,2005) and retain the
expression of cellular markers for NPCs. Therefore, special techniques, like viral-labeling of NPCs and
glioblastoma cells (Assanah et al.,2006) or inoculation of glioblastomas into transgenic mice with
reporter-gene activity in endogenous NPCs (Glass et al.,2005), were necessary to distinguish the tumor-
associated neural precursor cell population from the glioblastoma mass. These genetically labeled,
glioblastoma-associated NPCs were characterised, e.g. with cytochemical markers and by
electrophysiology, to be bona fide progenitor cells. Viral labeling of proliferative cells in the SVZ of
transgenic mice revealed that NPCs accumulate in many cellular layers around gliomas and that the
tumor-associated NPCs are diverted from their physiological migratory path to the olfactory bulb
(Walzlein et al.,2008).

A range of in vitro and in vivo experiments indicates that NPCs exert antitumorigenic actions. Cultivated
NPCs release soluble factors that can attenuate glioblastoma proliferation (Chen et al.,2009; Staflin et
al.,2004,2009; Suzuki et al.2005) cause glioblastoma cell death (Glass et al.,2005; Walzlein et al.,2008)
and induce the differentiation of glioblastoma stem cells {Chirasani,2010; p. 243). In mouse glioblastoma
models, using orthotopic implantation of glioblastoma cells, it was shown that co-implantation of
cultivated NPCs improves survival (Glass et al.,2005; Staflin etal.,2004). The tumor-suppressing capacity
of endogenous NPCs may explain why glioma growth is attenuated in younger animals and why younger
mice outlive older animals after glioblastoma inoculation into the brain. Adult neurogenesis, describing
the presence, proliferation, and differentiation of stem and precursor cells in the adult brain, declines
with aging (Kowalczyk et al.,2004; Tropepe et al.,1997). Consequentially, experimental glioblastomas
attract large numbers of NPCs only in the young brain, while this effect is strongly reduced in the older
brain (Walzlein et al.,2008). The number of glioblastoma associated NPCs inversely correlates with
tumor-size and survival. This may highlight a direct tumor-suppressor effect of the NPCs: first,
implantation of NPCs together with glioblastomas in older mice restores the survival-rate to the level of
young mice and second, NPC-released factors can directly attenuate glioblastoma proliferation, mediate
tumor cell death, and cause glioblastoma stem cell differentiation (see above).

It will be interesting to determine whether human NPCs have an antitumorigenic capacity that is similar
to the effects described for rodent models, especially since age is the most important prognostic factor
for glioblastoma occurrence and outcome in humans. It is well established that primary glioblastoma are
diagnosed mainly in older patients and are virtually unknown in infants (Ohgaki and Kleihues,2007).
However, adult neurogenesis in primates and humans (as in rodents) also declines massively with aging
and may be at a very low level after puberty (Gould et al.,1999; Knoth et al.,). This creates the somewhat
paradoxical situation that a stem cell-related disease like glioblastoma (originating from mutant brain
stem cells (Sanai et al.,2005)), occurs at a time when there are only very few stem cells left in the brain.
This may partly be explained by a long time-span needed to form a full-blown tumor from a single,
mutant stem cell (Hanahan and Weinberg,2000). However, the antitumorigenic properties of
endogenous NPCs may also contribute to protect the young human brain from primary glioblastomas.
With a view to the clinical application of the observed antitumor effects of NPCs, it will be important to
characterize the molecular nature of the NPC-secreted factors. A first NPC-derived paracrine tumor
suppressor, namely bone morphogenetic protein-7 (BMP7) that induces the differentiation of human
glioma stem cells, was recently identified (Chirasani,2010; p. 243).
THE TUMOR PERIVASCULAR NICHE

The tumor vasculature contributes an additional large compliment of nonmalignant cells to gliomas.
Gliomas are highly vascular tumors and emerging evidence indicates that the endothelial cells, pericytes,
and astrocytes that form the neurovascular unit function to support tumor progression. This
microanatomical structure has received increasing attention in recent years following the discovery that
the perivascular niche (PVN) is the stem cell niche in both normal and malignant neural tissue.

The vasculature in gliomas is characterized by endothelial hyperplasia and microvascular proliferation

(MVP) that are histologic hallmarks of high-grade gliomas (Wen and Kesari,2008). The presence of MVP
is associated with tumor transition to a malignant phenotype. Microvascular proliferating structures
have been characterized to derive from hyperproliferative activity in a combination of endothelial,
pericytic and vascular smooth muscle cells (Wesseling et al.,1995). MVP structures represent regions of
angiogenesis, which is critical for tumor progression and is particularly important for brain tumors
especially malignant gliomas that are among the most vascularized/angiogenic tumors. These angiogenic
regions of which, the perivascular niche is a part, have emerged as important locations responsible for
the maintenance of brain tumor stem cell-like (BTSC) populations (Calabrese etal.,2007; Charles et
al.,2010; Hambardzumyan et al.,2008).

In neurogenic niches of the normal brain, neural stem cells (NSCs) are concentrated in regions around
the vasculature that is thought to promote NSCs activity (Mirzadeh et al.,2008; Shen et al.,2008;
Tavazoie etal.,2008). In addition, a multitude of molecular signaling events from endothelial cells and
other stromal cells within the perivascular microenvironment, work collectively to regulate the activity
and support the stem cell properties of nearby NSCs. A similar organization of cell types and signaling
events appear to exist within the PVN of brain tumors where endothelial derived niche factors and
signaling from other stromal cells appear to regulate the stem cell-like properties of resident BTSCs
(Calabrese et al.,2007). The PVN contains several stromal cell types, other than endothelial cells, that
potentially make significant contributions to support tumor growth (Fig. 1).

Figure 1. The glioblastoma microenvironment. The microenvironment of glioblastoma is composed of

several stromal cell types which are believed to make distinct contributions to tumor progression and
invasion. These cells include but are not limited to astrocytes, macrophages, pericytes, fibroblasts, and
endothelial cells.

Surrounding the vasculature are large-body, GFAP-expressing astrocytes similar in structure to reactive
astrocytes seen in other types of brain pathology. In addition, there are smooth muscle actin-expressing
fibroblastic pericytes that intimately associate with tumor endothelia. As described in the first section of
this review, macrophages are also located in this region and are recognized to play a significant role in
tumor progression of many tumor types (Joyce and Pollard,2009). A collective understanding of
individual role played by each of these cell types provides important insights into brain tumor
mechanisms.

In addition, the PVN is a primary location for tumor cells with stem cell-like characteristics (Hadjipanayis
and Van Meir,2009). BTSCs are intimately associated with the vasculature across multiple brain tumor
types (Calabrese et al.,2007). Endothelial-derived factors also maintain self-renewal properties of GBM
stem cells in vitro (Folkins et al.,2007). When cultured with primary endothelial cells, BTSCs, relative to
their non-BTSC counterparts, preferentially associate with endothelial cells. These endothelial cells
enhanced BTSC self-renewal and accelerated their tumorigenic capacities (Calabrese et al.,2007). These
data suggest that cell to cell signaling in the PVN of brain tumors play an important role in brain tumor
progression. Furthermore, this fact highlights the need to examine the existing molecular signaling
pathways among resident cell types within the PVN that function to regulate BTSC behavior. Insights
gained from improved understanding of the mechanisms that control BTSC activity will provide a
rational for new therapies that target the microenvironment.

CELLS OF THE PVN

Recruitment of Stromal Cells to the Microenvironment of Brain Tumors

Development of the brain tumor vascular niche, (perivascular niche) environment, involves recruitment
of bone marrow derived (BMD) cells. Some of these BMD cells include endothelial progenitor cells that
mature into endothelia of the vascular lumen; pericyte progenitor cells (PPCs) that envelop the
vasculature and mature into pericytes and vascular smooth muscle (VSM) cells; and CD45+ vascular-
associated cells, some of which give rise to perivascular macrophages and microglia that are described in
the first section of this review.

Perivascular Pericytes and Vascular Smooth Muscle Cells in the Brain Tumor Microenvironment

Pericytes and VSM cells wrap around the vascular tube and their primary role has traditionally been
associated with maintaining stability and controlling hemodynamic processes in the vasculature (Cleaver
and Melton,2003; Sims,2000). In the normal brain during blood vessel formation pericytes and VSM cells
are recruited by endothelial cells expressing PDGF-B, to stabilize and develop the vascular tube
(Hellstrom et al.,1999; Hirschi et al.,1998).

In the context of tumor development, recruitment of pericytes and VSM cells to the tumor PVN is critical
for structural stability of the vascular niche and for survival of tumor endothelial cells (Song etal.,2005).
Indeed, early reports describe the extensive contribution of pericytes and VSM cells to the formation of
microvascular proliferating structures in glioblastomas (Wesseling et al.,1995). NG2-expressing pericytes
have been implicated in malignant glioma progression (Chekenya et al.,2002a). Abnormalities in
pericytes, particularly the dissociation of pericytes from the vasculature, contribute to increased tumor
vascular permeability (Hashizume et al.,2000; Morikawa et al.,2002). Other studies have implicated a
role of pericyte recruitment to brain tumors to promote angiogenesis (De Palma et al.,2005) and to be
critical components for establishment of the brain tumor neovascular tree (Bababeygy et al.,2008). In a
related study in glioblastomas, HIF-1α was identified to play a role in the recruitment of perictyte
progenitors to the PVN to promote glioblastoma neovascularization (Du et al.,2008).

Astrocytes in the Microenvironment of BrainTumors

Astrocytes have traditionally been considered to provide structural support in the brain, with additional
roles in homeostatic and communicative functions. Astrocytes localized to the PVN regions of the brain
ensheath endothelial cells through end-feet processes where they play a critical role in blood brain
barrier maintenance (Kim et al.,2006).

In the context of the brain tumor microenvironment, astrocytes have been implicated in the progression
of brain tumors. Reactive astrocytes are frequently associated with glioma cells in the CNS (Le et
al.,2003). Tumor-associated astrocytes have been demonstrated to mediate glioblastoma cell invasion
via activation of proMMP2 (Le et al.,2003), a metalloproteinase that plays a critical role in glioma
invasion (Uhm et al.,1996). Some reports have identified sonic hedgehog (SHH)-expressing reactive
astrocytes to be highly concentrated in the perivascular regions of gliomas. In one study, reactive
astrocytes were found to be closely associated with increasing grade in PDGF-induced gliomas (Becher
et al.,2008). Other reports suggest that stromal cell-derived factor-1 (SDF-1)/CXCR-4 signaling, may
induce aberrant glioma cell proliferation (Barbero et al.,2002) as this signaling pathway is elevated in
human glioblastoma (Rempel et al.,2000). Since astrocytes have been shown to secrete SDF-1 (Bajetto
et al.,1999a,b), the ligand for CXCR-4, it is conceivable that astrocytes may be involved in driving
abnormal tumor proliferation by activating the pathway in adjacent glioma cells.

In addition, the capacity for astrocytes to produce neurotrophic factors that function in tumor cell
invasion has implicated them in the promotion of GBM growth (Hoelzinger et al.,2007). Recently,
astrocyte elevated gene-1 (AEG-1) initially identified in primary human fetal astrocytes (Kang et al.,2005;
Su et al.,2002) was implicated in metastatic progression and invasion of brain tumors. AEG-1 is elevated
in adult astrocytes (Kang et al.,2005; Lee et al.,2006). This protein is frequently over-expressed in human
brain tumors (Emdad et al.,2007). Gene expression and protein levels were elevated in several human
brain tumors including malignant gliomas and suppression of AEG-1 activity diminished brain tumor
growth in mice (Emdad et al.). Reports suggest that the effects of AEG-1 on brain tumor invasion and
metastasis involve the activities of MMP-2 and MMP-9 (Emdad et al.; Liu et al.).

Fibroblasts in the Brain Tumor Microenvironment

Stromal fibroblasts activated by their associated tumor cells have been identified by several reports to
play a key role in the induction of angiogenesis and metastasis in melanoma (Gallagher et al.,2005;
Goldstein etal.,2005) and pancreatic cancers (Hwang et al.,2008). Fibroblasts have also been shown to
drive the proinvasive activity of colon cancer cells (De Wever et al.,2004). A similar role within the
microenvironment of brain tumors has also been suggested. Co-cultures of brain-derived human
fibroblasts involving interactions with glioblastoma cells induced production and activation of matrix
metalloproteinase MMP2, and its activators MT1-MMP and MT2-MMP (Sameshima et al.,2000). These
metalloproteinases have all been shown to be involved in the progression of gliomas (Belien et al.,1999;
Sawaya et al.,1996; Uhm et al.,1996).

Endothelial Cells in the Brain Tumor Microenvironment

Endothelial cells have emerged as critical participants in the progression of brain tumors. In addition to
their traditional roles as the source of oxygen and nutrients for tumor cells, the multivariate
contributions of endothelial cells to brain tumor progression include secretion of factors that help
maintain stem cell-like characteristics of BTSC populations. Furthermore, these cells are also important
mediators of tumor angiogenesis.

Brain Tumor Stem-like Cells

In contrast to the aforementioned PVN cells that are not neoplastic per se, there are tumor cells that live
in the niche as well. They are resistant to therapy, give rise to recurrence, and they express many stem
cell markers. In addition, they respond to the niche by activating stem cell signaling pathways described
below.

BTSCs, like their NSC counterparts (Tavazoie et al.,2008), are intimately associated with endothelial cells
of the perivascular niche. Recent reports have identified endothelial cells as important sources of niche
derived factors for BTSCs. One of the pioneering studies suggesting that endothelial-derived cues might
activate glioblastoma cells came from a study that utilized time-lapse microscopy of rat glioblastoma
cells invading into rat brain slices. The authors demonstrated the capacity for glioblastoma cells to travel
along blood vessels, where they rapidly intercalated with endothelial cells, temporarily pausing along
vascular branch points to proliferate (Farin et al.,2006). This interplay between endothelial cells and
brain tumor cells has also been demonstrated in human glioblastomas and other malignant gliomas. For
example, BTSCs expressing nestin and CD133 associate with the tumor endothelium across several
different brain tumor subtypes. In fact, the population of vessel associated BTSCs strongly correlated
with increasing grade of these tumors. Furthermore, in co-culture studies BTSCs were seen to
preferentially align themselves with the vasculature, in comparison to their nonstem cell counterparts.
In addition, endothelial derived factors were observed to drive the neural stem cell forming capability of
these BTSCs and accelerated their tumorigenic capacities (Calabrese et al.,2007). A related study by
Folkins et al. (2007) also demonstrated acceleration in the tumorsphere forming abilitiy of glioma cells
by endothelial-derived factors. These studies draw attention to the critical role played by the
endothelium in brain tumor progression, and underscore the need to identify the endothelial-derived
cues responsible for maintaining BTSCs.
Nitric oxide (NO) is one such niche derived factor with the capacity to enhance the self-renewal
characteristics of BTSCs residing the PVN of brain tumors. NO activates notch signaling in a population of
BTSCs, to enhance their self-renewal characteristics in vitro and their tumorigenic capacities in vivo.
Further, eNOS, an enzyme that synthesizes NO from the vascular endothelium, is elevated in the
platelet-derived growth factor (PDGF)-subset of gliomas and suppression of eNOS activity, which
corresponded to a decrease in Notch signaling in these tumors, prolonged survival of tumor bearing
mice (Charles et al.,2010). It remains to be determined whether maintenance of cancer stem cell
populations by NO signaling from the tumor endothelium is conserved across other brain tumor sub-
types and other types of cancers.

The critical contribution of endothelial cells to tumor neovascularization is well established (De Palma
and Naldini,2006; Rafii et al.,2002). In brain tumors, vasculogenesis and angiogenesis are common
mechanisms utilized for the genesis of new blood vessels. Vasculogenesis was traditionally considered
an embryonic process but has since been identified to occur in several tumor types (Lyden et al.,2001)
including brain tumors (Santarelli et al.,2006; Zhang et al.,2009). Vasculogenesis involves the de novo
formation of blood vessels that utilize bone marrow-derived endothelial progenitor cells (Jain et
al.,2007). Angiogenesis is the production of new blood vessels by endothelial cells from preexisting
vessels and is considered to be the primary mode of vessel formation in gliomas (Tate and Aghi,2009).
Endothelial cells play a major role in brain tumor angiogenesis. During the angiogenic process,
degradation of the vessel membrane and extracellular matrix (ECM) facilitates endothelial cell activation
and migration towards tumor cells expressing proangiogenic factors (Tate and Aghi,2009). Activated
endothelial cells upregulate factors involved in cell surface adhesion and migration to ultimately
promote cell adhesion, migration, and survival (Brooks et al.,1994; Gladson,1996). In addition, activated
endothelial cells also secrete PDGF, which recruits pericytes to newly developing vessels, thus facilitating
the establishment of a basement membrane (Ferrara and Kerbel,2005).

In summary, the perivascular niche is a complex environment. It is composed of multiple cell types,
some neoplastic cells derived from the tumor itself and other non-neoplastic cells derived from the
stroma. The interplay between these cell types regulates differentiation and tumor progression.

CROSS-TALK IN THE BRAIN TUMOR PVN MICROENVIRONMENT

Thus far we have described the common cell types found in the microenvironment of brain tumors and
highlighted potential contributions each cell type makes to facilitate brain tumor progression. We will
now examine the signaling interactions between these various cell types, which are mediated through
paracrine cross-communication among cells, and which integrate to collectively support tumor
development and progression.

Tumor–Stromal Communication in the Brain Tumor Microenvironment (Immune Cells)

Tumor cells exploit the surrounding stromal environment through the recruitment of nonmalignant cells
that provide physiological resources to facilitate tumor progression. They are a major source of secreted
factors that recruit inflammatory cells into the tumor microenvironment. These factors include
cytokines, growth factors, chemokines, and colony stimulating factors that help in tumor initiation,
angiogenesis, proliferation, and invasion (Pollard,2004). Inter-cellular communication between microglia
and glioblastoma cells has been shown to facilitate invasion. Microglia was observed to secrete
proMMP2, the inactive form of the matrix metalloproteinase. Glioblastoma cells then provided the
necessary soluble factors used to activate the enzyme (Markovic et al.,2005), which mediates
breakdown of the ECM required for invasion. Microglia in the glioma microenvironment is also a primary
source of interleukin 1β (IL-1β), which can enhance gene expression of TGF-β (Naganuma et al.,1996).
Increased transcription of TGF-β can lead to suppression of antiglioma responses by inhibiting the
proliferation of lymphocytes, reducing immune cell activation, blocking antitumor activity, and inhibiting
antigen presentation (Letterio and Roberts,1998). TGF-β can also lead to angiogenesis (through VEGF
expression), proliferation (through EGFR expression), and invasion (through MMP-9 production)
(Watters et al.,2005). Additional modes of interaction between glioma cells and microglia are suggested
by the fact that glioma cells can secrete EGF to stimulate microglial motility (Nolte et al.,1997), and via
their expression of EGFR, can potentially also proliferate in response to EGF secretion by microglia.

Pericyte/VSM–Endothelial Cell Interactions in the Tumor Microenvironment

Pericyte-endothelial interactions play an important role in normal and pathological angiogenesis (Diaz-
Flores et al.,2009; Ozerdem and Stallcup,2004). TGF-β signaling plays an important role during vascular
development and is known to maintain proper endothelial pericyte/VSM cell interactions. TGF-β
signaling in endothelial cells further drives the expression, synthesis and release of TGF-β to nearby VSM
cells to influence their differentiation (Carvalho et al.,2004; Wurdak et al.,2005). Studies suggest the
importance of juxtaposed intercellular communication between endothelial cells and pericytes/VSM
cells for activation of the TGF-β pathway (Antonelli-Orlidge etal.,1989; Sato et al.,1990).

Reciprocal communication between endothelial cells and pericytes also involve angiopoietin-Tie2
signaling that is required for vessel stabilization (Armulik etal.,2005). Paracrine signaling between
pericytes and endothelial cells is suggested by the general expression of the Tie2 receptor on endothelial
(Sato et al.,1995) cells, while its ligand Ang1 is mainly expressed on juxtaposed perivascular pericytes
(Sundberg etal.,2002). In-vivo studies in mice demonstrate that the Ang1-Tie2 signaling axis is necessary
for the stability and maturation of blood vessels. Ang1 and Tie2 deficient mice demonstrate impaired
angiogenesis and defective pericyte coverage (Sato et al.,1995; Suri et al.,1996), whereas over-
expression of Ang1 generates an enhanced stabilized vasculature (Suri et al.,1998; Thurston et al.,1999).

Endothelial cells recruit PDGF-expressing pericytes that function to stabilize the outer lining of newly
developing vasculature for angiogenesis (Gerhardt and Betsholtz,2003). Indeed, the expression of PDGF-
B is observed mainly at sites where active angiogenesis occurs (Hellstrom etal.,1999,2001). Endothelial
cells secrete PDGF-B, which communicates with pericytes through PDGFR-β expressed by these cells.
This results in proliferation and migration of pericytes/VSM cells during maturation of vessels
(Betsholtz,2004; Hoch and Soriano,2003). Studies show deletion of the retention motif of pdgfb
specifically in the endothelium, results insignificant pericyte deficiency in the vasculature (Bjarnegard et
al.,2004; Enge et al.,2002), while similar depletion of pdgfb in hematopoetic or neuronal cells had no
effect on the vasculature (Buetow et al.,2001; Engeet al.,2003). These interactions highlight the critical
role of endothelium-derived PDGF in pericyte recruitment.

Additional reports suggest that immature NG2-expressing pericytes may play a role in brain tumor
angiogenesis by sequestration of angiotensin, which is known to negatively regulate endothelial cell
proliferation and migration (Chekenya et al.,2002b). Tumor vessels vary in their extent of pericyte
coverage. Moreover, tumor vasculature with pericyte coverage is better protected than naked regions
of the endothelium against antiangiogenic therapies targeted to the endothelium (Baluk etal.,2005).
These observations engendered the belief that combined therapies against the tumor endothelia and
pericytic cells might be effective approaches for therapy. One study tested this hypothesis and observed
synergistic antitumor effects in vivo (Bergers et al.,2003). In all, these studies suggest that pericyte
interaction with the microvascular endothelium mediates maturation, proliferation and stabilization of
the vasculature, consistent with its suggested role in promoting MVP in malignant gliomas.

Astrocytic-related Interactions in the Brain Tumor Microenvironment

Astrocytes are closely associated with the vascular endothelium and they secrete a number of
neurotrophic factors, including transforming growth factor (TGF-α), CXCL12, SIP, and GDNF. These
neurotrophic factors have been described as capable of driving the invasive properties of GBM cells
(Hoelzinger et al.,2007). Reports suggest that glioma-induced remodeling within the tumor
microenvironment through disruption of the BBB facilitates tumor invasion and involve abnormal
astrocyte-endothelial interactions (Lee et al.,2009).

Tumor-astrocytic interactions are an important mechanism by which glioma cells facilitate tumor
invasion. For example, one study found marked increases in MMP2 production following cross-
communication between astrocytes and glioblastoma cells. This study utilized co-cultures of
glioblastoma cells with astrocytes to demonstrate enhancement of the invasive capacities of
glioblastoma cells through production of proMMP (Le et al.,2003). This effect was mediated by the
urokinase-type plasminogen activator (uPA)-plasmin cascade. Astrocytes produced significant amounts
of proMMP2, the inactive form of the matrix metalloprotease MMP, as well as uPA and its receptor
uPAR, both required for plasminogen cleavage. Plasminogen provided by glioblastoma cells were
cleaved to produce plasmin, which cleaved proMMP2 to its active form, MMP2. Furthermore, analysis of
resected human glioblastoma specimens revealed elevated levels of plasminogen, suggesting that a
similaar mechanism may exist in vivo.

Astrocytes also mediate activation of developmental pathways like the sonic hedgehog (SHH) pathway
in the glioma PVN. SHH signaling is known to play an important role in BTSC self-renewal and growth of
gliomas (Clement etal.,2007; Stecca and Ruiz i Altaba,2005). Hedgehog signaling was shown to be
activated in gliomas and was correlated with increasing grade. Moreover, SHH and GFAP-expressing
reactive astrocytes localized to the glioma PVN lay adjacent to nestin-expressing stem-like of the PVN
(Becher et al.,2008). Since glioma stem-like cells express patched, the receptor for SHH, it is conceivable
that cross communication between reactive astrocytes and BTSCs may exist to drive BTSC
characteristics.

Brain Tumor-Cell Endothelial-Cell Signaling

In the brain tumor microenvironment, there is considerable cross-talk between endothelial cells and
brain tumor cells, which include BTSCs, as well the nontumor initiating tumor cell populations. Some of
these involve paracrine interactions between endothelial cells and nearby progenitor cells, through the
release of endothelial-derived soluble factors. The identities of these factors is slowly beginning to
emerge and some of these reportedly include, BDNF (Leventhal et al.,1999), PEDF (Ramirez-Castillejo et
al.,2006), VEGFC (Le Bras et al.,2006). These paracrine factors are released from the endothelium to
enhance NSC proliferation and neurogenesis. Reciprocal communication from NSCs to endothelial cells
also involves the paracrine factor BDNF and VEGF (Li et al., 2006). In addition, VEGF has also been shown
to mediate BTSC intercellular cross-talk with tumor endothelia to induce angiogenesis (Bao et al.,2006).

The Notch signaling pathway mediates intercellular communication within the PVN microenviroment of
gliomas. Notch signaling is a critical developmental pathway that controls neural stem cell fate and
suppresses radial glia differentiation (Ehebauer et al.,2006). The notch signaling pathway is
constitutively active in many high-grade gliomas and has been associated with progression of gliomas
(Hulleman et al.,2009; Jeon et al.,2008; Shih and Holland,2006; Zhang et al.,2008). Notch signaling from
endothelial cells to hematopoetic stem cells (HSC) via expression of notch ligands has been shown to
drive the self-renewal characteristics and in vivo repopulation of long-term HSCs that lie adjacent to the
vasculature (Butler et al.,2010). Whether a similar mechanism is conserved in gliomas remains to be
seen. However, recent data using a PDGF-driven mouse model of proneural glioma implicates tumor
endothelia as the source of NO, which was demonstrated to activate notch signaling in PVN BTSCs.
Tumor endothelial cells, which highly express eNOS, the enzyme required for NO synthesis in the
endothelium, lies adjacent to nestin-expressing BTSCs associated with the PVN. Activated notch
signaling by NO in BTSCs was shown to accelerate tumor progression (Charles etal.,2010). This finding
identifies potentially new targets for the rational design of drugs against the PVN.

Malignant gliomas are among the most vascularized tumor types. Notch signaling from glioblastoma
cells to the tumor endothelia has also been demonstrated to drive angiogenesis in gliomas. The notch
ligand, Delta-like 4 (DLL4), is upregulated in endothelial and glioblastoma cells. In a mouse model of
glioblastomas, DLL4 expression in tumor cells was demonstrated to drive tumor angiogenesis via
activation of the Notch pathway in endothelial cells (Li et al.,2007).

Defects in growth factor signaling pathways are a common molecular hallmark of malignant gliomas
(Wen and Kesari,2008). This often leads to activation of pathways downstream of growth factor
signaling, such as the PI3K/Akt signaling pathway, which is activated in approximately 70% of gliomas
(Holland,2001). The PI3K/Akt pathway is an important regulator of glioma cell survival and studies
indicate that the pathway is activated in the BTSC populations of brain tumors. For instance, BTSCs of
gliomas have been shown to be more heavily dependent on PI3K/Akt signaling than their nonstem cell
counterparts (Eyler et al.,2008). In addition, this pathway was shown to mediate radioresistance of
BTSCs located in the PVN of medulloblastomas (Hambardzumyan et al.,2008). Although it is unclear
what the specific cell of origin responsible for transducing the PI3K/Akt signal in BTSCs is, the pathway is
active in the niche and plays a significant role in supporting BTSC functions.

THERAPEUTIC IMPLICATIONS OF THE STROMAL COMPONENTS OF GLIOMAS

Many of the signaling pathways that appear to play critical parts in the biology of the perivascular niche
are already being targeted therapeutically for the tumor as a whole. These pathways include PI3K,
notch, and SHH. Other pathways that may be more specific for the perivascular region are NO, cGMP
analogs, and PKG. Many of these compounds are also under development for vascular disease states but
may be translatable to oncology based on more recent findings.

In addition to signaling pathways that could be addressed therapeutically, the stromal cells themselves
may be considered as potential targets as they exert significant influence on the tumor
microenvironment. In the case of cells of the monocyte lineage, we need to better understand what role
these cells play in either tumor formation or maintenance. If it turns out that cells of the monocytic
lineage play a critical role in maintenance of the tumor or its resistance to therapy, only then will
targeting these cells in formed tumors be useful.

Astrocytes will be difficult to target as they do not proliferate and are inherently resistant to cytotoxic
therapies. Moreover, just as for monocytes, we do not have evidence at this point whether they serve a
critical function in the maintenance of these tumors.

Endothelial cells in general may not be good targets; however, the tumor vasculature is already being
targeted effectively in the clinical setting. Anti-angiogenic agents targeting VEGF such as avastin are
showing some therapeutic responses. The reasons for these responses are not clear at this point.
However, it is possible that a part of the therapeutic response to VEGF blockade may be the disruption
of the perivascular niche and loss of support for the BTSC populations in these tumors.

REFERENCES

Aboody KS,Brown A,Rainov NG,Bower KA,Liu S,Yang W,Small JE,Herrlinger U,Ourednik V,Black
PM,Breakefield XO,Snyder EY. 2000. Neural stem cells display extensive tropism for pathology in adult
brain: Evidence from intracranial gliomas. Proc Natl Acad Sci USA 97: 12846–12851.

Aboody KS,Najbauer J,Danks MK. 2008. Stem and progenitor tumor selective gene therapy. Gene Ther
15: 739–752.

Alterman RL,Stanley ER. 1994. Colony stimulating factor-1 expression in human glioma. Mol Chem
Neuropathol 21: 177–188.
Antonelli-Orlidge A,Saunders KB,Smith SR,D'Amore PA. 1989. An activated form of transforming growth
factor beta is produced by cocultures of endothelial cells and pericytes. Proc Natl Acad Sci USA 86:
4544–4548.

Armulik A,Abramsson A,Betsholtz C. 2005. Endothelial/pericyte interactions. Circ Res 97: 512–523.

Assanah M,Lochhead R,Ogden A,Bruce J,Goldman J,Canoll P. 2006. Glial progenitors in adult white
matter are driven to form malignant gliomas by platelet-derived growth factor-expressing retroviruses. J
Neurosci 26: 6781–6790.

Assanah MC,Bruce JN,Suzuki SO,Chen A,Goldman JE,Canoll P. 2009. PDGF stimulates the massive
expansion of glial progenitors in the neonatal forebrain. Glia 57: 1835–1847.

Bababeygy SR,Cheshier SH,Hou LC,Higgins DM,Weissman IL,Tse VC. 2008. Hematopoietic stem cell-
derived pericytic cells in brain tumor angio-architecture. Stem Cells Dev 17: 11–18.

Badie B,Bartley B,Schartner J. 2002. Differential expression of MHC class II and B7 costimulatory
molecules by microglia in rodent gliomas. J Neuroimmunol 133: 39–45.

Badie B,Hunt K,Economou JS,Black KL. 1994. Stereotactic delivery of a recombinant adenovirus into a C6
glioma cell line in a rat brain tumor model. Neurosurgery 35: 910–915; discussion 915–916.

Badie B,Schartner JM. 2000. Flow cytometric characterization of tumor-associated macrophages in

experimental gliomas. Neurosurgery 46: 957–961; discussion 961–962.

Bajetto A,Bonavia R,Barbero S,Florio T,Costa A,Schettini G. 1999a. Expression of chemokine receptors in
the rat brain. Ann N Y Acad Sci 876: 201–209.

Bajetto A,Bonavia R,Barbero S,Piccioli P,Costa A,Florio T,Schettini G. 1999b. Glial and neuronal cells
express functional chemokine receptor CXCR4 and its natural ligand stromal cell-derived factor 1. J
Neurochem 73: 2348–2357.

Baluk P,Hashizume H,McDonald DM. 2005. Cellular abnormalities of blood vessels as targets in cancer.
Curr Opin Genet Dev 15: 102–111.

Bao S,Wu Q,Sathornsumetee S,Hao Y,Li Z,Hjelmeland AB,Shi Q,McLendon RE,Bigner DD,Rich JN. 2006.
Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor.
Cancer Res 66: 7843–7848.

Barbero S,Bajetto A,Bonavia R,Porcile C,Piccioli P,Pirani P,Ravetti JL,Zona G,Spaziante R,Florio T,Schettini
G. 2002. Expression of the chemokine receptor CXCR4 and its ligand stromal cell-derived factor 1 in
human brain tumors and their involvement in glial proliferation in vitro. Ann N Y Acad Sci 973: 60–69.

Becher OJ,Hambardzumyan D,Fomchenko EI,Momota H,Mainwaring L,Bleau AM,Katz AM,Edgar

M,Kenney AM,Cordon-Cardo C,Blasberg RG,Holland EC. 2008. Gli activity correlates with tumor grade in
platelet-derived growth factor-induced gliomas. Cancer Res 68: 2241–2249.
Belien AT,Paganetti PA,Schwab ME. 1999. Membrane-type 1 matrix metalloprotease (MT1-MMP)
enables invasive migration of glioma cells in central nervous system white matter. J Cell Biol 144: 373–
384.

Bergers G,Song S,Meyer-Morse N,Bergsland E,Hanahan D. 2003. Benefits of targeting both pericytes and
endothelial cells in the tumor vasculature with kinase inhibitors. J Clin Invest 111: 1287–1295.

PubMed,ChemPort

Bertrand I,Mannen H. 1960. Étude des réactions vasculaires dans les astrocytomes. Revue neurol 102:
3–19.

Betsholtz C. 2004. Insight into the physiological functions of PDGF through genetic studies in mice.
Cytokine Growth Factor Rev 15: 215–228.

Bettinger I,Thanos S,Paulus W. 2002. Microglia promote glioma migration. Acta Neuropathol 103: 351–
355.

Bjarnegard M,Enge M,Norlin J,Gustafsdottir S,Fredriksson S,Abramsson A,Takemoto M,Gustafsson

E,Fassler R,Betsholtz C. 2004. Endothelium-specific ablation of PDGFB leads to pericyte loss and
glomerular, cardiac and placental abnormalities. Development 131: 1847–1857.

Brooks PC,Clark RA,Cheresh DA. 1994. Requirement of vascular integrin alpha v beta 3 for angiogenesis.
Science 264: 569–571.

Buetow BS,Crosby JR,Kaminski WE,Ramachandran RK,Lindahl P,Martin P,Betsholtz C,Seifert RA,Raines

EW,Bowen-Pope DF. 2001. Platelet-derived growth factor B-chain of hematopoietic origin is not
necessary for granulation tissue formation and its absence enhances vascularization. Am J Pathol 159:
1869–1876.

Butler JM,Nolan DJ,Vertes EL,Varnum-Finney B,Kobayashi H,Hooper AT,Seandel M,Shido K,White

IA,Kobayashi M,Witte L,May C,Shawber C,Kimura Y,Kitajewski J,Rosenwaks Z,Bernstein ID,Rafii S. 2010.
Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic
stem cells. Cell Stem Cell 6: 251–264.

Calabrese C,Poppleton H,Kocak M,Hogg TL,Fuller C,Hamner B,Oh EY,Gaber MW,Finklestein D,Allen
M,Frank A,Bayazitov IT,Zakharenko SS,Gajjar A,Davidoff A,Gilbertson RJ. 2007. A perivascular niche for
brain tumor stem cells. Cancer Cell 11: 69–82.

Carvalho RL,Jonker L,Goumans MJ,Larsson J,Bouwman P,Karlsson S,Dijke PT,Arthur HM,Mummery CL.
2004. Defective paracrine signalling by TGFbeta in yolk sac vasculature of endoglin mutant mice: A
paradigm for hereditary haemorrhagic telangiectasia. Development 131: 6237–6247.
Charles N,Ozawa T,Squatrito M,Bleau AM,Brennan CW,Hambardzumyan D,Holland EC. 2010.
Perivascular nitric oxide activates notch signaling and promotes stem-like character in PDGF-induced
glioma cells. Cell Stem Cell 6: 141–152.

Chekenya M,Enger PO,Thorsen F,Tysnes BB,Al-Sarraj S,Read TA,Furmanek T,Mahesparan R,Levine

JM,Butt AM,Pilkington GJ,Bjerkvig R. 2002a. The glial precursor proteoglycan, NG2, is expressed on
tumour neovasculature by vascular pericytes in human malignant brain tumours. Neuropathol Appl
Neurobiol 28: 367–380.

Chekenya M,Hjelstuen M,Enger PO,Thorsen F,Jacob AL,Probst B,Haraldseth O,Pilkington G,Butt A,Levine
JM,Bjerkvig R. 2002b. NG2 proteoglycan promotes angiogenesis-dependent tumor growth in CNS by
sequestering angiostatin. FASEB J 16: 586–588.

Chen FX,Ren WW,Yang Y,Shen D,Zong Y,Xu S,Duan Y,Qian Y,Ji Y. 2009. Reciprocal effects of conditioned
medium on cultured glioma cells and neural stem cells. J Clin Neurosci 16: 1619–1623.

Chirasani SR,Sternjak A,Wend P,Momma S,Campos B,Herrmann IM,Graf D,Mitsiadis T,Herold-Mende

C,Besser D,Synowitz M,Kettenmann H,Glass R. 2010. Bone morphogenetic protein-7 release from
endogenous neural precursor cells suppresses the tumourigenicity of stem-like glioblastoma cells. Brain
133(Pt 7): 1961–1972.

Cleaver O,Melton DA. 2003. Endothelial signaling during development. Nat Med 9: 661–668.

Clement V,Sanchez P,de Tribolet N,Radovanovic I,Ruiz i Altaba A. 2007. HEDGEHOG-GLI1 signaling
regulates human glioma growth, cancer stem cell self-renewal, and tumorigenicity. Curr Biol 17: 165–
172.

De Palma M,Naldini L. 2006. Role of haematopoietic cells and endothelial progenitors in tumour
angiogenesis. Biochim Biophys Acta 1766: 159–166.

De Palma M,Venneri MA,Galli R,Sergi Sergi L,Politi LS,Sampaolesi M,Naldini L. 2005. Tie2 identifies a
hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a
mesenchymal population of pericyte progenitors. Cancer Cell 8: 211–226.

De Wever O,Nguyen QD,Van Hoorde L,Bracke M,Bruyneel E,Gespach C,Mareel M. 2004. Tenascin-C and
SF/HGF produced by myofibroblasts in vitro provide convergent pro-invasive signals to human colon
cancer cells through RhoA and Rac. FASEB J 18: 1016–1018.

Diaz-Flores L,Gutierrez R,Madrid JF,Varela H,Valladares F,Acosta E,Martin-Vasallo P,Diaz-Flores LJr. 2009.
Pericytes. Morphofunction, interactions and pathology in a quiescent and activated mesenchymal cell
niche. Histol Histopathol 24: 909–969.

Du R,Lu KV,Petritsch C,Liu P,Ganss R,Passegue E,Song H,Vandenberg S,Johnson RS,Werb Z,Bergers G.
2008. HIF1alpha induces the recruitment of bone marrow-derived vascular modulatory cells to regulate
tumor angiogenesis and invasion. Cancer Cell 13: 206–220.
Ehebauer M,Hayward P,Arias AM. 2006. Notch, a universal arbiter of cell fate decisions. Science 314:
1414–1415.

Emdad L,Sarkar D,Lee SG,Su ZZ,Yoo BK,Dash R,Yacoub A,Fuller CE,Shah K,Dent P,Bruce JN,Fisher PB.
2010. Astrocyte elevated gene-1: A novel target for human glioma therapy. Mol Cancer Ther 9: 79–88.

Emdad L,Sarkar D,Su ZZ,Lee SG,Kang DC,Bruce JN,Volsky DJ,Fisher PB. 2007. Astrocyte elevated gene-1:
Recent insights into a novel gene involved in tumor progression, metastasis and neurodegeneration.
Pharmacol Ther 114: 155–170.

Enge M,Bjarnegard M,Gerhardt H,Gustafsson E,Kalen M,Asker N,Hammes HP,Shani M,Fassler

R,Betsholtz C. 2002. Endothelium-specific platelet-derived growth factor-B ablation mimics diabetic
retinopathy. EMBO J 21: 4307–4316.

Enge M,Wilhelmsson U,Abramsson A,Stakeberg J,Kuhn R,Betsholtz C,Pekny M. 2003. Neuron-specific

ablation of PDGF-B is compatible with normal central nervous system development and astroglial
response to injury. Neurochem Res 28: 271–279.

Eyler CE,Foo WC,LaFiura KM,McLendon RE,Hjelmeland AB,Rich JN. 2008. Brain cancer stem cells display
preferential sensitivity to Akt inhibition. Stem Cells 26: 3027–3036.

Farin A,Suzuki SO,Weiker M,Goldman JE,Bruce JN,Canoll P. 2006. Transplanted glioma cells migrate and
proliferate on host brain vasculature: A dynamic analysis. Glia 53: 799–808.

Ferrara N,Kerbel RS. 2005. Angiogenesis as a therapeutic target. Nature 438: 967–974.

Folkins C,Man S,Xu P,Shaked Y,Hicklin DJ,Kerbel RS. 2007. Anticancer therapies combining
antiangiogenic and tumor cell cytotoxic effects reduce the tumor stem-like cell fraction in glioma
xenograft tumors. Cancer Res 67: 3560–3564.

Gallagher PG,Bao Y,Prorock A,Zigrino P,Nischt R,Politi V,Mauch C,Dragulev B,Fox JW. 2005. Gene
expression profiling reveals cross-talk between melanoma and fibroblasts: Implications for host-tumor
interactions in metastasis. Cancer Res 65: 4134–4146.

Gerhardt H,Betsholtz C. 2003. Endothelial-pericyte interactions in angiogenesis. Cell Tissue Res 314: 15–
23.

Gladson CL. 1996. Expression of integrin alpha vs. beta 3 in small blood vessels of glioblastoma tumors. J
Neuropathol Exp Neurol 55: 1143–1149.

Glass R,Synowitz M,Kronenberg G,Walzlein JH,Markovic DS,Wang LP,Gast D,Kiwit J,Kempermann

G,Kettenmann H. 2005. Glioblastoma-induced attraction of endogenous neural precursor cells is
associated with improved survival. J Neurosci 25: 2637–2646.
Goldstein LJ,Chen H,Bauer RJ,Bauer SM,Velazquez OC. 2005. Normal human fibroblasts enable
melanoma cells to induce angiogenesis in type I collagen. Surgery 138: 439–449.

Gould E,Reeves AJ,Fallah M,Tanapat P,Gross CG,Fuchs E. 1999. Hippocampal neurogenesis in adult Old
World primates. Proc Natl Acad Sci USA 96: 5263–5267.

Graeber MB,Scheithauer BW,Kreutzberg GW. 2002. Microglia in brain tumors. Glia 40: 252–259.

Hadjipanayis CG,Van Meir EG. 2009. Tumor initiating cells in malignant gliomas: Biology and implications
for therapy. J Mol Med 87: 363–374.

Hambardzumyan D,Becher OJ,Rosenblum MK,Pandolfi PP,Manova-Todorova K,Holland EC. 2008. PI3K

pathway regulates survival of cancer stem cells residing in the perivascular niche following radiation in
medulloblastoma in vivo. Genes Dev 22: 436–448.

Hanahan D,Weinberg RA. 2000. The hallmarks of cancer. Cell 100: 57–70.

Hanisch UK,Kettenmann H. 2007. Microglia: active sensor and versatile effector cells in the normal and
pathologic brain. Nat Neurosci 10: 1387–1394.

Hashizume H,Baluk P,Morikawa S,McLean JW,Thurston G,Roberge S,Jain RK,McDonald DM. 2000.
Openings between defective endothelial cells explain tumor vessel leakiness. Am J Pathol 156: 1363–
1380.

Held-Feindt J,Hattermann K,Muerkoster SS,Wedderkopp H,Knerlich-Lukoschus F,Ungefroren H,Mehdorn

HM,Mentlein R. 2010. CX3CR1 promotes recruitment of human glioma-infiltrating
microglia/macrophages (GIMs). Exp Cell Res 316: 1553–1566.

Hellstrom M,Gerhardt H,Kalen M,Li X,Eriksson U,Wolburg H,Betsholtz C. 2001. Lack of pericytes leads to
endothelial hyperplasia and abnormal vascular morphogenesis. J Cell Biol 153: 543–553.

Hellstrom M,Kalen M,Lindahl P,Abramsson A,Betsholtz C. 1999. Role of PDGF-B and PDGFR-beta in
recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in
the mouse. Development 126: 3047–3055.

Hirschi KK,Rohovsky SA,D'Amore PA. 1998. PDGF, TGF-beta, and heterotypic cell-cell interactions
mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth
muscle fate. J Cell Biol 141: 805–814.

Hoch RV,Soriano P. 2003. Roles of PDGF in animal development. Development 130: 4769–4784.

Hoelzinger DB,Demuth T,Berens ME. 2007. Autocrine factors that sustain glioma invasion and paracrine
biology in the brain microenvironment. J Natl Cancer Inst 99: 1583–1593.

Holland EC. 2001. Gliomagenesis: Genetic alterations and mouse models. Nat Rev Genet 2: 120–129.
Hulleman E,Quarto M,Vernell R,Masserdotti G,Colli E,Kros JM,Levi D,Gaetani P,Tunici P,Finocchiaro
G,Baena RR,Capra M,Helin K. 2009. A role for the transcription factor HEY1 in glioblastoma. J Cell Mol
Med 13: 136–146.

Hwang RF,Moore T,Arumugam T,Ramachandran V,Amos KD,Rivera A,Ji B,Evans DB,Logsdon CD. 2008.
Cancer-associated stromal fibroblasts promote pancreatic tumor progression. Cancer Res 68: 918–926.

Jain RK,di Tomaso E,Duda DG,Loeffler JS,Sorensen AG,Batchelor TT. 2007. Angiogenesis in brain
tumours. Nat Rev Neurosci 8: 610–622.

Jeon HM,Jin X,Lee JS,Oh SY,Sohn YW,Park HJ,Joo KM,Park WY,Nam DH,DePinho RA,Chin L,Kim H. 2008.
Inhibitor of differentiation 4 drives brain tumor-initiating cell genesis through cyclin E, notch signaling.
Genes Dev 22: 2028–2033.

Joyce JA,Pollard JW. 2009. Microenvironmental regulation of metastasis. Nat Rev Cancer 9: 239–252.

Kang DC,Su ZZ,Sarkar D,Emdad L,Volsky DJ,Fisher PB. 2005. Cloning and characterization of HIV-1-
inducible astrocyte elevated gene-1, AEG-1. Gene 353: 8–15.

Kim JH,Park JA,Lee SW,Kim WJ,Yu YS,Kim KW. 2006. Blood-neural barrier: intercellular communication at
glio-vascular interface. J Biochem Mol Biol 39: 339–345.

Knoth R,Singec I,Ditter M,Pantazis G,Capetian P,Meyer RP,Horvat V,Volk B,Kempermann G. 2010.
Murine features of neurogenesis in the human hippocampus across the lifespan from 0 to 100 years.
PLoS One 5: e8809.

Kokovay E,Goderie S,Wang Y,Lotz S,Lin G,Sun Y,Roysam B,Shen Q,Temple S. 2010. Adult SVZ lineage cells
home to and leave the vascular niche via differential responses to SDF1/CXCR4 signaling. Cell Stem Cell
7: 163–173.

Kowalczyk A,Filipkowski RK,Rylski M,Wilczynski GM,Konopacki FA,Jaworski J,Ciemerych MA,Sicinski

P,Kaczmarek L. 2004. The critical role of cyclin D2 in adult neurogenesis. J Cell Biol 167: 209–213.

Le Bras B,Barallobre MJ,Homman-Ludiye J,Ny A,Wyns S,Tammela T,Haiko P,Karkkainen MJ,Yuan L,Muriel
MP,Chatzopoulou E,Breant C,Zalc B,Carmeliet P,Alitalo K,Eichmann A,Thomas JL. 2006. VEGF-C is a
trophic factor for neural progenitors in the vertebrate embryonic brain. Nat Neurosci 9: 340–348.

Le DM,Besson A,Fogg DK,Choi KS,Waisman DM,Goodyer CG,Rewcastle B,Yong VW. 2003. Exploitation of
astrocytes by glioma cells to facilitate invasiveness: a mechanism involving matrix metalloproteinase-2
and the urokinase-type plasminogen activator-plasmin cascade. J Neurosci 23: 4034–4043.

Lee J,Lund-Smith C,Borboa A,Gonzalez AM,Baird A,Eliceiri BP. 2009. Glioma-induced remodeling of the
neurovascular unit. Brain Res 1288: 125–134.
Lee SG,Su ZZ,Emdad L,Sarkar D,Fisher PB. 2006. Astrocyte elevated gene-1 (AEG-1) is a target gene of
oncogenic Ha-ras requiring phosphatidylinositol 3-kinase and c-Myc. Proc Natl Acad Sci USA 103: 17390–
17395.

Letterio JJ,Roberts AB. 1998. Regulation of immune responses by TGF-beta. Annu Rev Immunol 16: 137–
161.

Leventhal C,Rafii S,Rafii D,Shahar A,Goldman SA. 1999. Endothelial trophic support of neuronal
production and recruitment from the adult mammalian subependyma. Mol Cell Neurosci 13: 450–464.

Li J,Tan J,Zhuang L,Banerjee B,Yang X,Chau JF,Lee PL,Hande MP,Li B,Yu Q. 2007. Ribosomal protein S27-
like, a p53-inducible modulator of cell fate in response to genotoxic stress. Cancer Res 67: 11317–11326.

Liu L,Wu J,Ying Z,Chen B,Han A,Liang Y,Song L,Yuan J,Li J,Li M. 2010. Astrocyte elevated gene-1
upregulates matrix metalloproteinase-9 and induces human glioma invasion. Cancer Res 70: 3750–3759.

Li JC,Li R. 2007. RAV12 accelerates the desensitization of Akt/PKB pathway of insulin-like growth factor I
receptor signaling in COLO205. Cancer Res 67: 8856–8864.

Li JL,Sainson RC,Shi W,Leek R,Harrington LS,Preusser M,Biswas S,Turley H,Heikamp E,Hainfellner

JA,Harris AL. 2007. Delta-like 4 Notch ligand regulates tumor angiogenesis, improves tumor vascular
function, and promotes tumor growth in vivo. Cancer Res 67: 11244–11253.

Lyden D,Hattori K,Dias S,Costa C,Blaikie P,Butros L,Chadburn A,Heissig B,Marks W,Witte L,Wu Y,Hicklin
D,Zhu Z,Hackett NR,Crystal RG,Moore MA,Hajjar KA,Manova K,Benezra R,Rafii S. 2001. Impaired
recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor
angiogenesis and growth. Nat Med 7: 1194–1201.

Markovic DS,Glass R,Synowitz M,Rooijen N,Kettenmann H. 2005. Microglia stimulate the invasiveness of
glioma cells by increasing the activity of metalloprotease-2. J Neuropathol Exp Neurol 64: 754–762.

Mirzadeh Z,Merkle FT,Soriano-Navarro M,Garcia-Verdugo JM,Alvarez-Buylla A. 2008. Neural stem cells

confer unique pinwheel architecture to the ventricular surface in neurogenic regions of the adult brain.
Cell Stem Cell 3: 265–278.

Mora R,Regnier-Vigouroux A. 2009. Autophagy-driven cell fate decision maker: activated microglia
induce specific death of glioma cells by a blockade of basal autophagic flux and secondary
apoptosis/necrosis. Autophagy 5: 419–421.

Morikawa S,Baluk P,Kaidoh T,Haskell A,Jain RK,McDonald DM. 2002. Abnormalities in pericytes on blood
vessels and endothelial sprouts in tumors. Am J Pathol 160: 985–1000.
Naganuma H,Sasaki A,Satoh E,Nagasaka M,Nakano S,Isoe S,Nukui H. 1996. Modulation of transforming
growth factor-beta secretion from malignant glioma cells by interleukin-1 beta. Neurol Med Chir (Tokyo)
36: 145–150.

Nolte C,Kirchhoff F,Kettenmann H. 1997. Epidermal growth factor is a motility factor for microglial cells
in vitro: Evidence for EGF receptor expression. Eur J Neurosci 9: 1690–1698.

Ohgaki H,Kleihues P. 2007. Genetic pathways to primary and secondary glioblastoma. Am J Pathol 170:
1445–1453.

Okada M,Saio M,Kito Y,Ohe N,Yano H,Yoshimura S,Iwama T,Takami T. 2009. Tumor-associated
macrophage/microglia infiltration in human gliomas is correlated with MCP-3, but not MCP-1. Int J Oncol
34: 1621–1627.

Ozerdem U,Stallcup WB. 2004. Pathological angiogenesis is reduced by targeting pericytes via the NG2
proteoglycan. Angiogenesis 7: 269–276.

Platten M,Kretz A,Naumann U,Aulwurm S,Egashira K,Isenmann S,Weller M. 2003. Monocyte

chemoattractant protein-1 increases microglial infiltration and aggressiveness of gliomas. Ann Neurol
54: 388–392.

Pollard JW. 2004. Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev
Cancer 4: 71–78.

Rafii S,Heissig B,Hattori K. 2002. Efficient mobilization and recruitment of marrow-derived endothelial
and hematopoietic stem cells by adenoviral vectors expressing angiogenic factors. Gene Ther 9: 631–
641.

Ramirez-Castillejo C,Sanchez-Sanchez F,Andreu-Agullo C,Ferron SR,Aroca-Aguilar JD,Sanchez P,Mira

H,Escribano J,Farinas I. 2006. Pigment epithelium-derived factor is a niche signal for neural stem cell
renewal. Nat Neurosci 9: 331–339.

Rempel SA,Dudas S,Ge S,Gutierrez JA. 2000. Identification and localization of the cytokine SDF1 and its
receptor, CXC chemokine receptor 4, to regions of necrosis and angiogenesis in human glioblastoma.
Clin Cancer Res 6: 102–111.

Sameshima T,Nabeshima K,Toole BP,Yokogami K,Okada Y,Goya T,Koono M,Wakisaka S. 2000. Glioma
cell extracellular matrix metalloproteinase inducer (EMMPRIN) (CD147) stimulates production of
membrane-type matrix metalloproteinases and activated gelatinase A in co-cultures with brain-derived
fibroblasts. Cancer Lett 157: 177–184.

Sanai N,Alvarez-Buylla A,Berger MS. 2005. Neural stem cells and the origin of gliomas. N Engl J Med 353:
811–222.
Santarelli JG,Udani V,Yung YC,Cheshier S,Wagers A,Brekken RA,Weissman I,Tse V. 2006. Incorporation of
bone marrow-derived Flk-1-expressing CD34+ cells in the endothelium of tumor vessels in the mouse
brain. Neurosurgery 59: 374–382.

Sato TN,Tozawa Y,Deutsch U,Wolburg-Buchholz K,Fujiwara Y,Gendron-Maguire M,Gridley T,Wolburg

H,Risau W,Qin Y. 1995. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel
formation. Nature 376: 70–74.

Sato Y,Tsuboi R,Lyons R,Moses H,Rifkin DB. 1990. Characterization of the activation of latent TGF-beta
by co-cultures of endothelial cells and pericytes or smooth muscle cells: A self-regulating system. J Cell
Biol 111: 757–763.

Sawaya RE,Yamamoto M,Gokaslan ZL,Wang SW,Mohanam S,Fuller GN,McCutcheon IE,Stetler-Stevenson

WG,Nicolson GL,Rao JS. 1996. Expression and localization of 72 kDa type IV collagenase (MMP-2) in
human malignant gliomas in vivo. Clin Exp Metastasis 14: 35–42.

Shen Q,Wang Y,Kokovay E,Lin G,Chuang SM,Goderie SK,Roysam B,Temple S. 2008. Adult SVZ stem cells
lie in a vascular niche: a quantitative analysis of niche cell-cell interactions. Cell Stem Cell 3: 289–300.

Shih AH,Holland EC. 2006. Notch signaling enhances nestin expression in gliomas. Neoplasia 8: 1072–
1082.

Sims DE. 2000. Diversity within pericytes. Clin Exp Pharmacol Physiol 27: 842–846.

Song S,Ewald AJ,Stallcup W,Werb Z,Bergers G. 2005. PDGFRbeta+ perivascular progenitor cells in
tumours regulate pericyte differentiation and vascular survival. Nat Cell Biol 7: 870–879.

Staflin K,Honeth G,Kalliomaki S,Kjellman C,Edvardsen K,Lindvall M. 2004. Neural progenitor cell lines
inhibit rat tumor growth in vivo. Cancer Res 64: 5347–5354.

Staflin K,Zuchner T,Honeth G,Darabi A,Lundberg C. 2009. Identification of proteins involved in neural
progenitor cell targeting of gliomas. BMC Cancer 9: 206.

Stecca B,Ruiz i Altaba A. 2005. Brain as a paradigm of organ growth: Hedgehog-Gli signaling in neural
stem cells and brain tumors. J Neurobiol 64: 476–490.

Su ZZ,Kang DC,Chen Y,Pekarskaya O,Chao W,Volsky DJ,Fisher PB. 2002. Identification and cloning of
human astrocyte genes displaying elevated expression after infection with HIV-1 or exposure to HIV-1
envelope glycoprotein by rapid subtraction hybridization, RaSH. Oncogene 21: 3592–3602.

Sundberg C,Kowanetz M,Brown LF,Detmar M,Dvorak HF. 2002. Stable expression of angiopoietin-1 and
other markers by cultured pericytes: phenotypic similarities to a subpopulation of cells in maturing
vessels during later stages of angiogenesis in vivo. Lab Invest 82: 387–401.
Suri C,Jones PF,Patan S,Bartunkova S,Maisonpierre PC,Davis S,Sato TN,Yancopoulos GD. 1996. Requisite
role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 87: 1171–
1180.

Suri C,McClain J,Thurston G,McDonald DM,Zhou H,Oldmixon EH,Sato TN,Yancopoulos GD. 1998.
Increased vascularization in mice overexpressing angiopoietin-1. Science 282: 468–471.

Suzuki T,Izumoto S,Wada K,Fujimoto Y,Maruno M,Yamasaki M,Kanemura Y,Shimazaki T,Okano

H,Yoshimine T. 2005. Inhibition of glioma cell proliferation by neural stem cell factor. J Neurooncol 74:
233–239.

Suzuki Y,Funakoshi H,Machide M,Matsumoto K,Nakamura T. 2008. Regulation of cell migration and
cytokine production by HGF-like protein (HLP)/macrophage stimulating protein (MSP) in primary
microglia. Biomed Res 29: 77–84.

Tate MC,Aghi MK. 2009. Biology of angiogenesis and invasion in glioma. Neurotherapeutics 6: 447–457.

Tavazoie M,Van der Veken L,Silva-Vargas V,Louissaint M,Colonna L,Zaidi B,Garcia-Verdugo JM,Doetsch F.
2008. A specialized vascular niche for adult neural stem cells. Cell Stem Cell 3: 279–288.

Thurston G,Suri C,Smith K,McClain J,Sato TN,Yancopoulos GD,McDonald DM. 1999. Leakage-resistant
blood vessels in mice transgenically overexpressing angiopoietin-1. Science 286: 2511–2514.

Tropepe V,Craig CG,Morshead CM,van der Kooy D. 1997. Transforming growth factor-alpha null and
senescent mice show decreased neural progenitor cell proliferation in the forebrain subependyma. J
Neurosci 17: 7850–7859.

Uhm JH,Dooley NP,Villemure JG,Yong VW. 1996. Glioma invasion in vitro: regulation by matrix
metalloprotease-2 and protein kinase C. Clin Exp Metastasis 14: 421–433.

Walzlein JH,Synowitz M,Engels B,Markovic DS,Gabrusiewicz K,Nikolaev E,Yoshikawa K,Kaminska

B,Kempermann G,Uckert W,Kaczmarek L,Kettenmann H,Glass R. 2008. The antitumorigenic response of
neural precursors depends on subventricular proliferation and age. Stem Cells 26: 2945–2954.

Watters JJ,Schartner JM,Badie B. 2005. Microglia function in brain tumors. J Neurosci Res 81: 447–455.

Wen PY,Kesari S. 2008. Malignant gliomas in adults. N Engl J Med 359: 492–507.

Wesseling P,Schlingemann RO,Rietveld FJ,Link M,Burger PC,Ruiter DJ. 1995. Early and extensive
contribution of pericytes/vascular smooth muscle cells to microvascular proliferation in glioblastoma
multiforme: an immuno-light and immuno-electron microscopic study. J Neuropathol Exp Neurol 54:
304–310.
Wurdak H,Ittner LM,Lang KS,Leveen P,Suter U,Fischer JA,Karlsson S,Born W,Sommer L. 2005.
Inactivation of TGFbeta signaling in neural crest stem cells leads to multiple defects reminiscent of
DiGeorge syndrome. Genes Dev 19: 530–535.

Zhang HR,Chen FL,Xu CP,Ping YF,Wang QL,Liang ZQ,Wang JM,Bian XW. 2009. Incorporation of
endothelial progenitor cells into the neovasculature of malignant glioma xenograft. J Neurooncol 93:
165–174.

Zhang XP,Zheng G,Zou L,Liu HL,Hou LH,Zhou P,Yin DD,Zheng QJ,Liang L,Zhang SZ,Feng L.,Yao LB,Yang
AG,Han H,Chen JY. 2008. Notch activation promotes cell proliferation and the formation of neural stem
cell-like colonies in human glioma cells. Mol Cell Biochem 307: 101–108.