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The FASEBjo urnal • Review
Tumor exploits alternative strategies to
achieve vascularization
Benedetta Bussolati, Cristina Grange, and Giovanni Camussi 1
Department of Internal Medicine, Research Center for Experimental Medicine and Center for
Molecular Biotechnology, Department of Biomedical Sciences and Human Oncology, University of
Torino, Turin, Italy
ABSTRACT Neoangiogenesis is crucial for solid tumor
growth and invasion, as the vasculature provides
metabolic support and access to the circulation. Current
antiangiogenic therapies have been designed on
the assumption that endothelial cells forming the tumor
vasculature exhibit genetic stability. Recent studies
demonstrate that this is not the case. Tumor endothelial
cells possess a distinct phenotype, differing from
normal endothelial cells at both molecular and functional
levels. This challenges the concept that tumor
angiogenesis exclusively depends on normal endothelial
cell recruitment from the surrounding vascular
network. Indeed, recent data suggest alternative strategies
for tumor vascularization. It has been reported that
tumor vessels may derive from an intratumor embryoniclike
vasculogenesis. This condition might be due to
differentiation of normal stem and progenitor cells of
hematopoietic origin or resident in tissues. Cancer stem
cells may also participate in tumor vasculogenesis by
virtue of their stem and progenitor cell properties.
Finally, normal endothelial cells might be reprogrammed
to a proangiogenic or dedifferentiated phenotype
by genetic information transmitted from the
tumor trough apoptotic bodies, or following mRNA
and microRNA transfer by exosomes and microvesicles.
In this review, we discuss the different
aspects of intratumor angiogenesis and vasculogenesis,
the known mechanisms involved, and the possible
implications for the response to antiangiogenic therapy.Bussolati,
B., Grange, C., Camussi, G. Tumor exploits
alternative strategies to achieve vascularization. FASEB
J. 25, 2874-2882 (2011). www.fasebj.org
Key Wards: angiogenesis • vascul.ogenesis • exosomes • antiangiogenic
therapy
TIIE PROCES OF BLOOD VESSEL formation in the embryo,
defined as vasculogenesis, relies on endothelial
differentiation ofmultipotent cells located in the dorsal
aorta. These include hemangioblasts, which generate
both endothelial and hematopoietic cells (I), and mesangioblasts,
precursors of endothelial cells and mesodennal
derivatives such as bone, muscle, and cartilage (2). The
primitive vasculature created by vasculogenesis is
then remodeled by a process called angiogenesis. In
postnatal life, tissue remodeling and regeneration
2874
have been ascribed mainly to angiogenesis, which
involves preexisting vessels (3). However, more recently,
it has been found that vasculogenesis can also
occur in the adult and particularly during tumor
vascularization ( 4, 5).
A wealth of evidence indicates that tumor blood
vessels differ significantly from normal vessels in their
structural organization and endothelial propertie .
This finding suggests that tumor vascularization depends
on mechanisms distinct from tl1e simple recruitment
from adjacent tissue of preexisting blood vessels.
The recapitulation of an embryonic-like vasculogenesis
may allow a de novo fom1ation of vessels within the
tumor. This process involves endothelial differentiation
of normal or malignant adult cells bearing stem and
progenitor properties leading to generation of endothelial
cells with abnormal characteristics. In this context,
the tumor microenvironment is thought to play a
pivotal role, either by altering normal endothelial cells
present in the tumor, or by activating angiogenic
programs in stem and progenitor cells. Therefore,
tumor vascularization is a complex scenario due to the
concomitant activity of different mechanisms, which
may in tum vary according to tumor type, grade, and
therapeutic response, as well as from patient to patient.
This review analyzes different strategies involved in
the formation of new vessels in tum ors in light of the
altered characteristics of tumor endothelial cells
(TECs).
ALTERED CHARACTERISTICS OF TECS
Morphologically, tumor blood vessels are irregular,
dilated, and tortuous. Thi.s chaotic organization results
in the absence of distinct venules, arterioles, and capillaries
and results in tl1e formation of a va.scular tumor
network that is often leaky and hemorrhagic (for
review, see ref. 6). Tumor pericytes are also implicated
in the abnormal nature of tumor vessels. Pericytes
around tumor vessels are loosely attached to endothelial
cells, have abnormal shapes, or present long cyto-
1 Con-espondence: Departrnent of internal Medicine, Corso
Dogliotti 14, 10126, Torino, Italy. E-mail: giovanni.carnussi@w1ito.it
doi: 10.1096/fj.10-180323
0892-6638/l l/0025-2874 © FASEB
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plasmic processes away from the vessel wall ( 6, 7).
Strategies to enhance pericyte coverage may prevent
tumor vessel leakage, dilatation, and tortuosity and may
promote vessel stabilization and normalization (7). In
addition, several studies indicate that tumor vessels are
lined by altered endothelial cells. The information on
TEC genotype, phenotype, and function were obtained
by isolating and, in some instances, culturing TECs
from different human and experimental tumors. As a
matter of comparison, the studies generally used normal
endothelial cells from the same tissue, or angiogenic
endothelial cells derived from a proliferative
nonnal endothelium, such as that of the corpus luteum
or placenta.
Genetic alterations
The most remarkable abnormality identified in TECs is
chromosomal instability (8). Aneuploidy, i.e., the presence
of an abnormal chromosome number, is a common
characteristic of tumor cells. Hida et al. (9) first
demonstrated that unlike normal endothelial cells,
which remain diploid in long-term culture, freshly
isolated TECs-or at least a fraction of them-are
aneuploid, with the latter exacerbated in culture. These
aneuploid cells showed structural abeJTations, such as
nonreciprocal translocations, missing chromosomes,
and marker chromosomes, and have multiple cent:rosomes
(9). Hida et al. ( 10) suggested a causal relationship
between the centrosomal abnormalities and the
presence of extra chromosomes, as the defects in
centrosome function may contribute to loss of polarity
and altered segregation of chromosomes during cell
division. These changes were observed in TECs from
renal carcinomas, by fluorescence in situ hybridization
analysis of both tissue and isolated cells ( 11). The
different cytogenetic profile found in individual TECs
in these studies supports the heterogeneity of this
population rather than suggesting a clonal origin. In
human hematopoietic tumors, TECs consistently show
genetic alterations common to the tumor of origin
(12-14). In particular, TECs exhibit chromosomal
translocations in B-cell lymphomas ( 13), the BCR/ ABL
fusion gene in leukemias ( 12), and the myeloma specific
13q14 chromosomal deletion (14). Moreover, in
tumors of the nervous system, TECs present the same
genetic amplification or chromosomal aberrations of
the tumor of origin (15, 16). In human xenografts of
renal carcinoma, melanoma, and liposarcoma, murine
TECs are aneuploid, bearing alterations similar to
those obse1ved in human TECs (9). This observation
remains unexplained. It cannot be ascribed to cell
fusion among tumor and endothelial cells, as no human
DNA was present in murine TECs. The researchers
speculated that the tumor microenvironment may produce
factors capable of inducing genetic instability, or
loss of tumor suppressors and/or check point activity,
resulting in aneuploidy ( I 0). Altogether, these data
suggest two different explanations for the origin of
TECs. The first is that they originate from a common
ALTERNATIVE STRA HGIES FOR TUMOR VASCULARIZATION
progenitor of tumor and endothelial cells targeted by
the neoplastic transformation; the second is tl1at the
effect of tl1e tumor microenvironment leads to genetic
instability.
Expression of TEC specific and embryonic genes
Serial analysis of gene expression showed that TECs
express genes not shared by normal tissue blood vessels
( 17). These genes have been named tum or endothelial
markers (TEMs). St Croix et al. (18) first isolated
endothelial cells from colon carcinoma and demonstrated
tl1at they express genes distinct from normal
colonic mucosa) endothelium. Subsequent studies allowed
the identification of tumor-specific sets of up-regulated
genes on TECs freshly isolated from primary
human breast, glioma, colon, and ovai;an carcinomas
in comparison with the nonnal tissue ( 19-23). TEMs
were shown to be relatively specific for the tumor of
origin and to display a prognostic value (24). However,
TEMs were found on otl1er angiogenic endothelial
cells, such as those of placenta and corpus luteum, and
finally, only TEM8 was found to be restricted to tumor
vessels (25).
The expression of embryonic markers is a general
characteristic of tumor cells. However, embryonic
genes are also expressed by the tumor-derived endothelial
cells (26, 27). We have demonstrated that TECs
from renal carcinomas share with tl1e tumor the expression
of the embryonic renal transcription factor PAX2
(28). PAX2 contributes to both apoptosis resistance
and proangiogenic properties of TECs. PAX2 induction
in normal endothelial cells conferred a proinvasive,
proangiogenic phenotype similar to that of tumorderived
endothelial cells, suggesting a role for PAX2 as
an oncoangiogene (29). TECs from human renal carcinomas
also expressed the embryonic form of HLA
antigen, HLA-G (26). HLA-G is a marker of embryonic
cytotrophoblast cells expressed by placental vessels and
is involved in feto-matemal tolerance. It has been
suggested that HLA-G may also be involved in the
ability of tumors to escape immune smveillance.
The expression of embryonic markers by TECs may
indicate tliat they 01;ginate from nonnal adult or
malignant stem and progenitor cells differentiated into
endothelial cells. Indeed, TECs were shown to display
stem cell properties through the ability to undergo
osteocytic differentiation, a characteristic feature of
mesenchymal stem cells (30).
Functional alterations
TECs were shown to be resistant to senescence in vitro,
which is typically seen in normal endothelial cells, and
able to proliferate for prolonged periods in culture in
serum-free media (9, 26, 31). These properties might
be related to an autocrine loop involving the production
of growth factors, such as angiopoietin-1 and
vascular endothelial growth factor (VEGF)-D, and the
expression of their angiogenic receptors (26). The
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increased VEGF expression in TECs has recently been
ascribed to the effect of RNA binding Hu antigen
proteins (HuR) on VEGF mRNA with consequent
stabilization (32). Indeed, the presence of HuR RNAstabilizing
proteins in the cell cytoplasm correlates with
tumor cell malignancy and may confer an angiogenic
phenotype on TECs. Moreover, up-regulation of the
epidermal growth factor receptor has been described in
TECs of distinct origins and has been suggested to be
critical for their proliferation (33). A relevant molecular
alterntion in TECs is the constitutive activation of
Akt. Depletion of Akt abrogates resistance to apoptosis
and the proangiogenic phenotype of TECs in vitro and
in viva (34). Moreover, Akt activation reduced thrombospondin-
1 expression in renal TECs (34). As thrombospondin-
1 is a potent physiological inhibitor of angiogenesis,
strategies aimed at inhibiting the PI3K/
Akt/mTOR pathway may restore a normal quiescent
endothelial phenotype in TECs by promoting thrombospondin-
1 syntl1esis. Loss of the endogenous angiogenesis
inhibitor endostatin has been also implicated
by Mangieri et al. (35) in the angiogenic switch of
multiple myeloma.
TECs from different tumors (melanoma, liposarcoma,
glioma, renal, breast, and hepatocellular carcinoma)
were also reported to be more resistant tl1an
nonnal cells to serum starvation and to cytotoxic drugs,
such as vincristine, doxorubicin (26, 36), or temozolomide
(37). In addition, TECs displayed enhanced resistance
to antiangiogenic drugs, such as Sorafenib (a
tyrosine kinase inhibitor of VEGF and PDGF receptors
and c-kit; ref. 38). These data indicate tl1at TEC resistance
is not cancer related but rather a common
characteristic of TECs, which are less sensitive to drugs
per se. Therefore, one of the mechanisms involved in
acqui1ing resistance to antiangiogenic therapies might
depend on the expansion of neovessels with specifically
altered endothelial characteristics (Fig. 1).
ORIGIN OF TECS
The abnormal phenotype and function of TECs
raises the problematic question of their origin. It can
be postulated that TECs derive from normal endothelium
of tumor adjacent vessels, with changes in
their phenotype due to the tumor microenvironment
(Fig. 2). In particular, endothelial cells might undergo
epigenetic alterations by transfer of genetic
material [mRNAs, microRNAs (miRNAs), oncogenes]
mediated by exosomes and microvesicles released
from tumor cells. Moreover, tumors may activate
alternative strategies of vascularization, such as
vasculogenesis (Fig. 2). In this setting, endothelial
cells may derive from differentiation of bone marrowderived
circulating stem cells, tissue-resident normal
stem cells, or tumor stem cells.
PHENOTYPIC GENETIC
ALTERATIONS ALTERATIONS
Embryonic
Phenotype
VR 1-2-3
HLA-G
PAX-2
TEMs
tie-2 VEGF·D
Ang-1
Pro-angiogenic
Phenotype
FUNCTIONAL
ALTERATIONS
Drug resistance
‘-,
Aneuploidy
Tumor specific mutations
1 TPS-1
T Endostatin
Figure 1. Schematic representation of the altered characteristics
of TECs. TECs express an embryonic phenotype, as
shown by the presence of embryonic HLA (HLA-G) and
PAX2 transcription factor and genetic alterations including
aneuploidy and tumor-specific mutations. TECs overexpress
proangiogenic growth factors and receptors, suggesting an
autocrine loop of maintenance of an activated phenotype.
The functional alterations are sustained by a constitutive
enhanced Pl3K/ Akt pathway and a down-regulation of antiangiogenic
factors, such as thrombospondin-1 (TSP-1) and
endostatin, and determine resistance to chemotherapeutic
and antiangiogenic drugs.
Reprogramming of normal endothelial cells by
transfer of genetic material mediated by exosomes
and microvesicles
Microvesicles include a heterogeneous population of
vesicles released as exosomes from the endosomal
compartment or as shedding vesicles from the cell
surface (39). Due to their heterogeneity in the biological
fluids, we refer to these structures collectively as
microvesicles (MVs). MVs are actively released from
tumor cells during their growth and spreading. It is
now recognized that MVs constitute an integral part of
the intercellular microenvironment, a cting as vehicle
for information transfer. In particular, MVs may reprogram
target cells by transferring various bioactive molecules,
including membrane receptors, proteins, bioactive
lipids, and R As. Epigenetic changes induced by
MVs depend on tl1e delivery of specific subsets of
mRNA and miRNAs. The potential for MVs to reprogram
recipient cells was first established by Ratajczak
el al. ( 40). Several subsequent studies indicate that
mRNA delivered by MVs can be translated into the
corresponding proteins by the target cells ( 41, 42).
Moreover, MVs contain selected patterns of miRNA
that can be transferred to target cells, inducing epigenetic
changes in bystander cells ( 43, 44). Recent studies
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Origin of Tumor Endothelial Cells
[ TUMOR MICROENVIRONMEN~ I STEM/PROGENITORc EL;–]
~DOTH ELAI LD IFFERENTIATI~
Epigenetic Reprogramming
EPC
Uptake of
Microvesicles
TUMOR
STEM CELL
r.
Bone Marrow-<lerlved
STEM CELL
Figure 2. Possible origin of TECs. TECs may
de1;ve from normal endothelial cells undergoing
epigenetic 1-eprogramming within the tumor
microenvi1-onment, either by uptake of
mRNA and miRNA 1-eleased from tumor cells or
EPCs or by uptake of apoptotic bodies with
transfer of oncogenes. Alternatively, TECs may
originate from tissue resident or bone marrowde,;
ved stem cells undergoing endothelial differentiation,
or from tumor stem cells that can
produce both tumor cells and TECs. These
mechanisms suggest a contribution of vasculogenesis
to neoformed blood vessels in tumors.
Hypoxia, irradiation and antiangiogenic drugs
may favor the shift from nom1al angiogenesis to
an intratumor embryonic-like vasculogenesis. Tumor Endothelial Cells
hypoxia, irradiation, antl-angiogenic drugs
angiogenesis ————– vasculogenesis
suggest that tumor-derived MVs may cooperate to establish
a favorable tumor niche and to promote tumor
growth, invasiveness and progression ( 45-48).
Transfer of rnRNA and rniRNA err lransf er of turnor genes
Ratajczak et al. ( 49) demonstrated that embryonic stem
cell-derived MVs can reprogram hematopoietic progenitors
through horizontal transfer of mRNA and protein
delivery. Characterization of glioblastoma-derived MVs
showed that they contain a variety of mRNA and
miRNA transcripts related to cell migration, angiogenesis,
and proliferation. These MVs were taken up by
endothelial cells within the tumor ( 45). Moreover,
bioinformatic analysis of MVs shed by co lo rectal cancer
cells revealed that they contain cell cycle-related mRNAs
involved in the activation of endothelial cell proliferation
(50). We found that endothelial progenitor cell
(EPC)-derived MVs activate an angiogenic program in
nonnal quiescent endothelial cells (41). EPC-derived
MVs are incorporated in normal endothelial cells
through interaction with o.4- and [31-integrins expressed
on the MV surface ( 41). The interaction of MVs
with endothelial cells promoted survival, proliferation,
and organization into capillary-like structures in vitro,
and stimulation of their in viva organization into patent
vessels. This angiogenic effect correlated with transfer
of mRNA by MVs. Microarray analysis of MV mRNA
revealed that MVs transfer specific subsets of cellular
mRNA, such as mRNA associated with the PI3K/ AKT
and endothelial nitric oxide synthase (eNOS) signaling
pathway. Indeed, protein expression and functional
studies demonstrated that PI3K and eNOS play a critical
role in the angiogenic effect of MVs ( 41). Within
the tumor environment, cell damage induced by hyp-
ALTERNATIVE STRATEGIES FOR TUMOR VASCULARIZATION
oxia, chemotherapy, and irradiation may induce MV
secretion by tumor cells and promote angiogenic pathways
(47). Recently, Balaj et al. (51) demonstrated that
tumor-derived MVs contain retrotransposon elements
and amplified oncogene sequences. This observation
expands the repertoire of genetic infom1ation that can
be transferred by tumor MVs to endothelial cells. At
variance of MVs that are actively released by growing or
activated tumor cells, apoptotic bodies are released as a
consequence of tumor cell apoptosis, which may occur
after chemotherapy. Endothelial cell uptake of apoptotic
bodies derived from tumor cells may also be involved
in transfer of oncogenes. This mechanism described
for stromal cells may be involved in the
tumorigenic properties of stromal cells derived from
epithelial tumors (52, 53). It has been shown by BajKryworzeka
et al. (54) that tumor-derived MVs may
transfer tumor genes to bystander cells, such as monocytes.
Therefore, it is possible that a similar mechanism
is involved in transferring genes from tumor cells to
normal endothelial cells present in tumors, thus triggering
an angiogenic program.
Transfer of angiogenic proteins and oncogene products
Tumor-derived MVs may stimulate tumor growth and
metastasis by promoting endothelial cell migration,
invasion, and in viva neovascularization through transfer
of angiogenic proteins or oncogene products. For
instance, transfer of CDl47E by MVs shed by ovarian
cancer cells activates endothelial cells and stimulates
angiogenesis (55). The treatment of ovarian cancer
cells with small interfering RNA against CD147 reduces
the angiogenic potential of MVs, suggesting that the
proangiogenic properties of ovarian cancer cell MVs is
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dependent on a CD147-mediated mechanism (55).
Other studies have suggested that MY-induced endothelial
cell migration requires expression of sphingomyelin
(56) or tetraspanin by MYs (57). Tetraspanin is
a constitutive component of exosomes, which is enriched
in tumor MYs. In particular, tetraspanin 8
contributes to selective recruitment of proteins and
mRNA into exosomes and has been suggested to play a
central role in endothelial cell activation and angiogenesis
(57), and in the premetastatic niche (58).
Al-Nedawi et al. (59, 60) suggested that the angiogenic
switch in tumors may be induced by MY-mediated
transfer of the oncogenic EGFR. Glioma cell-derived
MYs contain a truncated and oncogenetic fonn of the
EGFR (EGFRvIII) that activates MAPK and AKT pathways
in normal endothelial cells via autocrine synthesis
ofVEGF. Moreover, tumor-derived MYs may propagate
angiogenic activation of quiescent endothelial cells by
transferring DII4 Notch ligand, with a consequent
inhibition of Notch signaling (61).
Role of bone marrow-derived or tissue-resident stem
cells in tumor vasculogenesis
Several studies indicate a contribution from bone marrow-
derived cells in tumor vascularization. Large numbers
of different cell populations have been implicated,
including classical EPCs and myelo-monocytic cells,
such as Tie-2+ monocytes, CD 11 b + myeloid cells,
VEGFRI + hemangiocytes, CD45+ /VE-cadherin + vascular
leukocytes, and tumor associated-macrophages (for
review, see ref. 62). The relative contribution of the
different populations, as well as their effective participation
in vasculogenesis, is still debated. Some studies
indicate a perivascular localization of bone marrowderived
cells without integration into those of the
tumor vessels, suggesting a paracrine action on resident
endothelial cells (63).
Other studies both in humans and in experimental
models suggest that bone marrow-derived cells, mainly
EPCs, undergo incorporation and differen tiation in
vessel-like structures ( 64-66). This finding is supported
by a recent report of a human renal carcinoma developed
in a kidney allograft of a sex-cross-matched patient,
showing presence ofY-chromosome-positive cells
in vessels of a Y-chromosome-negative tumor (67). Of
interest, it has been reported that the ischemic injury
due to tumor irradiation induces recruitment of bone
marrow-derived cells, primarily myelomonocytes, to the
tumor, restoring the irradiation-damaged vasculature
by a vasculogenic process (68). Moreover, it has been
suggested that epithelial-to-mesenchymal transition of
the tumor might induce differentiation of leukocytes
into endothelial-like cells through the effect ofpleiotrophin
(69).
Another possible explanation for the expression of
stem cell markers and an immature phenotype by
TECs is their origin from normal stem and progenitor
cells resident in tissue. In renal carcinomas, a
population of CD34-/CD133+ resident progenitor
cells similar to that described in normal renal tissue
is present. This CD 133+ population does not initiate
tumors, but when coinjected with turnor cells differentiates
into endothelial cells, promoting angiogenesis
and turnor development (70). Therefore, resident
stern cells and turnor cells may reciprocally
influence their behavior. The tumor rnicroenvironment
may favor activation of vasculogenic properties
in tissue resident stern cells.
Role of cancer stem cells in tumor vasculogenesis
Emerging evidence suggests that the capacity of a
turnor to grow and propagate resides in a small population
of tumor low-proliferating cells, termed cancer
stern cells, or turnor-initiating cells (71). These sternlike
cells are identified by their ability to give rise to
new, serially transplanta.ble turnors when xenografted
in small numbers into immunodeficient mice, and to
display stern or progenitor cell properties. In particular,
they are characterized by self-renewal and the capacity
to reestablish tumors that recapitulate the turnor of
origin. Thanks to their ability to undergo asymmetric
division, turnor stem cells may generate progeny
that can differentiate to produce turnor cells with
heterogeneous phenotypes (72). Moreover, it has been
shown that melanoma-derived stern cells can differentiate
in vitro into multiple mesenchymal lineages, such
as adipocytic, osteocytic and chondrocytic lineages
(73). This pluripotency may include the potential to
generate TECs. Indeed, the differentiation of cancer
stern cells into endothelial cells and the consequent
involvement in tumor vascularization has been recently
described in different turnors. Shen et al. (74) demonstrated
that precancer stem cells from leukernias can
differentiate into endothelial cells and contribute to
turnor vasculogenesis. In breast carcinomas, we demonstrated
that CD44+ /CD24- stern/progenitor cells cultured
as marnrnospheres differentiate not only into
epithelial cells, but also into endothelial cells, both in
vitro and in viva in transplanted tumors originating
from turn or stem cells (75). This capability is also
present in tumor stem cells derived from human renal
carcinomas (76), as well as from ovarian cancer, and is
absent in differentiated mature turnor cells (77). In
human renal cell carcinomas, a subset of turnor-initiating
cells expressing the mesenchymal stem cell marker
CD I 05 was shown to display stem cell properties, such
as clonogenic ability, expression of Nestin, Nanog,
Oct3-4 stem cell markers, and lack of differentiative
epithelial markers (76). This CD 105 + population has
the capacity to generate epithelial and endothelial cells
and serially transplantable tumors in viva (76). The
definitive proof that turnor stern cells are bipotent
relies on the ability of clones of tumor stem cells to
differentiate in vitro and in viva into both tumor epithelial
and endothelial cells (75, 76). More recently, the
ability to differentiate into endothelial cells has also
been reported for cancer stern cells present in neuroblastomas
(16, 78). In particular, only a fraction of stem
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cells, characterized by CD 133 and CD 144 coexpression
(78), or in a recent report, by Oct4 and tenascin C
coexpression (79), shows vasculogenic potential and is
selectively localized in proximity of tumor vessels (79).
The functional relevance of tumor vasculogenesis can
be inferred by xenograft experiments using injection of
Tie-2-thymidine kinase transduced glioblastoma neurospheres,
yielding tumors with thymidine kinase expressing
endothelial cells that could be killed by ganciclovir
administration. Interestingly, this strategy of targeting
of tumor-derived endothelium induced tumor reduction
and degeneration, suggesting a relevant role of
tum or stem cell-derived vasculogenesis ( 16).
Regarding the mechanisms involved in endothelial
differentiation of cancer stem cells, both VEGF-dependent
and independent mechanisms have been reported.
In particular, Wang et al. (78) showed that
VEGF blockade inhibits maturation but not differentiation
of cancer stem cells into endothelial cells, and
this, in turn, is blocked by NOTCHl inhibition. In
addition, Shen et al. (74) demonstrated a role for
hypoxia in the endothelial differentiation of leukemic
precancerous cells. We have also found that hypoxia
promotes endothelial differentiation of breast cancer
stem cells independently of VEGF (unpublished results).
This concept is of particular relevance in tumor
antiangiogenic therapy, as therapy-induced hypoxia
may switch the tumor vascularization from normal to
tumorigenic.
An alternative mechanism of tumor blood perfusion
implies the possibility that tumor cells form
channels connected to the tumor vasculature. This
finding was refered to as “vasculogenic mimicry” (80,
81). Since these structures are lined by cells lacking
endothelial phenotype and markers, it has been
suggested that tumor cells themselves organize in
channels. Alternatively, the process of tumor vasculogenic
mimicry could be interpreted as dependent
on tumor stem cells (82, 83), as a transitory step in
stem cell differentiation toward endothelial cells.
Indeed, the ability of aggressive tumor cells to express
vascular cell associated genes (82, 84) and to
organize into tubular structures (80) is explained by
the plasticity of cancer stem cells.
IMPLICATIONS FOR ANTIANGIOGENIC
THERAPY
Since angiogenesis is recognized to promote tumor
development and metastasis, intensive investigation
has been devoted to develop antiangiogenic strategies
for treatment of cancer. Most of these studies
were based on the consideration that an ideal antiangiogenic
drug may target different types of tumor,
assuming endothelial cells to be similar in different
tumor types and genetically stable. However, the
therapeutic efficacy of antiangiogenic drugs was not
successful as expected (85), and endothelial cells
acquired drug resistance (86). This setback is possi-
ALTERNATIVE STRATEGIES FOR TUMOR VASCULARIZATION
bly due to the fact that most antiangiogenic drugs
were tested on normal endothelial cells. It is now
clear that endothelial cells among different tumor
types are heterogeneous and that TECs substantially
differ from normal endothelial cells in gene profile
and behavior, as reviewed in this article. Furthermore,
TECs contradict the assumption that TECs are
genetically stable, as a number of genetic abnormalities
have been described ( I 0).
In light of the involvement of angiogenesis and
va sculogenesis in tumor vascularization, it can be
speculated that tumor cytotoxic therapies, radiotherapy,
and antiangiogenic drugs, by inducing tumor
hypoxia and/or epithelial mesenchymal transition,
may stimulate vasculogenesis. The involvement of
vasculogenesis following radiotherapy has been recently
reported in gliomas (68). Moreover, as tum or
hypoxia may promote cancer stem cell vasculogenesis,
it may enhance the number of endothelial cells
with a malignant phenotype, so rendering the vascular
network of the tumor progressively insensitive to
antiangiogenic therapy (Fig. 2). This might explain
the demonstration of increased metastasis in experimental
models of antiangiogenic tumor treatment
(87, 88). Similar results were obtained in the clinical
setting. After an initial inhibition of tumor growth
and prolongation of progression-free survival, VEGFtargeted
therapy has been associated with tumor
relapse and with more invasive metastatic disease
(89). Therefore, targeting angiogenesis and vasculogenesis
in tumors may be required to inhibit its
vascularization, growth, and invasion. In particular,
an improved knowledge of the relative contribution
of vasculogenesis to tumor vascularization is likely to
be critical for development of specific therapeutic
strategies. Studies aimed at interfering with endothelial
cell reprogramming, or with differentiation from
normal or tumor stem/precursor cells may represent
a viable approach to this issue. [!ii
The authors thank Dr·. Justin Mason (Imper·ial College,
London, UK) for the helpful suggestions and Danilo Bozzetto
for· art graphics. This study was supported by Associazione
Italiana per la Ricerca sul Cancro (AIRC), project IG8912; by
the Italian Ministry of University and Research (MIUR),
Prin08; and by Regione Piemonte, Nano-IgT, and Piattaforme
Biotecnologiche, Pi-Stem project.
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    Received Jar publication March 30, 2011.
    Accepted for publication May 19, 2011.
    2882 Vol. 25 September 2011 The FASEB Journal· www.fasebj.org BUSSOLA TI ET AL.