CELLULAR PLAYERS AND MECHANISMS IN TUMOR VASCULARIZATION

Endothelial Sprouting and Pericyte Coverage
Tumor blood vessels form and develop with tumors by several
mechanisms, the most studied of which is endothelial sprouting,
whereby new capillaries bud from nearby existing ones.
Sprouting can proceed through a phase of a vascular network
that is superfluous and undergoes pruning, or it can be guided
to hypoperfused regions as it is created, mostly by VEGF gradients.
37 The phases of endothelial sprouting have been carefully
described. 38 The basement membrane of postcapillary venules
is degraded at the location of the future endothelial sprout.
Through the resultant opening in the basement membrane, endothelial
cells migrate and form a cord of cells; this is followed
by the appearance of a lumen. According to another report, the
sprouting vessel sustains a lumen and continuous intracellular
junctions as it is produced, rather than going through a stage of
dedifferentiated cord of cells as commonly thought. 39
Endothelial cell migration is a major process in angiogenesis.
It is regulated by chemotaxis toward VEGF, basic fibroblast
growth factor (bFGF), and other angiogenic factors. It is also
controlled by haptotaxis, the migration toward a gradient of
immobilized ligands, which is dependent on integrin-ECM interactions.
Mechanotaxis is the sensing of sheer stress of blood
flow by cytoskeletal elements and migration in its direction and
is another mechanism that controls endothelial migration. 40,41
The last phase of the angiogenesis process is the recruitment
of pericytes and deposition of new basement membrane. 42
Pericytes of the parent blood vessel proliferate and migrate to
envelop the new vessel. 39 The platelet-derived growth factor
(PDGF) pathway is the major regulator of pericyte recruitment
and maintenance. PDGF-B is secreted mostly by endothelial
cells. Acting on PDGF receptor- (PDGFR- ) on pericytes,
it facilitates their recruitment to new blood vessels. Pericytes,
in turn, contribute to the stability and functionality of blood
vessels, partly through the secretion of VEGF. 43 Importantly,
tumor blood vessels that lack pericyte coverage are the first to
regress after VEGF pathway inhibition. 44 Tumor blood vessels
contain more than one subtype of pericytes, which vary in
their molecular marker expression and dependence on PDGF
signaling for tight adhesion to endothelial cells. 45
The origin of tumor vascular pericytes is thought to be
mesenchymal progenitor cells, 43 which are characterized by
Tie2 expression, 46 or bone marrow–derived hematopoietic
stem cells. 47 Some models indicate that endothelial cells and
pericytes share a common angioblast progenitor cell. 48 The
differences between vascular smooth muscle cells and pericytes
are not clear, suggesting that they are similar cell types in different
phases of phenotypic change. 3 Regardless of the origin
of these cells, the clinical activity of PDGFR inhibition in the
treatment of cancer indicates that pericytes are important in
the maturation and modulation of tumor angiogenesis 43 (see
Fig. 8.1 for a schematic representation of the major cell types
and niches that modulate tumor angiogenesis).
Vasculogenesis: Cells from the Bone Marrow
Vasculogenesis is the de novo formation of blood vessels from
vascular progenitor cells. Circulating bone marrow–originating
cells travel to specific foci and undergo in situ differentiation
to form mature components of blood vessels. Vasculogenesis
was initially thought to occur only in embryonic tissues, but
it has been found to occur in adults as well. Bone marrow–
derived endothelial progenitor cells (EPC) were found to be
mobilized (thus becoming CEPs) by GM-CSFs or ischemia in
experimental animals and travel to areas of ischemia. 49 Studies
in which mice underwent bone marrow transplantation from
mice that expressed a unique marker demonstrated that bone
marrow–derived cells contribute directly to blood vessel formation.
49,50 VEGFR-2 is critical for vasculogenesis in embryos 51
and adult tissues. 52 In addition, increased expression of stromal
cell– derived factor-1 (SDF-1, also called CXCL12)-in peripheral
blood and ischemic tissues, in parallel to reduced SDF-1/
CXCL12 expression in bone marrow, may enhance the recruitment
of CEPs to ischemic tissues. SDF-1/CXCL12 enhanced
the number of EPC in ischemic vessels by promoting their adhesion
through 2, 4, and 5 integrins to fibronectin and
collagen I. 53 CEPs have also been found in patients, in numbers
that were correlated with plasma levels of VEGF 165. 54
CEPs were also shown to contribute to the formation of
blood vessels in cancer. Id knockout mice display defective
angiogenesis, not allowing them to support the growth of implanted
tumors. 55 This phenotype was saved by transplantation
with wild-type bone marrow. Donor-derived, VEGFR-2-
positive CEPs formed most of the tumor blood vessels in this
model. Donor-derived, VEGFR-1-positive myeloid precursors
were also recruited to the tumors, where they were thought to
have secreted angiogenic cytokines. 52 The role of inflammatory
cells in angiogenesis will be discussed later. The expression of the
transcription factor Id1 in CEPs was essential for their contribution
to lung metastasis in another mouse model. 56 Systemic
17- estradiol administration contributed to the recruitment
of CEPs to tumors in a mouse model of breast cancer. 57 Blood
counts of CEPs may be a promising surrogate marker for angiogenesis
or vasculogenesis, possibly useful in the real-time assessment
of the efficiency of treatment targeting tumor blood
vessels. 24 For example, vascular-disrupting agents induced a
surge in the CEP blood concentration in a mouse cancer model.
Treatment with anti-VEGFR-2 antibody disrupted this surge
and augmented the efficacy of cancer eradication. Blocking the
CEP surge prevented the regrowth of tumor from the rim of
viable cells that typically remain when most of the tumor necrotizes.
58 Cancer-associated fibroblasts (CAFs), a prominent
component of the stromal reaction to cancer, contributed to
the recruitment of CEPs. 59 SDF-1/CXCL12 release by CAFs
is critical to this recruitment. 59 CEPs were found in the blood
of cancer patients and were demonstrated to respond to effective
systemic therapy. 60 Therefore, bone marrow–derived EPCs
may be one of the important manners by which tumors develop
their vascular supply.
The importance of EPCs is controversial, spurring disputes
in the scientific literature. 61,62 Estimations of their contributions
to tumor endothelium vary from significant (10% to 50%) to
negligible. 24,63 Studies finding no evidence of EPC contribution
to tumor endothelium were also reported. 61 A plausible
explanation for this variability was suggested when timedependent
changes in EPC contribution were evaluated. Using
high-resolution microscopy, aided by flow cytometry, bone
marrow–originating cells can be located in inoculated tumors
in mice that have undergone bone marrow transplantations
of GFP-positive cells. In this model, the proportion of EPCs
among endothelial cells was about 30% in the first 4 to 6 days of
tumor growth but dropped to less than 1% after 4 weeks. This
study also demonstrated bone marrow–derived cells close to endothelial
cells, suggesting that they are a source of EPC overestimation
in tumor vessels. 64 A study of cancers that developed in
bone marrow transplant recipients that are gender mismatched
with their donor revealed that about 5% of their tumor endothelial
cells are donor-originated.63 Importantly, almost all of the
CEC that had a significant proliferative capacity, were donororiginated.
65 This study suggests that even very low numbers of
EPC might contribute significantly to tumor vasculature.
The evaluation of CEPs in blood samples of cancer
patients is hampered by the low numbers of these cells in
circulation and the technical difficulties of their positive identification.
Identification methods include enrichment by cell
sorting and immunomagnetic beads. However, these methods
depend on specific surface markers, which are lacking. For example,
CD146 may be a specific marker of CEPs or CECs and
may be measurable in the serum of cancer patients. CD146
mRNA levels in serum were correlated with CECs in breast
cancer patients. 66 However, a fluorescence-activated cell sorting
analysis of blood mononuclear cells demonstrated that
CD146 was expressed mainly on a subpopulation of T cells. 67
Recently, tumor endothelial marker 1/endosialin/CD248 was
reported to be highly expressed in CEPs, suggesting a new
method of measuring or potentially eradicating blood CEPs. 68
An important property of CEPs that may be useful in their
identification is their ability to proliferate, but this would not
differentiate CEPs from hematopoietic progenitor cells. The
technical difficulties of detecting a minute subpopulation of
cells in the blood have not been satisfactorily solved. 24 The
results of studies of CEPs and CECs in the blood of cancer
patients must be interpreted cautiously.
Alternative Mechanisms of Enhanced Vascularization
In vessel co-option, tumors or metastatic foci develop
along existing blood vessels. In this way, tumors become vascularized
with no need for blood vessel formation. 69 Vessel co-option
occurs in the initial growth phase of tumors. As cancer cells proliferate,
the tumor outgrows its blood supply. The co-opted host
blood vessels then undergo regression, possibly as a host defense
mechanism. The endothelial cells of these vessels are detached
from their supporting cells, at which point they undergo apoptosis.
Angiopoietin 2 (Ang-2) expression is induced in the co-opted
vessels prior to their regression. Ang-2 is involved in physiologic
vessel remodeling in a VEGF-dependent manner (see succeeding
discussion). Co-option of existing vessels may be an important
method for obtaining a vascular supply in early tumors. Its extent
is controlled by the local production of VEGF, Ang-1, and Ang-2.
Intussusceptive microvascular growth, the longitudinal
separation of existing vessels into daughter vessels, is another
mechanism of enhancing blood supply to tumors. In this way,
the network’s complexity and efficiency improves, with no
need for endothelial cell proliferation.
Vasculogenic mimicry, which has been observed mostly
in melanomas, is the ability of cancer cells to transform into
endothelial-like cells in specific sites, thus forming blood vessels
made of cancer cells. 38 The importance of these alternative
mechanisms of vascular supply in cancer growth is not known.
Role of Immune System Cells in Angiogenesis
The stroma of cancer is infiltrated by immune system cells in
varying proportions. The role of the immune system in the
progression of cancer is not obvious but seems to be context
dependent. The immune system has a cancer-inhibitory effect,
as evidenced by the high risk of cancer in immunocompromised
patients. In addition, a correlation was found between
increased effector memory T-cell infiltration of the tumor
and a good prognosis in colon cancer patients. 70 However,
in many other settings, the immune system apparently contributes
to cancer progression. The adaptive immune system
can mount an antitumor response in some conditions. In contrast,
innate immune system cells may be more commonly recruited
by cancer cells and function in a pro-cancer manner. 71
Neutrophilic infiltration of tumors is common, but its clinical
significance is not clear. In bronchoalveolar carcinoma, it has
been associated with a poor prognosis, 72 but data on its importance
in other cancers are limited. Lung cancer– infiltrating
lymphocytes exert an anticancerous effect, as in colon cancer.
High densities of CD4 and CD8 lymphocytes in the
stroma of NSCLC tumors have been found to be associated
with a good prognosis. 73,74
Monocytes circulate in the blood; once recruited to sites
of tissue inflammation, they differentiate into macrophages.
Various chemoattractants play a role in the chemotaxis of
monocytes into tumors, possibly mostly to hypoxic areas of
tumors. 75 Macrophages constitute a major subset of the immune
system cells that populate the tumor stroma: tumorassociated
macrophages (TAMs). TAMs seem to promote the
progression of cancer. 76 Unlike classically activated macrophages
(M1 macrophages), TAMs have a poor antigen-presenting ability
and produce factors that suppress T-cell proliferation and activity.
The chemokines and chemokine receptors profile they express is
adapted for scavenging for debris, promoting cell migration and
angiogenesis, and repairing and remodeling wounded or damaged
tissues. Cancer microenvironment exposure to interleukin
(IL)-4 and IL-10 can induce monocytes to develop into TAMs
(otherwise known as polarized type II [ alternatively activated] or
M2 macrophages). 77 Proangiogenic monocytes that localize in
tumors are also characterized as Tie-2 expressors. 46 Macrophage
infiltration was found to be associated with vessel density in several
types of cancer. In a mouse model of breast cancer, depletion
of macrophages inhibited the angiogenic switch and cancer progression.
Increased macrophage infiltration was correlated with
earlier tumor progression. 78
IL-1 is a proangiogenic cytokine that depends on macrophage
recruitment to tumor sites for its angiogenic effect. 79
MMP9 release by macrophages, leading to mobilization of
VEGF, 80 is a mechanism in which the infiltration of macrophages
or other myelomonocytes 81 induces angiogenesis. In addition,
IL-8/CXCL8, another proangiogenic factor, was upregulated
in both cancer cells and macrophages when these two cell types
were cultured together, and IL-8/CXCL8 mRNA levels in lung
cancer specimens were correlated with MVD and poor patient
prognosis. 82 Relating an angiogenic response to inflammation,
which is common in cancer, IL-1 was shown to recruit VEGFexpressing
inflammatory cells. VEGFR-2 blockage prevented
this angiogenic response. 83 Regardless of the available data on
the contribution of macrophages to angiogenesis, tumor islet infiltration
by macrophages was a good prognostic factor in a study
of 175 NSCLC patients. On the other hand, stromal macrophage
infiltration was associated with a poor prognosis. 84 These
results were reproduced in a study of 199 NSCLC patients. 85
Importantly, the studies that showed a positive prognostic effect
of macrophages in lung tumors differed from earlier studies by
differentiating between stromal and tumoral macrophages.
Natural killer (NK) cells are another part of the innate
immune system that have important interactions with cancer
and cancer-induced angiogenesis. NK cells recognize and
lyse cancer cells and are thought to have important roles in
immune surveillance against cancer. IL-12 is an antiangiogenic
agent that depends on NK cell recruitment to affect cancer
angiogenesis. 86 NK cells secrete interferon- , causing inhibition
of endothelial cell proliferation; this is probably the major
mechanism of NK cells’ antiangiogenic effects. 87
Mast cells were found to be essential for tumor progression
in a mouse model of squamous cell carcinoma and were required
for neoangiogenesis. 88 Mast cell infiltration was correlated
with MVD in a study of NSCLC specimens. 89 However, mast
cell tumor infiltration was associated with a good prognosis
in NSCLC specimens. 84 More detailed studies of the role of
mast cells in lung cancer are required. In light of the inhibitory
effect of most anticancer treatments on the immune system,
further insight is required into the effect of immune system
cells on angiogenesis and cancer progression.
Tumor Stroma–Dependent Effects The stroma of
tumors is more than a mechanical scaffold; stromal cells seem
to be reprogrammed by cancer cells to participate in cancer
progression. A mouse model demonstrated activation of the
VEGF gene promoter in tumor stroma fibroblasts. 90 CAFs
also contribute to cancer progression, 91 apparently by activating
angiogenesis. Activation of hepatocyte growth factor
(HGF)–c-Met signaling is another role of the stroma in tumor
angiogenesis (see succeeding discussion). 92
MMPs are a family of Zn2 proteases that are produced
mostly by stromal fibroblasts and by cancer and endothelial
cells. These proteases have important roles in angiogenesis.
MMP2 and MMP9, for example, mediate the breakdown of
collagen type IV, a major component of the vascular basement
membrane. The mobilization of growth factors, including
VEGF 80 and additional angiogenic molecules from the ECM,
is another angiogenic activity of MMP. This mobilization may
be essential in the initial stages of cancer, becoming less important
as the tumor progresses and alternative sources of VEGF
become available. MMP9 was also shown to be required for
the recruitment of bone marrow–derived cells into the tumor
microenvironment, for the maturation of tumor vasculature,
and for pericyte coverage. 93 In contrast, at later stages of tumor
growth and MMP activity, the dominant end products of
MMP protein cleavage are antiangiogenic factors. 94 Although
MMPs are correlated with angiogenic activity in lung cancer,
general MMPs inhibition did not improve the clinical outcome
of NSCLC patients. 95 Modification of specific MMP(s)
might be required for impacting clinical end points.
Vascular Basement Membrane The ECM that envelops
endothelial cells, and within which pericytes are embedded,
is called the vascular basement membrane . The major collagen
that constitutes the basement membrane is collagen IV, which
has the unique ability to self-assemble into sheets. Additional
components are laminins, which bind cell membrane anchors
such as integrins on one side and ECM collagen on the other
side. The mature basement membrane signals differentiation
and reduced proliferation to adjacent endothelial cells. The
same protein constituents, while being deposited as a new basement
membrane, present different molecular moieties to the
cells around them. Through integrins, they provide proliferation
and migration signals. The ECM structural components
also include molecular messengers, such as endostatin, that can
be proteolytically released from collagen XVIII and functions
as an antiangiogenic effector. Arrestin, canstatin, and tumstatin
are also collagen-derived antiangiogenic molecules. On the
other hand, triple-helix fragments of collagen IV activate endothelial
cell migration. Therefore, specific molecules in the
ECM/basement membrane can convey different messages at
different phases of tumor growth. Deposited by the resident
cells, the ECM/basement membrane is an important manner
of cell–cell indirect communication. This adds another level of
complexity to the cellular events occurring in the process of
new blood vessel formation.