Magnetic resonance (MR) provides a unique combination of molecular and functional dynamic imaging with rational targeted therapy, offering broad clinical possibilities for the future. MR is particularly well suited for imaging various aspects of angiogenesis that may be adapted for the evaluation, monitoring, and treatment of an ever-expanding list of human disease. This review will focus on some of the current and emerging MR techniques for imaging angiogenesis that were presented at the 12th Scientific Meeting and Exhibition of the International Society for Magnetic Resonance in Medicine held in Kyoto, Japan, on May 15 to 21, 2004, and how they may impact on clinical practice and our understanding of the molecular pathophysiology of tumor angiogenesis.
is currently a third-year Resident in Diagnostic Radiology at
Thomas Jefferson University Hospital, Philadelphia, PA. She
received a BA in Biology and Chemistry from Transylvania
University, Lexington, KY, in 1993. She received her MD-PhD
degree in Molecular Genetics from Thomas Jefferson University,
Philadelphia, PA in 2000. She plans to complete a combined MRI
Fellowship in Body and Musculoskeletal Imaging at Thomas
Jefferson University, Philadelphia, PA and to continue her
research projects in cancer molecular imaging and image-guided
The strength of magnetic resonance (MR) in imaging the various
complex stages of angiogenesis is that it is a noninvasive method
that allows repetitive in vivo analyses to measure functional
parameters of the vasculature and/or adjacent tissue of interest
that may be combined with dynamic molecular information. In
addition, MR enables these analyses without the potentially harmful
effects of radiation that are unavoidable with CT and nuclear
medicine protocols. Unlike optical methods that have been used
extensively in the laboratory with genetically altered animal
models (such as transgenic and knockout mice) to decipher the
phenotypic consequences of various molecular pathways, MR is not
hampered by poor tissue penetration. MR also does not suffer from
the poor resolving capacity of nuclear medicine schemes and
maintains a relatively high spatial resolution for both superficial
and deep structures.
The challenge with MR imaging (MRI) lies within the relatively
low sensitivity compared with that of other nuclear imaging
modalities, such as positron emission tomography (PET). However,
this lack of aptitude for pure molecular imaging is offset by the
safety as well as the high spatial and temporal resolution of MRI,
allowing visualization of fine anatomic detail and quantification
of a variety of functional parameters to be repeatedly examined
over time. A combination of MRI and other modalities may be the
most synergistic way to integrate the strength of each modality.
Intriguingly, MR, as a single modality, has the most promise to
delivery precise anatomic detail (traditional imaging); to depict
specific biological molecules participating directly in the
regulation of a physiological process (molecular imaging); and to
detect and/or quantify the functional consequences of such
physiological process or a defined stimulus (functional imaging).
Angiogenesis imaging is just the arena to display the great
attributes and potential of MRI in a wide variety of disease
states. This review will give a brief background on the formation
of tumor vascularity and focus on a few of the current MR imaging
modalities for the depiction of angiogenesis that were highlighted
at the recent Scientific Meeting and Exhibition of the
International Society for Magnetic Resonance in Medicine (ISMRM)
held in Kyoto, Japan.
All tissues within the body receive nutrients, red blood cells,
immune cells, and other molecules from blood vessels that also
remove harmful waste products. This exchange of nutrients and waste
material occurs in a hierarchical and spatially organized vascular
network by diffusion and convection requiring cells to be located
within 100 to 200 µm from the blood vessels. A simplistic notion of
vascular development involves formation, stabilization, branching,
remodeling and pruning, and specialization. The nascent vascular
network functioning during embryonic development is formed by
vasculogenesis (de novo vessel formation from angioblasts or stem
cells) and angiogenesis (sprouting, bridging, and intussusceptive
growth from existing vessels).
Angiogenesis is a highly regulated phenomenon in physiologic
conditions and plays a critical role not only during embryonic
development but also in the reproductive cycle, ocular maturation,
and wound healing. Through angiogenesis, the body can meet the
ever-changing demands of the local metabolic and mechanical
microenvironment by altering the maturation and remodeling of the
vasculature. This is accomplished through dynamic response to
abnormal hydrostatic pressure or shear stress, hypoxia, low pH, and
the release of paracrines such as transforming growth factor-beta
(TGF-β& and platelet-derived growth factor (PDGF) from
activated platelets in response to tissue injury.
Deregulated vessel growth and remodeling impacts upon a wide
variety of issues in health and disease (Table 1). Excessive
angiogenesis not only contributes to psoriasis, rheumatoid
arthritis, blindness, and cancer, as historically recognized, but
also includes a growing list of common human illnesses such as
asthma, obesity, atherosclerosis, and certain infectious diseases.
Anomalous vessel regression and inadequate vessel growth is not
only related to myocardial ischemia and stroke but can lead to
hypertension, pre-eclampsia, neurodegeneration, respiratory
distress, osteoporosis, and other debilitating chronic diseases.
This ever-growing list of disease processes linked to altered
angiogenesis underscores the need to develop safe, noninvasive,
dynamic imaging to better understand the regulatory mechanisms
involved in this process and how they can be manipulated in certain
disease states. Although this review will focus mainly on imaging
angiogenesis in tumors, one can easily see the far-reaching
potential of imaging angiogenesis and how these same techniques may
be applied to other pathologic states.
Tumor cells located more than 100 µm away from blood vessels
become hypoxic. If angiogenesis is not activated, tumor clones will
be constrained to a maximum 1 to 1.5 mm diameter.
Clones within hypoxic regions may remain dormant for months to
years before they switch to an angiogenic phenotype.
Vascular co-option is confined only in the tumor periphery;
therefore, gradual tumor expansion results in progressive central
hypoxia. Hypoxia induces the expression of pro-angiogenic factors
through hypoxia-inducible factor-alpha (HIF-α), and if the level of
activators exceeds that of inhibitors the angiogenic balance will
be weighted toward the angiogenic switch, resulting in endothelial
cells leaving quiescence and entering into an activated angiogenic
Tables 2 and 3 give a detailed, although not exhaustive, list of
some of the key molecules involved in promoting and inhibiting
angiogenesis. The most prominent of the hypoxia-induced activating
angiogenic factors are vascular endothelial growth factor (VEGF)
and VEGF receptor-2 (VEGFR-2, also known as Flk-1), which in
conjunction with angiopoietin-2 (ANG2), promote vascular remodeling
The presence of viable hypoxic cells is likely a reflection of the
development of hypoxia tolerance through clonal selection resulting
from modulation of cell death (apoptosis, or programmed cell death)
in the microenvironment. The hypoxic microenvironment selects cells
capable of surviving in the absence of normal oxygen availability.
This is supported by the selection of cancer cells with TP53
mutations (the wild type TP53 gene is a tumor suppressor gene known
as the "guardian of the genome," which prevents replication of
damaged/mutated DNA by regulating the cell cycle G2/M checkpoint,
apoptosis, and certain pathways of angiogenesis) for their ability
to withstand hypoxic conditions.
More than 50% of human cancers contain mutations in TP53.
Alternatively, the stable hypoxic microenvironments in solid tumors
may play a positive role in tumor growth by providing angiogenic
and metastatic signals, thus allowing prolonged survival in the
absence of oxygen and generation of a persistent angiogenic signal.
A variety of situations can provoke an unbalanced shift toward
proangiogenic factors, such as metabolic and mechanical stresses,
hypoxia, and genetic mutations or altered expression of oncogenes
or tumor suppressor genes that can stimulate blood vessel growth,
and the exact mechanism behind this is still unknown.
Several sequential steps can be highlighted during tumor
angiogenesis. In mature (quiescent) capillaries, the vessel wall is
composed of an endothelial cell lining, a basement membrane, and a
layer of cells termed pericytes, which are related to vascular
smooth muscle and are adjacent to and partially surrounding the
endothelium. The pericytes share a common basement membrane with
the endothelial cells and occasionally make direct contact with
them through gap-junction connections. Initially, the pre-existing
capillaries or postcapillary venules undergo vasodilatation and
increased vascular permeability in response to elevated levels of
VEGF, allowing extra-vasation of plasma proteins. The plasma
proteins then lay down a provisional matrix for the activated
endothelial cells to migrate. In response to the binding of ANG2 to
a tyrosine kinase receptor selectively expressed in endothelial
cells (TIE2), the pericyte covering is loosened.
The activated endothelial cells secrete proteases, heparinase, and
other digestive enzymes that digest the basement membrane
surrounding the vessel. Degradation of basement membrane and the
local extracellular matrix (ECM) occurs as a mechanism of matrix
metalloproteinases (MMPs), a family of metalloendopeptidases
secreted by the tumor cells and the supporting cells. The
dissolution of extracellular matrix also allows the release of
proangiogenic factors from the matrix.
Cell projections pass through the space created between altered
endothelial cell junctions, and the newly formed sprout grows
toward the source of the stimulus. Alternatively, the cells may
respond to hypoxemic or nutritive stresses by intussusceptive
microvascular growth through the partitioning of the vessel lumen
by tissue pillars and interstitial tissue structures. Activated
endothelial cells invade the matrix and begin to migrate and
proliferate into the tumor mass. In this location, newly formed
endothelial cells organize into hollow tubes (canalization) and
create a new basement membrane for vascular stability. The newly
established fused blood vessels form the blood flow within the
tumor. The formation of the lumen during canalization is driven by
important interactions between the ECM and cell-associated surface
proteins such as hybrid oligosaccharides, platelet endothelial cell
adhesion molecules (PECAM-1, also known as CD31), galectin-2, and
vascular endothelial cadherin (VE-cadherin).
Vascular basement membrane components are involved in the
regulation of tumor angiogenesis.
Vascular maturation occurs with the recruitment of perivascular
cells, making the cells more resistant to VEGF withdrawal.
Figure 1 is a schematic representation of the classical angiogenic
switch involved in solid tumor progression and metastasis.
The hallmark of solid tumors is an abnormal vasculature with a
disorganized ultrastructure, chaotic blood flow, and leaky vessels.
Imbalances between the levels of pro- and antiangiogenic molecules,
which vary throughout the tumor microenvironment, account for the
continuous remodeling of the neovasculature and the variations in
permeability and blood flow between tumors, the primary tumor and
its metastatic deposits and within a given tumor both spatially and
Large caliber tumor vessels may have thin walls usually belonging
to capillaries or an incomplete basement membrane and an unusual
The vessels from which new vessels originate are characterized by
degradation of the basement membrane and decreased number of
pericytes and a thinned endothelial cell lining. The loss of
adherence between endothelial junctions as well as a discontinuous
basement membrane contribute to tumor vessel hyperpermeability.
The induction of vascular permeability is mediated by
vesiculovacuolar organelles, the redistribution of PECAM-1, and
VE-cadherin. Vascular permeability allows the extravasation of
plasma proteins that constitute a momentary scaffold for migrating
endothelial cells, as previously described.
Another common feature in tumor blood vessels is the presence of
focal hemorrhages forming static extravascular blood lakes that
occur spontaneously, mainly if the tumor cells express VEGF
isoforms. The human VEGFA gene generates 4 different isoforms (VEGF
, and VEGF
) from alternative exon splicing of which the predominant isoform
The structural aberrations described so far in tumor vessels are
also coupled to molecular and functional disorders, such as the
overexpression of growth factors, integrins, and the uptake of
The molecular basis of physiologic and pathologic angiogenesis
involves complex signaling pathways interlinking growth factor and
cytokine signaling pathways with proto-oncogenes, tumor suppressor
genes, molecules governing cell-cell and cell-matrix interactions,
cell cycle machinery, and apoptosis. This is further complicated by
the fact that several of the key players have different, almost
paradoxical functions, depending upon the molecular environmental
milieu in which they find themselves. Details of the current
understanding of this process are beyond the scope of this text,
and the reader is referred to several outstanding reviews for
Magnetic resonance angiography
Angiography is the most direct method to visualize the vascular
system in vivo. This is an excellent technique for depicting larger
arteries and veins. To overcome signal-to-noise limitations,
macromolecular intravascular contrast agents with a sustained
vascular enhancement phase (blood pool contrast agents) are
required to acquire the spatial resolution to evaluate the
microvasculature by offering a longer acquisition window.
This allows optimization of spatial resolution and signal-to-noise
ratio (SNR) by decreasing the bandwidth of the pulse sequence,
which increases the total scan time. Conventional MR contrast
agents would not be amenable to the prolonged scan times due to
rapid diffusion into the extravascular compartment, thus severely
compromising vessel-to-background contrast. Examples of such blood
pool agents that have been used for high-resolution MR angiography
(MRA) include high-loaded gadolinium albumin (HAS-[Gd-DTPA]
) with a molecular weight of 92 kDa
and polyamidoamine (PAMAM) dendrimer-based gadolinium (Gd) chelates
with molecular weights ranging from 58 to 467 kDa.
This typically allows a spatial resolution approaching 200 µm in
Spatial resolution can be improved to approximately 10 µm with
microMR; however, the temporal resolution is extremely poor. Such
impressive high-resolution scanning is obtained by using dedicated
high magnetic field animal scanners (>3T), which require
specialized pulse sequences to overcome magnetic susceptibility
artifacts generated from the increasing field strength.
Although extremely useful for research purposes, microMR has
essentially no clinical applications.
A dedicated radiofrequency receiver coil with high sensitivities
in combination with a blood pool contrast agent with high T1
relaxivity will further improve the SNR. Intriguing work has been
generated that takes advantage of such a design using a Gd-based
inter-mediate-sized macromolecular MR blood pool agent, Gadomer-17
(Schering AG, Berlin, Germany) in rodent tumor models.
Gadomer-17 is a polymeric complex with 24 Gd atoms bonded to a
dendritic backbone with a molecular weight of 30 kDa.
High-resolution 3-dimensional (3D) MRA utilizing a clinical 1.5T
MRI scanner allowed clear distinction of intratumoral blood vessels
(Figure 2). This morphologic characterization of the intratumoral
vasculature was obtainable with only minimal hardware and software
modifications, supporting ease of transition into the clinical
realm. The blood vessels displayed by MRA when correlated with
histologic analysis corresponded to vasculature with a diameter in
the range of 150 to 200 µm.
Important insights may be gained into tumor angiogenesis with in
vivo morphologic characterization of intratumoral blood vessels
that may be acquired dynamically and repetitively to access changes
with time and/or therapy. Unfortunately, these macromolecular
contrast agents are not yet available for clinical use. Although
the imaging features are favorable with increasing molecular weight
of the blood pool agents due to longer intravascular distribution,
eventual clearance may become a problem. Only drugs <70 kDa or
<7 nm in molecular size may be cleared by glomerular filtration,
depending on charge.
The long echo times needed to improve SNR and spatial resolution
come with a cost of increased pulsation and respiration artifacts.
This may prove particularly problematic when imaging certain
thoracic and intra-abdominal tumors. Even with the current
advances, not all angiogenic blood vessels are resolved. This
limitation is likely to improve as high magnetic field scanners
become more commonplace in academic and clinical practice.
Functional measurements of angiogenesis
A variety of functional parameters may be evaluated by MRI as
methods for imaging angiogenesis, including measurements of
permeability, blood volume, perfusion, and vasoreactivity.
Dynamic contrast-enhanced MRI is routinely employed at many
centers for clinical cancer imaging. To the extent that a tumor is
homogeneous, this may provide a meaningful estimation of vascular
permeability. Variations in leakage are averaged out from region to
region due to the large volume (1 Z 1 Z 3 mm) of signal represented
by each pixel in MRI. Each pixel, therefore, represents several
tumor microenvironments. The signal is a complex summation not only
of vascular permeability but also of vascular surface area,
interstitial pressure, and blood flow. Measurement variability and
sensitivity to motion further complicate the issue.
Nonetheless, dynamic contrast-enhancement MRI parameters (higher
enhancement amplitude and shorter transport rate K2 [outflux
transport rate]) have been shown to be better prognostic indicators
of patient survival than are measurements of mean vessel density or
VEGF for uterine cervical cancer.
Typical MRI approaches to study the permeability of tumor
neovasculature consist not only of dynamic imaging with
extracellular fluid contrast agents combined with kinetic analysis
but also time-resolved T1-weighted MRI of the uptake of
macromolecular contrast agents, typically relative molecular weight
) >20,000, which correlate with variations in angiogenesis. Low
molecular weight Gd chelates rapidly extravasate at sites of
However, because they also leak from normal vasculature, it may be
difficult to differentiate angiogenic from normal vessels.
Macromolecular intravascular MR contrast agents, such as iron oxide
particles or Gd bound to albumin or to dendrimers, provide a more
stringent test of the endothelial barrier function of angiogenic
vessels, because they are better retained by normal vessels than
are low molecular weight contrast agents.
Prolonged acquisition times and concerns for potential toxicity
have limited clinical acceptance of these methods to
Medium molecular weight agents such as NMS60 (
~2000) show 50% lower leakage rates than does Gd-DTPA but yield 20%
higher peak contrast signal intensity due to their greater
molecular relaxivity [9.1 versus 3.5 (s Z mM) -1 at 1.5T and 37˚C].
Vessel hyperpermeability is linked to angiogenesis through VEGF,
a potent vascular permeability factor and key survival factor for
endothelial cells in immature neovasculature.
In many situations, hyperpermeability detected by contrast-enhanced
MRI can be used to map VEGF activity, as has been shown by spatial
coregistration of high levels of VEGF expression by
immunohistochemistry to regions of high permeability to
The contrary has also been shown, that the suppression of VEGF
expression by antibodies,
androgen ablation (inhibition of hormonally induced expression),
and inhibitors for the VEGF receptor all significantly reduce
vascular permeability, as measured by contrast-enhanced MRI.
Of course, caution must be taken, as few things in nature are
absolutes. Hyperpermeability often shows a strong correlation with
VEGF-induced angiogenesis but does not serve as an unqualified
proxy for VEGF or angiogenesis. In addition, VEGF-induced
angiogenesis may occur without hyperpermeability upon Src
or in the presence of elevated levels of angiopoietin-1.
Dynamic contrast-enhanced perfusion MRI can be used to create
maps of cerebral and/or tumor microcirculation to derive blood
volume, transit time, clearance, extraction fraction, blood flow,
and permeability surface area product (PSP) by following the
passage of a tracer through the tumor vasculature using the same
concepts as those employed for the cerebrovascular system.
Signal changes from the passage of tracer through a region of
interest form the basis of perfusion MRI. Indicator-dilution
methods originally developed for the radioisotope measurements of
blood flow and volume form the basis of the analysis of whether the
tracer is endogenous (arterial water) or exogenous (deuterium
oxide, gadopentetate dimeglumine) and either nondiffusible
(gadopentetate dimeglumine) or freely diffusible (arterial water,
Tofts and Kermode
developed an entirely unique approach founded on pharmacokinetic
modeling, primarily to measure vascular permeability. This approach
is well suited to evaluate tumor angiogenesis and will likely prove
to be a powerful tool in the management of cancer and other disease
processes linked to perturbations in angiogenesis (Table 1). The
precise details of diffusible tracer kinetics are beyond the scope
of this text, and the reader is referred to excellent reviews for
Measurements are a relative rather than an absolute
quantification of blood volume. Nonetheless, measurement of the
blood volume and, more importantly, the changes over time are
reflective of de novo growth of blood vessels. The overall tumor
vascularity is depicted by cerebral blood volume (CBV), which is an
indirect assessment of angiogenesis.
MR measurements of blood volume/relative CBV (rCBV) have been shown
to correlate with histologic determination of microvessel density
and in a variety of noncerebral tumors of the body.
This allows MR perfusion imaging to currently serve as an adjuvant
to conventional imaging in the clinical realm. Malignant gliomas
are the most common primary brain tumors. According to the World
Health Organization, 3 histotypes are recognized: diffuse
astrocytoma (grade II) shows increased cellularity and modest
pleomorphism; anaplastic astrocytoma (grade III) shows greater
cellularity with atypia and mitotic activity but lacks necrosis;
glioblastoma multiforme (grade IV, the most common primary brain
tumor accounting for 12% to 15% of all intracranial neoplasms) in
addition to the criteria of grade III, shows endothelial
hyperplasia and necrosis (ineffective angiogenesis).
Vascular morphology is a critical prognostic indicator in gliomas
that determines malignant potential and survival, underscoring its
importance in determining therapy. Several studies have found a
statistically significant correlation between tumor rCBV and glioma
Figure 3 depicts an increased blood volume in an enhancing mass
with central necrosis consistent with the pathologic diagnosis of
glioblastoma multiforme. Figure 4 shows how perfusion imaging can
be used to distinguish between tumor necrosis and tumor recurrence.
The combination of CBV maps and conventional MR images can be used
for preoperative grading of gliomas, guiding stereotactic biopsy,
differentiating between recurrent tumor and delayed radiation
necrosis, and monitoring tumor response to therapy.
Perfusion may also be evaluated by arterial spin labeling
saturation transfer. Vascular function is mapped by following water
as a tracer for per-fusion.
This has shown utility not only in physiologic models of
but also in the clinical realm as an indicator for tumor
angiogenesis in cervical carcinoma.
This has the advantage of not requiring the administration of an
extrinsic tracer; therefore, no contrast medium affects the
physical, physiologic, or chemical properties of the blood.
A few studies, however, have reported a weak correlation between
contrast-enhanced MRI and histologic microvessel density.
This may be explained in part by the larger diameter of the tumor
vessels resulting in an increased blood volume fraction without an
increased number of vessels as determined by microvessel density.
Furthermore, steady-state (R1)-based MRI measurements using blood
pool agents tend to overestimate blood volume in regions of
angiogenesis because of the extravasation of even the high
molecular weight contrast agents in these hyperpermeable vessels.
This limitation may be overcome by taking advantage of the signal
loss in susceptibility-weighted images due to the paramagnetic
property of deoxyhemoglobin in blood, thus serving as an intrinsic
MR contrast. This may be used to establish the apparent vessel
density, allowing a qualitative determination of vessel density.
Because no contrast material is administered, repetitive
measurements and detailed kinetic analysis may be made.
The increased vessel diameter in tumors can also be taken
advantage of because 2 relaxation mechanisms, R2 and R2*, have
different sensitivities to the structural difference. Maps of
∆R2*/∆R2 (the ratio of gradient-echo and spin-echo relaxation rate
changes) induced by a high molecular weight contrast agent provide
an indication of the average vessel size in a voxel and show
enlarged vessel diameters in tumors.
The chaotic nature and disorganization/tortuosity of tumor
vasculature is apparent in the abnormal recirculation of exogenous
The effectiveness of angiogenesis may be determined by showing
the ability of the vessel to transfer oxygen. This may be imaged by
intrinsic contrast MRI sensitized to blood oxygenation and
deoxy-hemoglobin content by monitoring R2* relaxation
(blood-oxygenation-level- dependent contrast). The paramagnetic
effect of deoxyhemoglobin within red blood cells allows the
measurement of changes in MRI signal intensity in functional
vessels in response to changes in blood oxygenation by inhalation
of 95% oxygen (hyperoxia).
Conversely, signal changes in response to hypercapnia (5% carbon
]) can be used to map vascular maturation. Vasoreactivity induced
(by the transition from air to 95% O
to air with 5% CO
) is conferred to new blood vessels by recruitment of pericytes and
smooth muscle cells.
MRI of vasoreactivity maps to vessels that are positive for
histologic staining with endothelial markers and with alpha-smooth
muscle actin for staining the contractile perivascular cells.
This phenomenon comes about by perivascular cells controlling
hematocrit and capillary blood flow, as confirmed by intravital
This allows MRI to reproduce the protective role of maturation in
maintaining vascular survival upon VEGF withdrawal
; such information is crucial in rational treatment strategies
using antiangiogenic agents.
Monitoring treatment response
As detailed earlier, MR offers a wide variety of methods to not
only study the preclinical mechanisms of angiogenesis but also to
noninvasively evaluate the efficacy of potential therapies.
MRI is an attractive tool to monitor neoadjuvant chemotherapy
response in invasive ductal breast carcinoma. For the past 20
years, neoadjuvant chemotherapy was introduced to treat patients
with locally advanced breast cancer,
which represents approximately 20% of all breast cancer diagnosed
The goal of this therapy is to treat distant metastasis
and to decrease the size of inoperable tumors so that conservative
breast surgery may be performed.
Adequate methods to monitor response may allow the early detection
of resistant tumors prompting changes in the therapeutic regiment.
In addition, surgical timing may be adjusted to that of the maximal
Contrast-enhanced MRI has shown promise in providing qualitative
and quantitative information that reflects early changes in tumors
due to chemotherapy.
Figure 5 depicts the response of an aggressive invasive ductal
breast carcinoma with local lymph nodal metastasis to neoadjuvant
chemotherapy. MRI is able to depict changes associated with the
regression of angiogenesis after chemotherapy. In particular, the
extraction flow product (EFP), as determined on dynamic
contrast-enhanced MRI, serves as a measure of blood flow and
microvascular permeability, has been shown to provide functional
information regarding changes in tumor angiogenesis due to
neoadjuvant chemotherapy and may be useful in monitoring tumor
Of course, no single technique should be used in a vacuum. In
the heterogeneous environment of tumors there are often mismatches
between spatial patterns of vascular permeability and blood volume
that complicate using a single parameter as a surrogate measure of
Coupling nuclear magnetic resonance spectroscopic data, dynamic
contrast-enhanced mapping of blood volume and permeability as well
as MRI mapping of pH provides insight into the relationship between
metabolic heterogeneity and tumor vascularity.
This may be used to define common regulatory pathways that
determine the final tumor microenvironment by linking specific
patterns to various parameters.
This can be taken a step further by imaging tumors and then
excising the tumors with concordant orientation, sectioning, and
coregistration. The tumors can then be examined histologically, or
RNA can be extracted for microarray analysis from specific tumor
regions by using laser capture microdissection to query for
molecular differences within imaged tumors to help explain the
heterogeneous microenvironment. This allows correlation at the
functional, metabolic, and molecular levels.
Such techniques can be used not only to discover future molecular
targets but also to understand mechanisms of resistance to
Dynamic contrast-enhanced MRI with the intravascular contrast
agent albumin-Gd-DTPA has recently been used to detect the effects
of the antiangiogenic agent TNP-470 on vascular volume and
permeability by using a rodent prostate carcinoma model.
TNP-470 is a fumagillin derivative that inhibits endothelial cell
which targets methionine aminopeptidase-2 (MetAP2)
and has been shown to inhibit tumor growth in preclinical models
and clinical studies.
Figure 6 depicts the multislice maps of vascular volume,
permeability surface area product and hematoxylin- and
eosin-stained histological sections, as well as the triplanar views
from 3D reconstructed maps of the tumors pre- and posttreatment
with TNP-470. The treated tumors show marked reduction of
detectable vascular volume and PSP as well as extensive necrosis by
histologic analysis. In the treated tumors, the vascular volume and
PSP were mainly detected around the periphery of the tumor. As seen
in prior studies,
a spatial discordance was detected between regions of high vascular
volume and high permeability, especially along the peripheral
region of the treated tumors with distinct regions showing a
pattern of reduced vascular volume but increased permeability
compared to control (nontreated) tumors. Interestingly, the VEGF
levels in TNP-470-treated tumors were higher than those in the
nontreated tumors. This suggests that the hypoxia induced by the
decreased vascular volume resulted in a compensatory increase of
VEGF and permeability after treatment with TNP-470.
This serves as a powerful example of the ability of MRI to detect
changes in tumor vascular characteristics. Serial MR scans over a
time course of treatment are likely to provide further insight into
the changes in tumor vasculature induced by various forms of
MR may be used not only to monitor antiangiogenic agents but
also to aid in their design and implementation. Several drug
companies at the most recent ISMRM meeting presented preliminary
data on using MRI to evaluate and select appropriate candidate
agents for further development. For some of the agents, the
antiangiogenic effects on tumor vasculature occurred rapidly and
were transient, some lasting no more than 6 hours. Such data can be
used to develop appropriate dosing schedules. Depending on the
level of vascular maturation that may be shown by functional MRI,
as detailed previously, one may be able to predict which
angiogenesis inhibitors or combination thereof may be most
effective for a particular tumor, because the efficacy of
angiogenesis inhibitors depends on the tumor stage.
Several molecular targets have been identified that are involved
in the regulation of angiogenesis (Tables 2 and 3). Promising work
has been shown with animal models in which MR contrast agents have
been specifically delivered to pathologic lesions with increased
angiogenesis. This has been accomplished by targeting structural
proteins such as matrix fibrin and extradomain β-fibronectin, as
well as endothelial receptors such as ICAM-1 and integrin α
, which are overexpressed at an early stage of angiogenesis.
The inherent problem with MRI and molecular targeting in
angiogenesis is that the targets are usually present in the
picomolar to low micromolar range.
In addition, the tissue concentration of the target agent is in
equilibrium with the blood, resulting in continuous redistribution
into the blood from the target. For one to visualize specific
binding to the target, the blood concentration must be low. This is
further compounded in humans by a high dilution of the percentage
of dose/gram tissue bound to the target, which is <30% in small
animals but <1% in humans, based on data from radiolabeled
High doses are therefore necessary for current Gd-based contrast
agents with relaxivities below 30 L/mmol*s even with high affinity
compounds to overcome the high dilution and reach target tissue
concentrations at or above the detection limit of MR (0.5 mmol/kg).
Often bulky contrast material is used to provide sufficient
sensitivity. Unfortunately, the biological effects of these
macromolecular imaging contrast agents upon signal transduction
pathways through the imaged receptor-ligand system have not been
studied in detail. Competition assays between the native ligand and
molecular contrast material are crucial to determine if the
detected signal represents an over- or underestimation of receptor
levels if such a strategy were to be used for mapping receptor
density for available target for therapy and/or tracking changes in
quantity for response to various forms of antiangiogenic
Several ingenious mechanisms are being employed to overcome
these limitations. Imaging agents are under development with
increased relaxivity to reduce the target tissue concentrations
necessary for detection. Amplification schemes can be designed to
target enzymes and/or transporters that result in the accumulation
of high concentrations of the imaging agent within the target
tissue and/or modify the agent over time.
Much excitement was generated at the ISMRM meeting regarding the
recent success of Dr. Patrick Winter and colleagues in developing
-targeted paramagnetic nanoparticles to noninvasively detect and
characterize biochemically and morphologically early angiogenesis
induced by minute solid tumors in vivo utilizing the Vx-2 rabbit
tumor model with a commercially available 1.5T MRI scanner by using
current clinical imaging techniques. This novel approach of
ligand-directed paramagnetic molecular imaging platform technology
consists of a lipid-encapsulated liquid perfluorocarbon
nanoparticle (approximately 250 nm nominal diameter) capable of
carrying enormous paramagnetic ion payloads (>90,000
atoms per particle) for high detection and sensitivity at a dose of
0.5 mL of nanoparticles per kilogram.
The work stems from an improvement in an earlier design in which
the nanoparticles were coupled to monoclonal antibodies specific
-integrin by avidin-biotin interactions that allowed MR in vivo
detection at 4.7T of angiogenic vessels stimulated within a rabbit
corneal micropocket model by exogenous basic fibroblast growth
In lieu of this older model, improvements were made by covalently
attaching the nanoparticle to a small arginine-glycine-aspartic
acid (RGD)-peptidomimetic compound that increased the number of
ligands per particle 10-fold. The payload of each α
-integrin-bound nanoparticle increased by 50%, resulting in a 400%
improvement in contrast signal enhancement compared with the
earlier model results. In addition, sensitivity was improved by
using a nanoparticle contrast agent with an "ultraparamagnetic" MR
character with a molecular T1 relaxivity of >1,800,000 (s × mM)
Even though several molecular markers of physiologic and
pathologic angiogenesis have been identified, integrin α
remains an attractive, well-defined biomarker that is relatively
selective for activated endothelial cells. During tumor
angiogenesis, there is selective expression of the adhesion
receptor integrin α
. In addition, the survival of new and activated endothelial cells
is increased by a specific signal triggered by the binding of
to its receptor. Mature/quiescent endothelial cells essentially do
not express the integrin α
The potential clinical implications of this work are astounding.
This may improve detection and quantification of occult tumors and
metastases. Using this as a model system, researchers may construct
several different ligand-directed paramagnetic nanoparticles to
detect, quantify, and biochemically and morphologically
characterize various biomarkers of tumor or other pathologic states
of neovasculature. Patients may be segmented a priori into
appropriate antiangiogenic therapy protocols and a noninvasive
means provided to monitor the effectiveness of antiangiogenic
therapeutic regimens that may be tailored to the individual
patient's needs as they change over the course of therapy. In
addition, what can be specifically targeted for diagnosis can also
be targeted for therapy. The ligand-directed paramagnetic
nanoparticles can be designed to entrap various drugs or genetic
material for site-specific drug/gene therapy to targeted cells
with simultaneous confirmation by
Gene-based drugs are highly active and most applicable to
nanoparticle vectors where the amount of gene injected is on the
order of micrograms and milligrams; therefore, a large volume of
nanoparticles is not required for delivery. Furthermore,
fluorine spectroscopy of the perfluorocarbon core may be used to
quantify the delivered therapeutic dose.
Stem cells and cellular imaging
Certain tumors require the recruitment of bone-marrow-derived
endothelial and hematopoietic precursor cells for tumor
This spurs an already avid interest in using MRI to monitor the in
vivo behavior of stem cells and cell tracking. During the ISMRM
meeting, Dr. Frank detailed a new and exciting research model
recently developed to label endothelial precursor cells with
superparamagnetic iron oxide nanoparticles (SPIO).
He and his colleagues have developed an efficient labeling system
using protamine sulfate complexed to ferumoxides for cellular
imaging with MRI.
The SPIO nanoparticles are incorporated with endosomes. The
iron-oxide-labeled cells appear as hypointense areas in tissues
with an associated susceptibility artifact or an amplification of
the decreased signal intensity on iron-sensitive T2-weighted and
T2*-weight-ed gradient echo images.
This process allows for the direct imaging of neovascularization of
the tumors at the cellular level, as depicted in Figure 7. The
high-resolution 3D image protocol used in Figure 7, using a
field-of-view of 3.2 Z 2.1 Z 2.1 cm, requires 20 hours for image
acquisition. However, 2-dimensional images may be obtained in 20
minutes with satisfactory resolution.
This particular combination for magnetic cellular imaging should
facilitate translation of the approach to clinical trials, because
protamine sulfate is FDA-approved for reversal of heparin anticoag-
and SPIO is FDA-approved for in vivo human use as an MRI contrast
Insight may be further gained into the pathogenesis of tumor
angiogenesis with such a strategy combined with directed mutational
analysis. The future applications include not only imaging of cell
trafficking of various stem cells (not only endothelial precursor
cells) into tissues, but may also aid in the development of novel
cell-based strategies for tissue repair and/or replacement of
This is a particularly promising therapeutic approach for other
disease processes characterized by insufficient angiogenesis (Table
Diagnostic imaging is evolving to take advantage of the insights
provided by molecular biology that has continued to grow at an
exponential rate since the completion of the human genome project.
Molecular and functional imaging not only serves as a powerful
research tool to study disease pathways under controlled genetic or
environmental perturbations, but also promises to be an invaluable
vehicle for the translation of bench top research to the bedside,
directly affecting patient care and outcome. The potential is vast,
considering that with the exception of trauma, all human disease
has a fundamental molecular pathophysiologic basis. Even with
trauma, however, the body's healing response occurs via a tightly
regulated pathway controlled at the molecular level. Adjunctive
molecular and functional imaging may improve the sensitivity of
diagnostic imaging in early disease states, allowing more prompt
intervention and providing additional information with respect to
drug, genetic, or stem cell treatment, development, and monitoring
as well as image-guided, site-specific, targeted delivery.
Angiogenesis is the perfect venue to display the vast talents of MR
in this brave new world of imaging and molecular medicine while
impacting on a large variety of issues in health and disease.
The author thanks Don Mitchell, MD and Lisa Tartaglino, MD for
guidance throughout residency and this project, in particular. A
special gratitude goes to Adam Flanders, MD; Scott Enochs, MD, PhD;
and Cathy Piccoli, MD for providing many of the images used in this
paper as well as to the authors who agreed to reprint their
published works. Appreciation is also extended to Pier Paolo
Claudio, MD, PhD, for critical review of the manuscript.