Noninvasive Visualization of the Activated
avb3 Integrin in Cancer Patients by Positron
Emission Tomography and [18F]Galacto-RGD
Roland Haubner1[¤a*, Wolfgang A. Weber1[¤b, Ambros J. Beer1,2, Eugenija Vabuliene1, Daniel Reim1, Mario Sarbia3,
Karl-Friedrich Becker3, Michael Goebel4, Ru ¨diger Hein5, Hans-Ju ¨rgen Wester1, Horst Kessler6, Markus Schwaiger1
1 Nuklearmedizinische Klinik und Poliklinik, Technische Universita ¨t Mu ¨nchen, Germany,
3 Institut fu ¨r Pathologie, Technische Universita ¨t Mu ¨nchen, Germany, 4 Klinik fu ¨r Orthopa ¨die und Sportorthopa ¨die, Technische Universita ¨t Mu ¨nchen, Germany, 5 Klinik und
Poliklinik fu ¨r Dermatologie und Allergologie, Technische Universita ¨t, Mu ¨nchen, Germany, 6 Department Chemie, Lehrstuhl II fu ¨r Organische Chemie, Technische Universita ¨t
Mu ¨nchen, Garching, Germany
2 Institut fu ¨r Ro ¨ntgendiagnostik, Technische Universita ¨t Mu ¨nchen, Germany,
Competing Interests: The authors
have declared that no competing
Author Contributions: R. Haubner,
W.A. Weber, H.J. Wester, H. Kess-
ler, and M. Schwaiger conceived
and designed the experiments. R.
Haubner, A.J. Beer, E. Vabuliene,
D. Reim, K.F. Becker, M. Goebel,
and R. Hein performed the ex-
periments. R. Haubner, W.A.
Weber, M. Sarbia and A.J. Beer
analyzed the data. R. Haubner,
W.A. Weber, A.J. Beer, H.J. Wester
and M. Schwaiger contributed to
the writing of the paper.
Academic Editor: Peter Ell, Insti-
tute of Nuclear Medicine, United
Citation: Haubner R, Weber WA,
Beer AJ, Vabuliene E, Reim D, et al.
(2005) Noninvasive visualization of
the activated avb3 integrin in
cancer patients by positron emis-
sion tomography and [18F]Galacto-
RGD. PLoS Med 2(3): e70.
Received: October 4, 2004
Accepted: January 28, 2005
Published: March 29, 2005
Copyright: ? 2005 Haubner et al.
This is an open-access article dis-
tributed under the terms of the
Creative Commons Attribution
License, which permits unre-
stricted use, distribution, and re-
production in any medium,
provided the original work is
Abbreviations: [18F]FDG, [18F]fluo-
rodeoxyglucose; PET, positron
emission tomography; p. i., post
injection; SUV, standardized up-
* To whom correspondence should
be addressed. E-mail: Roland.
[ These authors contributed
equally to this work.
¤a Current address: Universita ¨ts-
klinik fu ¨r Nuklearmedizin, Medizi-
nische Universita ¨t Innsbruck,
¤b Current address: Department of
Molecular and Medical Pharma-
cology, David Geffen School of
Medicine, University of California,
Los Angeles, California, United
States of America
A B S T R A C T
The integrin avb3 plays an important role in angiogenesis and tumor cell metastasis, and is
currently being evaluated as a target for new therapeutic approaches. Several techniques are
being studied to enable noninvasive determination of avb3 expression. We developed
expression with positron emission tomography (PET).
18F-labeled glycosylated avb3 antagonist, allowing monitoring of avb3
Methods and Findings
Here we show by quantitative analysis of images resulting from a small-animal PET scanner
that uptake of [18F]Galacto-RGD in the tumor correlates with avb3 expression subsequently
determined by Western blot analyses. Moreover, using the A431 human squamous cell
carcinoma model we demonstrate that this approach is sensitive enough to visualize avb3
expression resulting exclusively from the tumor vasculature. Most important, this study shows,
that [18F]Galacto-RGD with PET enables noninvasive quantitative assessment of the avb3
expression pattern on tumor and endothelial cells in patients with malignant tumors.
Molecular imaging with [18F]Galacto-RGD and PET can provide important information for
planning and monitoring anti-angiogenic therapies targeting the avb3 integrins and can reveal
the involvement and role of this integrin in metastatic and angiogenic processes in various
PLoS Medicine | http://www.plosmedicine.orgMarch 2005 | Volume 2 | Issue 3 | e700244
Open access, freely available online P PL Lo oS S MEDICINE
Cell–cell and cell–matrix interactions play essential roles in
tumor metastasis and angiogenesis. Integrins are one of the
main classes of receptors involved in these processes. In
addition to having adhesive functions, integrins transduce
messages via various signaling pathways and influence
proliferation and apoptosis of tumor cells, as well as of
activated endothelial cells. One prominent member of this
receptor class is the integrin avb3. It has been demonstrated
that avb3 is an important receptor affecting tumor growth,
local invasiveness, and metastatic potential [1,2]. This integrin
is expressed on various malignant tumors and mediates
adhesion of tumor cells on a variety of extracellular matrix
proteins, allowing these cells to migrate during invasion and
The integrin avb3 is also highly expressed on activated
endothelial cells during angiogenesis . In contrast, ex-
pression of avb3 is weak in resting endothelial cells and most
normal organ systems . On activated endothelial cells, the
receptor mediates migration through the basement mem-
brane during formation of the new vessel, which is essential
for sufficient nutrient supply of the growing tumor. Inhib-
ition of the avb3-mediated cell–matrix interaction has been
found to induce apoptosis of activated endothelial cells. Thus,
the use of avb3 antagonists is currently being evaluated as a
strategy for tumor-specific anti-cancer therapies [6,7,8].
Owing to the weak expression on non-activated endothelial
cells, treatment with avb3 antagonists does not affect
preexisting blood vessels. Inhibition of blood vessel forma-
tion in tumor models using avb3 antagonists not only blocks
tumor-associated angiogenesis, but in some cases results in
tumor regression .
However, avb3 antagonists can induce apoptosis not only
of activated endothelial cells but also of avb3-positive tumor
cells , resulting in a direct cytotoxic effect on tumor cells.
Moreover, blocking of the receptor expressed on tumor cells
can reduce invasiveness and spread of metastases .
Furthermore, avb3-binding molecules have been successfully
used to ‘‘target’’ a variety of therapeutic agents to the tumor
tissue. These include chemotherapeutic agents , cDNA-
encoding anti-angiogenic genes , and T lymphocytes .
These encouraging experimental studies have already led
to initial clinical trials evaluating the use of avb3 antagonists
(e.g., vitaxin  and cilengitide ) in patients with various
malignant tumors [17,18,19,20]. Currently available imaging
techniques are limited in monitoring treatment with this class
of drugs. Anti-tumor activity is generally assessed by
determining the percentage of patients in whom a significant
reduction in tumor size is achieved during a relatively short
period of therapy (‘‘response rate’’). Thus, this method may
not be applicable for a form of therapy that is aimed at
disease stabilization and prevention of metastases. New
methods are urgently needed for planning and monitoring
treatments targeting the avb3 integrin.
Based on cyclo(-Arg-Gly-Asp-DPhe-Val-) , a variety of
radiolabeled avb3 antagonists for single photon emission
tomography and positron emission tomography (PET) have
been developed (for review see [22,23]). [18F]Galacto-RGD
(arginine–glycine–aspartic acid), a glycosylated cyclic penta-
peptide, resulted from a consequent tracer optimization 
based on the first-generation peptide [125I]-3-iodo-DTyr4-
cyclo(-Arg-Gly-Asp-DTyr-Val-)  and showed high affinity
and selectivity for the avb3 integrin in vitro, receptor-specific
accumulation in avb3-positive tumors, and high metabolic
stability in a murine tumor model, as well as rapid,
predominantly renal elimination [26,27]. Here we describe
how [18F]Galacto-RGD allows quantification of avb3 expres-
sion in vivo, show that tumor-induced angiogenesis can be
monitored in a murine tumor model, and for the first time, to
our knowledge, demonstrate that this class of tracers can be
used in patients for noninvasive determination of avb3
Synthesis of the labeling precursor and subsequent
labeling was carried out as described . For application in
patients, after high-performance liquid chromatography the
collected fraction was evaporated to dryness; 0.5 ml of
absolute ethanol and 10 ml of phosphate-buffered saline
(pH 7.4) were added; and the product was passed through a
Millex GV filter (Millipore, Eschborn, Germany). Quality
control of the product was carried out according to the
demands of the local regulatory authorities.
Murine Tumor Models
For in vivo evaluation, xenotransplanted human melanoma
models (M21 and M21-L) and a human squamous cell
carcinoma model (A431) were used. The M21 cell line
expressing avb3 [25,28] acted as a positive control and the
M21-L cell line, a stable variant cell line of M21 failing to
transcribe the av gene, as a negative control . Cell culture
conditions for M21 and M21-L cells are described elsewhere
. Similar protocols were used for A431 cells.
The experimental protocol involving animals was ap-
proved by the Committee of Veterinarian Medicine of the
State of Bavaria; handling of animals was performed
according to the standards set by the Committee of
In order to study the correlation between avb3 expression
and tumor uptake of [18F]Galacto-RGD, we injected mice
subcutaneously with mixtures of M21 and M21-L cells. Pilot
experiments had indicated that injection of 1.5 3 106M21
cells leads within 4 wk to the formation of tumors with a
diameter of approximately 8 mm. To obtain similarly sized
M21-L tumors, it was necessary to inject 63106cells. In order
to study tumors with approximately 10%, 25%, 50%, and
75% M21 cells, we injected mice with the following mixtures
of M21 and M21-L cells: 1.53105/ 5.43105, 3.83105/ 4.63
106, 7.5 3 105/ 3 3 106, and 1.1 3 106/ 1.5 3 106. Four weeks
after inoculation, nude mice were injected with 7.4 MBq of
[18F]Galacto-RGD and scanned at the small-animal PET.
Subsequently, tumors and other organs of interest were
dissected, immediately counted, cut in two pieces, and frozen
for further workup.
For experiments with the squamous cell carcinoma model,
approximately 106A431 cells were injected subcutaneously in
nude mice. Two weeks after inoculation, 7.4 MBq of
[18F]Galacto-RGD was injected, and mice were scanned in
the animal PET. Animals were sacrificed, and organs of
interest were dissected and subsequently weighed and
counted or used for immunohistochemical analysis.
PLoS Medicine | http://www.plosmedicine.orgMarch 2005 | Volume 2 | Issue 3 | e700245
Visualization of avb3 Expression by PET
For immunohistochemical investigation, frozen tumor
tissues from mice, as well as from patients, were sectioned
(6 lm) and stained using the biotinylated monoclonal anti-
avb3 antibody LM609 (1:100; Chemicon Europe, Hofheim,
Germany). For staining the murine b subunit, a monoclonal
hamster anti-mouse antibody (1:10; Chemicon Europe) and a
biotinylated mouse anti-hamster IgG secondary antibody
(1:200; Chemicon Europe) were used. Sections were processed
by peroxidase staining (peroxidase substrate KIT AEC, Vector
Laboratories, Burlingame, California, United States).
The frozen tumor tissue was homogenized and extracted
with lysis buffer (50 mM Hepes (pH 7.5), 150 mM NaCl, 10%
Glycerol, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM
Na4P2O7, 1 mM MSF, 10 lg/ml Aprotinin, 10 lg/ml Leupep-
tin). Protein concentration was determined according to
Bradford  and adjusted to equivalent values using lysis
buffer. After SDS-PAGE and transfer, immunoblotting was
carried out using a polyclonal rabbit anti-av antibody (1:500;
Chemicon Europe) and a125I-labeled polyclonal donkey anti-
rabbit IgG antibody (1:400; 477 kBq/lg, Amersham Buchler,
Braunschweig, Germany). For analysis, blots were placed on a
phosphor screen for 2 d. For readout out, a Molecular
Dynamics PhosphorImager 445 SI (Sunnyvale, California,
United States) was used.
PET Studies with a Small-Animal Scanner
PET imaging of tumor-bearing mice was performed using a
prototype small-animal positron tomograph (Munich Ava-
lanche Photodiode PET; ). The animal scanner consists of
two sectors, comprising three detector modules each, which
rotate around the animal for acquisition of complete
projections in one transaxial slice (30 angular steps). Each
module consists of eight small (3.733.7312 mm3) lutetium-
oxy-orthosilicate crystals read out by arrays of avalanche
photodiodes. List mode data are reconstructed using stat-
istical, iterative methods including the spatially dependent
line spread function. Reconstructed image resolution is 2.5
mm (full width at half maximum) in a transaxial field of view
of 7.5 cm, and the slice thickness is 2 mm. Ninety minutes
after the injection of approximately 7.4 MBq of [18F]Galacto-
RGD, animals were positioned prone inside the tomograph,
and a transaxial slice through the tumor region was measured
for 480 s
The study protocol was approved by the ethics committee
of the Klinikum Rechts der Isar (Protocol S1), and each
patient gave written and informed consent prior to the study
(Protocol S2). Nine patients were scanned (five female and
four male; age, 26–75 y), who suffered from either malignant
melanoma with lymph node metastasis (stage IIIb; patients 1–
3), malignant melanoma with distant metastasis (stage IV;
patients 4 and 5), chondrosarcoma (patient 6), soft tissue
sarcoma (patient 7), osseous metastasis of renal cell carcino-
ma (patient 8), or villonodular synovitis of the knee (patient
9). Patient selection was focused on melanoma and sarcoma
because there is considerable evidence that these tumor types
Diagnosis prior to scanning was made by biopsy (patients
6–8), by CT (patients 1, 2, and 4–8), by MRI (patient 9), and/or
by [18F]fluorodeoxyglucose ([18F]FDG)–PET (patients 1–4 and
8). After scanning, the diagnosis was confirmed by surgery
and histopathological examination of the resectioned speci-
men (patients 1, 2, 5, 6, 8, and 9) or by combined analysis of
morphological imaging, [18F]FDG PET, and the patient’s
clinical data and history (patients 3, 4, and 7).
For immunohistochemistry, sampled specimens (patients 1,
2, 5, 6, 8, and 9) were snap frozen in liquid nitrogen and
stored at?70 8C until staining was performed. Tissue samples
were taken within 1 wk after scanning from the tumor regions
with the maximum tracer uptake. Light microscopic evalua-
tion of the density of avb3-positve microvessels was
performed as described previously . Briefly, areas with
the highest density of avb3-positve microvessels were
identified using scanning magnification. Subsequently, avb3-
positve microvessels were counted in three adjacent micro-
scopic fields using a 403 magnifying lens and 103 ocular,
corresponding to an area of 0.588 mm2. Determination of
microvessel density was performed by one senior pathologist
(M. S.), who was blinded for the results of the corresponding
standardized uptake value (SUV) analysis of tracer accumu-
lation. Then the correlation between the mean values of the
vessel counts and the corresponding SUVs was analyzed.
PET scanning was performed using an ECAT Exact PET
scanner (Siemens-CTI, Knoxville, Tennessee, United States).
After injection of 144–200 MBq of [18F]Galacto-RGD, three
consecutive emission scans (starting at 7 6 2.7 min, 37 6 10.5
min, and 79 6 18.4 min post injection [p. i.]) from the body
stem and, if necessary, from tumor regions outside the body
stem were obtained. For one patient, only one scan starting
120 min. p. i. was carried out. Attenuation- and decay-
corrected images were reconstructed by using an ordered
subsets expectation maximization algorithm. The accumula-
tion of [18F]Galacto-RGD was evaluated by calculating the
mean SUV normalized to the patient’s body weight according
to the following formula : (measured activity concen-
tration [Bq/ml]3body weight [kg]) / injected activity [Bq]. The
axial slice of the lesion with the maximum activity accumu-
lation was chosen by visual estimation, a region of interest
with a diameter of 15 mm was selected on the lesion, and the
resulting mean SUV was used for further analysis. For lesions
smaller than 2 cm in diameter, a region of interest with a
diameter of 10 mm was used and the analysis was based on
maximum SUV rather than mean SUV, in order to minimize
partial volume effects, which could lead to an underestima-
tion of the SUV.
Dosimetry calculations are based on the MIRDOSE 3.0
program. Data from six patients were analyzed by selecting
regions of interest with a diameter of 1.5 cm on the source
organs (lung, liver, spleen, kidneys, muscle, bladder, intestine,
and heart [left ventricle]). Activity measurements (in Bec-
querels per cubic centimeter) were performed for all three
consecutive scans (mean time p.i. 6 standard deviation, 7 6
2.7 min, 37 6 10.5 min, and 79 6 18.4 min, respectively),
using a monoexponential fit for calculation of residence
times. The volume of the source organs lung, liver, spleen,
and kidneys was measured by CT volumetry (Siemens,
Forchheim, Germany) in four patients. For the other source
organs in these four patients and all organs in the remaining
two patients, standardized volume values of the source organs
adapted to the patient’s body weight were used.
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Visualization of avb3 Expression by PET
All quantitative data are expressed as mean þ/? one
standard deviation. The correlation between quantitative
parameters was evaluated by linear regression analysis and
calculation of Pearson’s correlation coefficient. Statistical
significance was tested by using analysis of variance (AN-
Correlation of Tracer Uptake with avb3 Expression
We have previously demonstrated, using a murine tumor
model in which the tumor cells are either avb3-positive
(human melanoma M21) or av-negative (human melanoma
M21-L), that [18F]Galacto-RGD shows receptor-specific accu-
mulation in the avb3-positive tumor . Here we studied the
correlation of [18F]Galacto-RGD uptake with the level of avb3
expression. We injected tumor cell mixtures containing
increasing amounts of avb3-positive M21 cells subcutaneously
into nude mice. Transaxial images of mice 4 wk after cell
inoculation and 90 min after tracer injection using a
prototype small-animal PET scanner  showed increasing
tracer uptake in the tumor corresponding with the percent-
age of receptor-positive cells (Figure 1A and 1B).
We validated these qualitative results by determining the
relative amount of the av subunit in the dissected tumors
through Western blot analysis (Figure 1C). These data were
correlated with the tumor/background ratios resulting from
the quantitative analysis of the PET images (Figure 1D), as
well as with the tumor/muscle ratios resulting from the
biodistribution analysis carried out after the PET study
(Figure 1D). Both analyses showed a significant correlation
between [18F]Galacto-RGD and relative av expression, thus
confirming the qualitative analysis by immunohistochemistry.
The systematic difference between tumor/background and
tumor/muscle ratios is due to the fact that the region of
interest used to define the tumor region in the PET images
will always contain not only tumor, but also normal tissues
with low [18F]Galacto-RGD uptake, such as muscle and lung.
This is due to the limited spatial resolution of the PET
scanner, which does not allow a sharp distinction between
tumor and normal tissue. Accordingly [18F]Galacto-RGD
uptake by the tumor tissue will be underestimated, and the
tumor/background ratio will be lower than the tumor/muscle
ratio. Furthermore, tissue sampling was performed 30 min
Figure 1. Preclinical Evaluation of [18F]Galacto-RGD
(A) Transaxial images of nude mice bearing tumors with increasing
amounts of avb3-positive M21 cells (0% [M21-L], 25%, 75%, and
100% [M21]) 90 min p. i. provided by a prototype small-animal PET
scanner show an increasing tracer uptake in the tumor and low
(B) Immunohistochemical staining of tumor tissue sections prepared
after PET imaging with an anti-human avb3 monoclonal antibody
(LM 609) indicate that there is a correlation between tracer uptake
and avb3 expression.
(C) Western blots of the dissected tumors show a band at 25 kDa that
corresponds with the mass of the av subunit under reductive
conditions, and indicate the increasing avb3 density in the murine
tumor model used.
(D) The correlation between receptor expression and [18F]Galacto-
RGD accumulation is confirmed by quantitative analysis based on the
tumor/background ratios and tumor/muscle ratios calculated from
the PET images and from the biodistribution studies, respectively,
and by the relative av expression in Western blot analyses.
PLoS Medicine | http://www.plosmedicine.orgMarch 2005 | Volume 2 | Issue 3 | e700247
Visualization of avb3 Expression by PET
after the start of the PET scan. Clearance of radioactivity
from the muscle tissue during this time period will also
systematically increase the tumor/muscle ratio compared to
the tumor/background ratio calculated from the PET images.
When correlating the weight of the tumor with the relative
av expression, we found a nonsignificant trend for lower av
expression in larger tumors (r = 0.34, p = 0.09). This is
probably related to the presence of necrotic regions in larger
tumors, which do not demonstrate av expression. Thus, it can
be excluded that the positive correlation between av
expression and [18F]Galacto-RGD uptake is due to systematic
differences in the size of tumors.
Noninvasive Determination of avb3 Expression on Endo-
To determine whether PET with [18F]Galacto-RGD allows
noninvasive determination of avb3 expression on activated
endothelial cells, we used A431 tumor xenografts. A431 cells
do not express avb3, but induce extensive angiogenesis when
subcutaneously transplanted into nude mice. . Immuno-
histochemical staining of tumor sections using a monoclonal
anti-human avb3 antibody confirmed that the tumor cells do
not express this integrin (Figure 2A). In contrast, staining
with a polyclonal antibody against the murine b3 subunit
demonstrated expression of b3 on endothelial cells of the
tumor vessels. Since aIIbb3, the only further integrin
containing a b3 subunit, is mainly expressed on platelets, it
can be excluded that staining depends on this receptor. Thus,
in this case, staining for the b3 subunit correlates with avb3
Transaxial images of tumor-bearing mice 90 min after
injection of [18F]Galacto-RGD showed a contrasting tumor on
the right flank of the mouse, reflecting avb3-targeted tracer
accumulation on endothelial cells of the tumor vasculature
(Figure 2B). Moreover, we demonstrated receptor-specific
tracer accumulation at the tumor site by injecting 18 mg of
the pentapeptide cyclo(-Arg-Gly-Asp-DPhe-Val-) per kilogram
of mouse 10 min prior to tracer injection. After blocking
tracer accumulation, we found approximately 25% of the
initial activity in the tumor (0.28 6 0.05% injected dose per
gram versus 1.07 6 0.33% injected dose per gram).
Studies in Humans
For the initial evaluation in humans, we imaged nine
patients (five with malignant melanomas, two with sarcomas,
one with osseous metastasis from renal cell carcinoma, and
one with villonodular synovitis) with approximately 185 MBq
of [18F]Galacto-RGD. For all patients, rapid, predominantly
renal excretion was observed, resulting in fast tracer
elimination from blood and low tracer concentration in
most of the organs. Besides the kidneys (SUV = 5.5 6 3.7; 79
min), the highest activity concentration was found in spleen
(SUV = 2.5 6 0.5; 79 min p.i.), liver (SUV = 2.4 6 0.5; 79 min
p.i.), and intestine (SUV = 2.1 6 0.8; 79 min p.i.). In tumor
lesions, tracer accumulation showed great heterogeneity, with
SUVs ranging from 1.2 to 10.0. The SUV in the villonodular
synovitis was 3.2. The radioactivity was retained in the tumor
tissue for more than 60 min (Table 1), whereas in all other
organs a decrease of activity concentration was observed over
time. Tumor/blood and tumor/muscle ratios 79 min p. i. were
3.8 6 2.6 and 8.8 6 6.0, respectively. Although for one
melanoma patient multiple lesions were detected by the
[18F]FDG scan, which indicates viable tumor cells, no activity
accumulation was found using [18F]Galacto-RGD (Figure 3A).
For other patients, however, similar uptake patterns were
observed for [18F]FDG and [18F]Galacto-RGD (Figure 3B). The
metabolite analysis of blood samples 10, 30, and 120 min p. i.
showed in the soluble fractions more than 96% intact tracer
(n = 4) over the whole observation period and confirmed our
preclinical data . An effective radiation dose of 18.0 6 3.2
lSv/MBq was calculated on the basis of the patient data (n =
5). The highest absorbed dose was found in the urinary
bladder wall (0.20 6 0.04 mGy/MBq).
Immunohistochemical staining of sections obtained from
Figure 2. Noninvasive Monitoring of avb3 Expression on the Tumor
(A) Immunohistochemical staining of tumor section using the anti-
avb3 monoclonal antibody LM609 demonstrates that squamous cell
carcinoma cells of human origin do not express the avb3 integrin. In
contrast, staining of section with an antibody against the murine b3
subunit indicates that the tumor vasculature is avb3-positive.
(B) Transaxial images of a nude mouse bearing a human squamous
cell carcinoma at the right shoulder (left) acquired at the small-
animal PET 90 min after tracer injection show a clearly contrasting
tumor. Tracer accumulation in the tumor (right, top image) can be
blocked by injecting 18 mg of cyclo(-Arg-Gly-Asp-DPhe-Val-) per
kilogram of mouse 10 min prior to tracer injection (right, bottom
image), indicating receptor-specific accumulation.
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Visualization of avb3 Expression by PET
tumor tissue after surgery using an anti-avb3 antibody
showed avb3 expression on the endothelial cells of the tumor
vasculature (6/6), and for two patients expression on the
tumor cells as well (2/6) (Figure 4). The density of avb3-
positive vessels showed wide variation intraindividually and
between individual cases. Light microscopic quantification
revealed between one (inflammation of the knee due to
previous operation) and 35 (soft tissue sarcoma of the knee,
same patient) avb3-positive vessels per microscopic field.
Moreover, in the six cases under analysis, density of
immunohistochemically determined avb3-positive vessels
was significantly associated with tracer accumulation as
determined by SUV analysis (r = 0.94, p = 0.005).
Recently, we demonstrated that radiolabeled RGD peptides
allow receptor-specific monitoring of avb3 expression in
murine tumor models [24,25,26,27,35]. Here we have trans-
lated these findings to the clinical setting and for the first
time, to our knowledge, demonstrated noninvasive imaging of
avb3 expression in patients with malignant tumors. Further-
more, we have shown that the activity accumulation in the
tumor correlates with the receptor density, determined by
immunohistochemistry and Western blotting. This indicates
that a noninvasive quantitative determination of avb3
expression is feasible. Furthermore, we have demonstrated
in a squamous cell carcinoma model that the sensitivity of
PET is adequate to image expression of avb3 in the tumor
vasculature. This indicates that PET with [18F]Galacto-RGD
can be applied to study avb3 expression during angiogenesis.
The correlation between [18F]Galacto-RGD uptake in the
tumor and av expression shows considerable scattering. This
is probably due to several factors. As for any imaging probe,
tumor uptake of [18F]Galacto-RGD is not only influenced by
the expression of the avb3 integrin, but also by unspecific
factors such as perfusion and vascular permeability. Further-
more, heterogeneous tracer uptake within a tumor, e.g., due
to the presence of necrotic areas, will influence the
correlation between [18F]Galacto-RGD uptake and av ex-
pression, since separate samples were used for measurements
of tracer uptake and quantitative assessment of av expression.
Finally, the present study evalutated [18F]Galacto-RGD uptake
only at a fixed time, 90 min p. i. Imaging of the dynamics of
[18F]Galacto-RGD accumulation in the tumor tissue and
tracer kinetic modeling may allow a better quantitative
assessment of avb3 expression by PET imaging, and this
approach should be evaluated in animal models as well as in
patients. Nevertheless, the significant correlation between the
uptake of [18F]Galacto-RGD at a fixed time after injection and
avb3 expression is very important for clinical studies, since it
suggests that estimates of avb3 expression levels may be
obtained from simple whole-body PET scans.
It has been shown that the highly bent integrin conforma-
tion is physiological and has low affinity for biological
ligands, such as fibrinogen and vitronectin. Inside-out and
outside-in signaling involve a switchblade-like opening to an
extended structure with high affinity for endogenous ligands,
as well as integrin antagonists (for overview see ). The
inside-out activation is induced by conformational changes in
the membrane-proximal regions of the a and b subunit (e.g.,
by intracellular proteins like talin). Outside-in signaling is
Table 1. SUVs Determined Approximately 5 min, 35 min, and 75
min Post Injection
6.7 6 2.7 min36.8 6 10.5 min79.4 6 18.4 mina
2.41 6 0.46
2.89 6 0.51
3.76 6 0.76
2.68 6 0.78
0.70 6 0.11
7.64 6 1.15
4.21 6 1.95b
1.52 6 0.40
2.53 6 0.43
2.90 6 0.43
2.32 6 0.58
0.56 6 0.05
5.79 6 1.74
4.10 6 2.14b
1.17 6 0.36
2.44 6 0.45
2.54 6 0.49
2.10 6 0.78
0.50 6 0.13
5.45 6 3.68
4.40 6 2.77c
Values are given as mean 6 standard deviation (n = 8, unless otherwise indicated).
an = 9.
bn = 7.
cn = 10.
Figure 3. Comparison of [18F]FDG and [18F]Galacto-RGD Scans
Coronal image sections, acquired 60 min p. i.
(A) Patient with malignant melanoma stage IV and multiple
metastases in liver, skin, and lower abdomen (arrows): marked uptake
of [18F]FDG in the lesions (left), but no uptake of [18F]Galacto-RGD
(B) Patient with malignant melanoma stage IIIb and a solitary lymph
node metastasis in the right axilla (arrow): intense uptake of both
[18F]FDG (left) and [18F]Galacto-RGD (right).
PLoS Medicine | http://www.plosmedicine.orgMarch 2005 | Volume 2 | Issue 3 | e700249
Visualization of avb3 Expression by PET
triggered by Mn2þ, which defines by quaternary rearrange-
ments a pathway for communication from the ligand-binding
site to the cytoplasmatic proximal segments. However, it is
also reported that cyclo(-Arg-Gly-Asp-DPhe-Val-), in addition
to binding to the high-affinity conformer, can bind to the
low-affinity conformation when used at concentrations far
above its dissociation constant, resulting in a similar
activation as found for Mn2þ. The nanomolar concentration
used in our radiotracer approach is approximately 10,000-
fold lower than that reported for the activation of the low-
affinity conformation. Thus, PET with [18F]Galacto-RGD is
expected to provide information not only about the
expression of avb3 but also about the functional status of
The glycopeptide [18F]Galacto-RGD showed high metabolic
stability in patients and rapid, predominantly renal elimi-
nation, resulting in good tumor/background ratios and, thus,
in high-quality images. Moreover, this finding confirms the
general advantage of the glycosylation approach
[24,26,37,38,39] in designing peptide-based tracers with
favorable imaging properties for clinical applications. An-
other approach to optimize pharmacokinetics is based on the
conjugation of polyethyleneglycol [40,41,42,43,44,45]. It has
been demonstrated that such polyethyleneglycolated peptides
also improve pharmacokinetics and tumor retention. How-
ever, a direct comparison of tracers resulting from the
different strategies has not yet been carried out.
The correlation between regional tracer uptake in the
lesion and density of avb3-positive vessels confirms that this
technique allows not only visualization but also noninvasive
quantitative assessment of the integrin expression. Interest-
ingly, our study demonstrated high both inter- and intra-
individual variances in tracer accumulation in the different
lesions, indicating a great diversity in receptor expression.
This finding demonstrates the value of noninvasive techni-
ques for appropriate selection of patients entering clinical
trials of avb3-targeting therapies. This is further emphasized
by the fact that in some cases no [18F]Galacto-RGD uptake
was found, despite viable tumor cells being detected via a
Furthermore, PET imaging with [18F]Galacto-RGD can be
applied to assess successful blocking of avb3 by therapeutic
agents, thereby providing essential information for the dose
and dose scheduling of avb3 antagonists. Further studies are
needed to demonstrate the impact of this new technique as a
novel prognostic indicator in cancer. However, the first
evidence of the prognostic value is given by Gasparini et al.
, who found avb3 expression in tumor vasculature ‘‘hot
spots’’ to be a significant prognostic factor predictive of
relapse-free survival in both node-negative and node-positive
avb3 is also found on endothelial cells during wound
healing, in restenosis, in rheumatoid arthritis, and in
psoriatic plaques. Thus, radiolabeled RGD peptides may be
used to characterize not only malignant tumors but also
inflammatory diseases. Most recently, we demonstrated in a
murine model for cutaneous delayed-type hypersensitivity
reaction that [18F]Galacto-RGD allows noninvasive assess-
ment of avb3 expression in inflammatory processes . Our
preliminary data from a villonodular synovitis show that avb3
expression on endothelial cells in this lesion can be
monitored in patients. Altogether, these findings indicate
that [18F]Galacto-RGD might also become a new biomarker
for disease activity in inflammatory processes.
The primary advantage of PET in imaging molecular
processes is its high sensitivity combined with high pene-
tration of the gamma radiation resulting from positron
decay. Thus, PET imaging allows quantification of regional
radioactivity concentrations in human studies. The optical
imaging approach has an even higher sensitivity, but suffers
from the low penetration of light in most tissues. This results
in a very limited ability to carry out tomographic imaging and
to quantify the optical signal. Thus, optical imaging is
currently limited to preclinical studies in mice, whereas
Figure 4. Correlation of Tracer Accumulation and avb3 Expression
(A–C) patient with a soft tissue sarcoma dorsal of the right knee joint.
(A) The sagittal section of a [18F]Galacto-RGD PET acquired 170 min
p. i. shows circular peripheral tracer uptake in the tumor with
variable intensity and a maximum SUV of 10.0 at the apical-dorsal
aspect of the tumor (arrow). (B) The image fusion of the [18F]Galacto-
RGD PET and the corresponding computed tomography scan after
intravenous injection of contrast medium shows that the regions of
intense tracer uptake correspond with the enhancing tumor wall,
whereas the non-enhancing hypodense center of the tumor shows no
tracer uptake. (C) Immunohistochemistry of a peripheral tumor
section using the anti-avb3 monoclonal antibody LM609 demon-
strates intense staining predominantly of tumor vasculature.
(D–F) patient with malignant melanoma and a lymph node metastasis
in the right axilla. (D) The axial section of a [18F]Galacto-RGD PET
acquired 140 min p. i. shows intense focal uptake in the lymph node
(arrow). (E) Image fusion of the [18F]Galacto-RGD PET and the
corresponding computed tomography scan after intravenous injec-
tion of contrast medium. (F) Immunohistochemistry of the lymph
node using the anti-avb3 monoclonal antibody LM609 demonstrates
intense staining predominantly of tumor cells and also blood vessels.
PLoS Medicine | http://www.plosmedicine.org March 2005 | Volume 2 | Issue 3 | e700250
Visualization of avb3 Expression by PET
PET can be performed in preclinical as well as in clinical
studies. Magnetic resonance imaging provides high spatial
resolution and can combine morphological and functional
imaging, but has approximately 1,000-fold lower sensitivity
compared with PET. Thus, PET is the most appropriate
technique for noninvasive determination of molecular
processes in patients at the current time. Obviously, the
patient is exposed to high-energy c-rays during this proce-
dure. However, based on our radiation dose estimates, the
effective radiation dose for a [18F]Galacto-RGD scan is in the
same range as for a [18F]FDG scan, an approved routine
examination in the clinic in many countries . In
preclinical studies, different targeted magnetic resonance
contrast agents have been evaluated, using either anti-avb3
antibody-conjugated polymerized liposomes  or nano-
particles , or nanoparticles linked with an avb3 peptido-
mimetic antagonist . In those studies, depending on the
contrast agent and animal model used, an average magnetic
resonance signal intensity enhancement between approxi-
mately 20% and 120% was found, a finding which has not yet
been confirmed in clinical studies. In our patient study using
[18F]Galacto-RGD and PET, a 9-fold higher activity accumu-
lation, on average, was found in the tumor than in muscle,
further indicating the currently superior properties of this
radiotracer for molecular imaging. Moreover, recent develop-
ments in combining PET with computed tomography or
future possibilities to combine PET with magnetic resonance
imaging will allow correlation of these processes with the
To further improve tumor retention of avb3 radioligands,
multimeric RGD peptides were recently introduced. Our
group developed different series of multimeric structures with
up to eight RGD units linked via different spacers [40,41,42].
These multimeric RGD peptides showed increased binding
affinities in vitro and improved tumor accumulation and
tumor/background ratios in rodents compared with the
monomeric compounds. These data and data from other
may be used for optimization of the performance of peptide-
based tracers. However, studies in patients will be necessary to
demonstrate the potential of this approach in clinical settings.
In summary, this new class of PET tracer may offer insights
into molecular processes during tumor development and
dissemination in preclinical as well as clinical settings, and
will be a helpful tool in planning and controlling novel avb3-
Protocol S1. Approval of Ethics Committee
Found at DOI: 10.1371/journal.pmed.0020070.sd001 (1.4 MB PPT).
Protocol S2. Patient Consent Form
Found at DOI: 10.1371/journal.pmed.0020070.sd002 (4.2 MB PPT).
We thank W. Linke, C. Bodenstein, J. Carlsen, B. Blechert, and C.
Schott for their excellent technical assistance. The RDS-cyclotron
and PET team, especially M. Herz, G. Dzewas, and C. Kruschke, are
gratefully acknowledged. We thank D. A. Cheresh of the Scripps
Institute, La Jolla, California, for providing the melanoma cell lines
M21 and M21-L. This work was financially supported by the Sander
Foundation (grant number 96.017.3) and by a grant from the
Mu ¨nchner Medizinische Wochenschrift. The funders had no role in
study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
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Background Tumor cells express many different molecules on their
surface. These cell membrane molecules are involved in a variety of
different processes, such as those that hold cells together, trigger cell
death, or determine whether the tumor spreads. Some of these
molecules can be tagged with radiolabeled compounds, called tracers.
These tracers can show where these molecules are found and how many
there are by methods such as PET and SPECT scans that don’t require a
biopsy, i.e., are not invasive. These methods can then be used for
planning treatment with anti-cancer drugs that bind these molecules
What Did the Investigators Do? They induced tumors in mice and
injected them with a tracer for one cell surface molecule—an integrin.
They showed that the amount of the molecule on the tumor could be
measured by the intensity of tracer seen on a PET scan. They also
showed that the same molecule was present on the new blood vessels
that tumors produce. In a small study of patients with various tumors,
including melanomas, the researchers found that the same tracer could
be used to measure the expression of the integrin on tumor cells as well
as on endothelial cells, such as those found in blood vessels, and hence
measure the amount of new vessels in the tumors.
What Does This Mean for Patients? This tracer could be useful to
determine integrin expression noninvasively, to determine how many
new vessels tumors have, to get information for planning anti-cancer
therapies targeting integrin, and to study response to anti-cancer drugs.
However, this study involved only nine patients, so much more work will
need to be done before such a technique is shown to be generally
Where Can I Get More Information? The National Cancer Institute has
information on melanomas for patients: http://www.nci.nih.gov/cancer-
Radiology Info explains PET scanning: http://www.radiologyinfo.org/
PLoS Medicine | http://www.plosmedicine.orgMarch 2005 | Volume 2 | Issue 3 | e700252
Visualization of avb3 Expression by PET