Comparison between 68Ga-bombesin (68Ga-BZH3) and the cRGD tetramer 68Ga-RGD4 studies in an experimental nude rat model with a neuroendocrine pancreatic tumor cell line.

Abstract

Receptor scintigraphy gains more interest for diagnosis and treatment of tumors, in particular for neuroendocrine tumors (NET). We used a pan-Bombesin analog, the peptide DOTA-PEG2-[D-tyr6, β-Ala11, Thi13, Nle14] BN(6-14) amide (BZH3). BZH3 binds to at least three receptor subtypes: the BB1 (Neuromedin B), BB2 (Gastrin-releasing peptide, GRP), and BB3. Imaging of ανβ3 integrin expression playing an important role in angiogenesis and metastasis was accomplished with a 68Ga-RGD tetramer. The purpose of this study was to investigate the kinetics and to compare both tracers in an experimental NET cell line.
This study comprised nine nude rats inoculated with the pancreatic tumor cell line AR42J. Dynamic positron emission tomography (PET) scans using 68Ga-BZH3 and 68Ga-RGD tetramer were performed (68Ga-RGD tetramer: n = 4, 68Ga-BZH3: n = 5). Standardized uptake values (SUVs) were calculated, and a two-tissue compartmental learning-machine model (calculation of K1 - k4 vessel density (VB) and receptor binding potential (RBP)) as well as a non-compartmental model based on the fractal dimension was used for quantitative analysis of both tracers. Multivariate analysis was used to evaluate the kinetic data.
The PET kinetic parameters showed significant differences when individual parameters were compared between groups. Significant differences were found in FD, VB, K1, and RBP (p = 0.0275, 0.05, 0.05, and 0.0275 respectively). The 56- to 60-min SUV for 68Ga-BZH3, with a range of 0.86 to 1.29 (median, 1.19) was higher than the corresponding value for the 68Ga-RGD tetramer, with a range of 0.78 to 1.31 (median, 0.99). Furthermore, FD, VB, K1, and RBP for 68Ga-BZH3 were generally higher than the corresponding values for the 68Ga-RGD tetramer, whereas k3 was slightly higher for 68Ga-RGD tetramer.
As a parameter that reflects receptor binding, the increase of K1 for 68Ga-BZH3 indicated higher expression of bombesin receptors than that of the ανβ3 integrin in neuroendocrine tumors. 68Ga-BZH3 seems better suited for diagnosis of NETs owing to higher global tracer uptake.

Full text

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ORIGINAL RESEARCH
Comparison between68Ga-bombesin (68Ga-BZH3)
and the cRGD tetramer68Ga-RGD4studies in an
experimental nude rat model with a
neuroendocrine pancreatic tumor cell line
Open Access
Caixia Cheng1*, Leyun Pan1, Antonia Dimitrakopoulou-Strauss1, Martin Schäfer2, Carmen Wängler3, Björn Wängler3,
Uwe Haberkorn1and Ludwig G Strauss1
Abstract
Objectives: Receptor scintigraphy gains more interest for diagnosis and treatment of tumors, in particular for
neuroendocrine tumors (NET). We used a pan-Bombesin analog, the peptide DOTA-PEG2-[D-tyr6, b-Ala11, Thi13,
Nle14] BN(6-14) amide (BZH3). BZH3 binds to at least three receptor subtypes: the BB1 (Neuromedin B), BB2
(Gastrin-releasing peptide, GRP), and BB3. Imaging of aνb3 integrin expression playing an important role in
angiogenesis and metastasis was accomplished with a68Ga-RGD tetramer. The purpose of this study was to
investigate the kinetics and to compare both tracers in an experimental NET cell line.
Methods: This study comprised nine nude rats inoculated with the pancreatic tumor cell line AR42J. Dynamic
positron emission tomography (PET) scans using68Ga-BZH3 and68Ga-RGD tetramer were performed (68Ga-RGD
tetramer: n = 4,68Ga-BZH3: n = 5). Standardized uptake values (SUVs) were calculated, and a two-tissue
compartmental learning-machine model (calculation of K1 - k4 vessel density (VB) and receptor binding potential
(RBP)) as well as a non-compartmental model based on the fractal dimension was used for quantitative analysis of
both tracers. Multivariate analysis was used to evaluate the kinetic data.
Results: The PET kinetic parameters showed significant differences when individual parameters were compared
between groups. Significant differences were found in FD, VB, K1, and RBP (p = 0.0275, 0.05, 0.05, and 0.0275
respectively). The 56- to 60-min SUV for68Ga-BZH3, with a range of 0.86 to 1.29 (median, 1.19) was higher than the
corresponding value for the68Ga-RGD tetramer, with a range of 0.78 to 1.31 (median, 0.99). Furthermore, FD, VB,
K1, and RBP for68Ga-BZH3 were generally higher than the corresponding values for the68Ga-RGD tetramer,
whereas k3 was slightly higher for68Ga-RGD tetramer.
Conclusions: As a parameter that reflects receptor binding, the increase of K1 for68Ga-BZH3 indicated higher
expression of bombesin receptors than that of the aνb3 integrin in neuroendocrine tumors.68Ga-BZH3 seems
better suited for diagnosis of NETs owing to higher global tracer uptake.
Keywords:68Ga-bombesin,68Ga-RGD tetramer, PET, kinetic modeling, neuroendocrine tumors
* Correspondence: c.cheng@dkfz.de
1Clinical Cooperation Unit Nuclear Medicine, German Cancer Research
Center, Heidelberg, Germany
Full list of author information is available at the end of the article
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© 2011 Cheng et al; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

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Introduction
During the past decade, the application of radiolabeled
somatostatin analogs in nuclear medicine for diagnostics
and therapy of neuroendocrine tumors has achieved
success and stimulated the research in receptor targeting
of additional tumor types [1]. Positron emission tomo-
graphy (PET) is the most efficient imaging method in
nuclear medicine because of its option of an absolute
activity determination, its better contrast resolution, and
its higher detection efficiency compared with conven-
tional g-cameras. PET with18F-fluorodeoxyglucose (18F-
FDG) is frequently used for oncologic applications to
assess tissue viability, thereby gain the staging and ther-
apy monitoring by qualitative analysis of SUV and quan-
titative evaluation based on the compartmental analysis
of kinetic parameters [2]. However, not all tumors are
18F-FDG avid, and in particular treated tumorous lesions
may demonstrate a low fluorodeoxyglucose (FDG)
uptake and can therefore not be delineated using FDG.
Therefore, new specific tracers are needed to enhance
the sensitivity and specificity of PET. One approach is
to study the expression of receptors to gain specificity.
Experimental data demonstrated enhanced bombesin
(BN) receptors in neuroendocrine tumors (NETs) [3-5].
Bombesin is an amphibian neuropeptide of 14 amino
acids that shows a high affinity for the human gastrin-
releasing peptide receptor (GRP-r, also known as BB2),
which is overexpressed on several types of cancer. In
addition, for the neuromedin B (BB1) and the bombesin
receptor subtype (BB3), bombesin also shows a high affi-
nity. Thus, radiolabeled BN and BN analogs may prove
to be specific tracers for diagnostic and therapeutic tar-
geting of GRP-r-positive tumors in nuclear medicine
[6-13]. We have reported68Ga-labeled bombesin may be
helpful for diagnostic reasons in a subgroup of patients
with GIST and recurrent gliomas [14,15].
The expression of GRP receptor in AR42J cell line has
been reported by other groups [16,17]. So far, the
expression of integrin aνb3 in AR42J cell line has not
been reported yet. However, the integrin aνb3 plays an
important role in angiogenesis and tumor metastasis. It
is expressed on activated endothelial cells as well as
some tumor cells [18]. Therefore, it is a promising ima-
ging target as a potential surrogate parameter of angio-
genic activity.
The68Ga-RGD tetramer68Ga-RGD4is a specific tra-
cer for the integrin aνb3 [19]. Herein, dynamic PET stu-
dies with68Ga-Bombesin were performed in AR42J
tumor-bearing mice to investigate the impact of comple-
mentary receptor scintigraphy on diagnosis and the
potential of a radionuclide treatment. Furthermore,
dynamic
comparison.
68Ga-RGD4 studies were performed for
Materials and methods
Synthesis of RGD4
Resins for peptide synthesis, coupling reagents, and
Fmoc-protected amino acids were purchased from
NovaBiochem. For analytical and semi-preparative high-
performance liquid chromatography (HPLC), an Agilent
1200 system was used. The columns used for chromato-
graphy were a Chromolith Performance (RP-18e, 100 to
4.6 mm, Merck, Germany) and a Chromolith (RP-18e,
100-10 mm, Merck, Darmstadt, Germany) column,
operated with flows of 4 and 8 mL/min, respectively.
ESI and MALDI were obtained with a Finnigan
MAT95Q and a Bruker Daltonics Microflex (Bruker
Daltonics, Bremen, Germany), respectively.
The compound (DOTA-comprising maleimide tetra-
mer (DOTA-Mal4)) was synthesized on solid support by
standard Fmoc solid-phase peptide synthesis as
described by Wellings et al. [20] on a standard rink
amide resin. After coupling of Fmoc-Lys(Mtt)-OH to
this resin (100 μmol), the Mtt-protecting group was
removed by successive incubation with 1.75% TFA in
DCM followed by coupling of tris-tBu-DOTA and
Fmoc-Lys(Fmoc)-OH under standard conditions. After
removal of both lysine Fmoc protecting groups using
deprotection times of twice 2 min and twice 5 min,
Fmoc-Lys(Fmoc)-OH was coupled twice. After removal
of all four lysine Fmoc protecting groups using depro-
tection times of twice 2 min and twice 10 min, maleimi-
dobutyric acid was coupled applying the standard
protocol. The product was cleaved from the solid sup-
port and deprotected using a mixture of TFA (trifluor-
oacetic acid)/TIS (triisopropylsilane)/H2O (95:2.5:2.5) for
45 min. The product was purified by semi-preparative
HPLC using a gradient of 0% to 30% MeCN in 6 min
and was obtained as a white solid upon lyophilization
(49.8 mg, 31.6 μmol, 32%). ESI-MS (m/z) for [M + H]+
(calculated): 1,576.76 (1,576.76) and [M + 2H]2+(calcu-
lated): 788.89 (788.88).
c(RGDfK)-PEG1-SH was synthesized on a preloaded
Fmoc-Asp(NovaSyn TGA)-Oall resin (100 μmol) to
which were subsequently coupled Fmoc-Gly-OH, Fmoc-
Arg(Pbf)-OH, Fmoc-Lys(Mtt)-OH, and Fmoc-D-Phe-OH
using standard coupling protocols. After allyl-deprotec-
tion, the peptide was cyclized and after removal of the
Mtt-protecting group by successive incubation with
1.75% TFA in DCM, Fmoc-PEG1-OH, and SATA (N-
succinimidyl-S-acetylthioacetate) were coupled. The pro-
duct was cleaved from the solid support and depro-
tected using a mixture of TFA (trifluoroacetic acid)/TIS
(triisopropylsilane)/H2O (95:2.5:2.5) for 45 min followed
by an incubation with a hydroxylamine-containing solu-
tion (H2O + 0.1%TFA/MeCN + 0.1%TFA/50% hydroxy-
lamine × HCl solution in water (750:750:25 μL)) for 5
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min. The product was purified by semi-preparative
HPLC using a gradient of 0% to 40% MeCN in 6 min
and was obtained as a white solid upon lyophilization
(33.2 mg, 40.4 μmol, 40%). ESI-MS (m/z) for [M + H]+
(calculated): 823.38 (823.37).
The conjugation of c(RGDfK)-PEG1-SH to DOTA-
Mal4was carried out to yield DOTA-comprising RGD
tetramer (DOTA-RGD4) as described before [21]. In
brief, a solution of c(RGDfK)-PEG1-SH (15.6 mg, 19.0
μmol) in phosphate buffer (500 μL, 0.1 M, pH 6.0) was
added to a solution of DOTA-Mal4(5 mg, 3.2 μmol) in
MeCN/phosphate buffer (0.1 M, pH 5.0) 1:1 (250 μL)
and the pH of the mixture was adjusted to 7.4 by the
addition of phosphate buffer (0.5 M, pH 7.4, approxi-
mately 100 μL). After 10 min, the product was purified
by semi-preparative HPLC using a gradient of 0% to
40% MeCN in 6 min and was obtained as a white solid
upon lyophilization (13.8 mg, 2.8 μmol, 89%). ESI-MS
(m/z) for [M + Kcomplexed+ 4H]4+(calculated): 1,227.05
(1,227.05) and (m/z) for [M + Kcomplexed+ Nasalt+ 4H]4
+(calculated): 1,232.55 (1,232.55).
DOTA is 1,4,7,10-tetraazacyclododecane-N,N’,N″,N’″-
tetraacetic acid. PEG is ethylene glycol (2-aminoethyl-
carboxy-methyl ether). RGD is a cyclic pentapeptide
containing the amino acid sequence D-Phe-Lys-Arg-
Gly-Asp. Figure 1 shows the chemical structure of68Ga-
RGD4.
Synthesis of BZH3
BZH3 was prepared according to the method described
by Schuhmacher et al. [14]. BZH3 is DOTA-PEG2-[D-
Tyr6-b-Ala11-Thi13-Nle14]BN(6-14) amide.
Radiolabeling of BZH3 and RGD4
68Ga was used for labeling of both tracers and was
obtained from a68Ge/68Ga generator, which consists of
a column containing a self-made phenolic ion-exchanger
loaded with68Ge and coupled in series with a small-
sized anion-exchanger column (AG 1-X8 Cl-, mesh 200
to 400, Bio-Rad, Hercules, CA, USA) to concentrate
68Ga during elution. This generator provides68Ga with
an average yield of 60% for > 1.5 years.68Ga-BZH3 and
68Ga-RGD4were prepared according to the method
described by Schuhmacher et al. and Jae Min Jeong et
al., respectively [22,23]. The specific activity (the amount
of radioactivity per peptide amount) of68Ga-BZH3 and
68Ga-RGD4were measured to be 28 and 22 MBq/nmol,
respectively, which is sufficient for an efficient receptor
imaging in vivo. Furthermore, a binding affinity of 4.973
μM (IC50) was obtained for68Ga-RGD4binding to
HN
O
O
HO
HN
O
NH
O
NH
NH
O
HN
NH
NH2
N
H
O
O
O
O
HN
O
S
H
N
NH
HN
H2N
O
O
HN
O
O
N
N
N
N
O
HO
O
O
O
O
HN
O
O
N
O
O
N
O
O
NH
O
HN
HN
O
N
O
O
O
N
O
O
H
N
O
O
HO
O
NH
O
NH
O
HN
N
H
O
H
N
NH
H2N
HN
O
O
O
NH
O
S
NH
O
O
O
OH
H
N
O
HN
O
N
H
HN
O
NH
HNNH2
NH
O
O
O
H
N
O
S
N
H
O
O
HO
NH
O
NH
O
HN
H
N
O
HN
NH
HN
NH2
O
O
O
O
NH
OS
68Ga
Figure 1 The chemical structure of68Ga-RGD4.
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aνb3, which indicated that68Ga-RGD4could be used as
PET tracer with aνb3-positive neuroendocrine pancrea-
tic tumor cell line.
Cell lines
The AR42J cell line, derived from a rat exocrine pan-
creas neuroendocrine tumor, was used. Cells were
obtained from the European Collection of Cell Cultures
and were grown in RPMI 1640 medium supplemented
with 2 mmol/L glutamine and 10% fetal calf serum.
Adherent cells were dislodged with trypsin/ethylenedia-
minetetraacetic acid (0.02%: 0.05%, w/v). For PET-stu-
dies, 5 × 106cells in 200 μl RPMI without supplements
were inoculated subcutaneously in the right hind leg of
Wistar rats.
PET
The study included nine AR 42 J tumor-bearing nude
rats. We grouped all rats according to PET tracers (B,
68Ga-BZH3 and R,68Ga-RGD4), used in dynamic PET
scanning, as noted in Table 1. Dynamic PET studies
were performed for 60 min after the intravenous appli-
cation of 10 to 30 MBq68Ga-RGD4or 20 to 40 MBq
68Ga-BZH3, using a 28-frame protocol (ten frames of 30
s, five frames of 60 s, five frames of 120 s, and eight
frames of 300 s). Two animals can be examined in par-
allel per scanning by a homemade injector (Figure 2). A
dedicated PET-CT system (Biograph™ mCT, 128 S/X,
Siemens Co, Erlangen, Germany) with an axial field of
view of 21.6 cm with TrueV, operated in a three-dimen-
sional mode, was used for all animal studies. The system
provides the simultaneous acquisition of 369 transverse
slices with a slice thickness of 0.6 mm. The animals
were positioned in the axial plane of the system to
maintain the best resolution in the center of the system.
All PET images were attenuation-corrected and an
image matrix of 400 × 400 pixels was used for iterative
image reconstruction (voxel size 1.565 × 1.565 × 0.6
mm) based on the syngo MI PET/CT 2009C software
version. After the end of the dynamic series an ultrahigh
resolution CT with 85 mA, 80 kV and a pitch of 0.85
cm was performed for attenuation correction of the
acquired dynamic emission data. The reconstructed
images were converted to SUV images based on the for-
mula [24]: SUV = Tissue concentration (becquerel per
gram)/[injected dose (becquerel per gram))/body weight
(gram)]. The SUV 55 to 60 min post-injection was used
for the assessment of both tracers. The SUV images
were used for all further quantitative evaluations.
Dynamic PET data were evaluated using the software
package PMOD (provided courtesy of PMOD Technolo-
gies Ltd., Zuerich, Switzerland) [25,26]. Areas with
enhanced tracer uptake on transaxial, coronal, and sagit-
tal images were evaluated visually. A volume of interest
consists of several regions of interest over the target
area. Irregular regions of interest were drawn manually.
A detailed quantitative evaluation of tracer kinetics
requires the use of compartmental modeling. A two-tis-
sue-compartment model was used to evaluate the
dynamic studies. This methodology is standard, particu-
larly for the quantification of dynamic18F-FDG studies
[27,28].
In animals, a partial volume correction must be
applied to the data due to the small size of the input
and tumor volumes of interest (VOIs). Herein, the
recovery coefficient was 0.85 for a diameter of 8 mm
and 0.32 for a diameter of 3 mm based on phantom
measurements as well as the recent parameter settings
used with the reconstruction software. For the input
function the mean values of the VOI data obtained from
the heart were used. We used a preprocessing tool,
which allowed a fit of the input curve by a sum of up to
three decaying exponentials. The learning-machine two-
tissue-compartment model was used for the fitting and
provided five parameters: the transport parameters for
tracer into and out of the cell, K1 and k2, the para-
meters for phosphorylation and dephosphorylation of
intracellular tracer, k3 and k4, and the fractional blood
Table 1 Quantitative PET parameters for the both tracers’ kinetics of68Ga-BZH3 and68Ga-RGD4
Group
VB
K1
k2
k3
k4 RBPFD SUV Number
Mean
SD
Median
Minimum
Maximum
Mean
SD
Median
Minimum
Maximum
0.1037
0.0443
0.0903
0.0603
0.1646
0.0582
0.0102
0.0574
0.0468
0.0713
0.3717
0.0718
0.3506
0.3055
0.4862
0.2739
0.0479
0.2728
0.2304
0.3195
0.4808
0.1312
0.5216
0.2768
0.6103
0.5386
0.0985
0.5721
0.3952
0.6152
0.1235
0.0534
0.1177
0.0580
0.2005
0.1097
0.0298
0.1180
0.0678
0.1352
0.0986
0.0524
0.1005
0.0493
0.1746
0.0443
0.0052
0.0442
0.0382
0.0504
0.0765
0.0310
0.0607
0.0506
0.1202
0.0453
0.0048
0.0442
0.0414
0.0514
1.1198
0.3800
1.1425
1.0665
1.1495
0.9961
0.1184
1.0393
0.8215
1.0841
1.0837
0.1943
1.1865
0.8628
1.2884
1.0172
0.2172
0.9886
0.7833
1.3084
5
5
5
5
5
4
4
4
4
4
B
R
68Ga-BZH3 (B, n = 5) and68Ga-RGD4(R, n = 4)
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volume, also called vessel density (VB), which reflects
the amount of blood in the VOI. Following compart-
ment analysis, we calculated the global influx of tracer
from the compartment data using the formula: influx
= (K1 × k3)/(k2 + k3). Compared to the standard itera-
tive method, the machine learning method has the
advantage of a fast convergence and avoidance of over
fitting [29]. The model parameters were accepted when
K1 - k4 was less than 1 and VB exceeded 0. The unit
for the rate constants K1 to k4 was 1/min. In the case
of68Ga-BZH3 and68Ga-RGD4, K1 is associated with
receptor binding, k2 with displacement from the recep-
tor, k3 with cellular internalisation, and k4 with
externalisation.
Besides the compartmental analysis, a non-compart-
mental model based on the fractal dimension was used.
The fractal dimension is a parameter of heterogeneity
and was calculated for the time-activity data of each
individual volume of interest. The values fro fractal
dimension vary from 0 to 2, showing the deterministic
or chaotic distribution of tracer activity. We used a sub-
division of 7 × 7 and a maximal SUV of 20 for the cal-
culation of fractal dimension [30].
Statistical analysis
Statistical evaluation was performed with Stata/SE 10.1
(StataCorp, College Station, TX, USA). Statistical evalua-
tion was performed using the descriptive statistics and
scatter plots. The classification analysis was performed
using the GenePET software [31]. The software applies the
support vector machines (SVM) algorithm and provides a
classification analysis by optimizing a hyperplane between
the target variables. The algorithm for selection or elimi-
nation of variables, the feature ranking, can be based on
different criteria, e.g., F test, Mann-Whitney test, or the
SVM ranking feature elimination (SVM RFE) approach
[32]. The SVM RFE algorithm computes a multidimen-
sional weight vector for the PET variables and the square
of the vector is used to calculate the ranking criteria. For
comparison between two tracers, the two-sided Wilcoxon
rank-sum test was applied for all PET parameters, SUV,
and the fractal dimension (FD), using a single parameter
analysis. P values < 0.05 were considered significant.
Results
Figure 3 is a representative set of time-activity data
obtained with a image-derived measured blood input


Figure 2 Experimental setting. Our experimental setting demonstrating two rats prior to positioning in the PET-CT scanner and homemade
injector (two animals can be examined in parallel per scanning).
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function, which illustrates the good statistical quality of
the data and model fit using nonlinear regression and
two-tissue-compartment model.
Table 1 presents the mean, median, minimum, and
maximum values as well as the standard deviation for
the SUV, FD, and kinetic values of all parameters for
both tracers (Table 1). In the whole paper, B and R
represent68Ga-BZH3 and68Ga-RGD4respectively. The
Wilcoxon rank-sum test was used to reveal statistically
significant differences between all variables.
Figure 4 shows an example of 3D fused PET-CT
images for68Ga-RGD4and68Ga-Bombesin. The68Ga-
BZH3 image clearly showed enhanced
uptake in the tumor area in the lower leg.68Ga-BZH3
68Ga-BZH3

Figure 3 Representative blood input function (filled yellow squares) and tumor tissue tracer concentrations. Obtained in a 60-min
acquisition sequence (left,68Ga-BZH3; right,68Ga-RGD4). Filled green squares are tracer concentrations for each image, while the smooth line
through the data is the two-tissue-compartment model fit to the data. A smooth line (interpolation) was drawn through the input function data
to illustrate the shape of the curve.
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uptake in the evaluated tumor lesions was generally
higher than68Ga-RGD4uptake.
Box plots of68Ga-BZH3 and68Ga-RGD4uptake (56-
to 60-min SUV) in tumor tissue and FD are presented
in Figure 5. The corresponding quantitative data and
the corresponding P values are presented in Table 1 and
2. The 56- to 60-min SUV for68Ga-BZH3, with a range
of 0.86 to 1.29 (median 1.19) was higher than the corre-
sponding value for68Ga-RGD4, with a range of 0.78 to
1.31 (median 0.99). However, there was no significant
difference in the median SUV between the two tracers.
Interestingly, the median FD for68Ga-BZH3 (1.1425)
was significantly higher as compared with68Ga-RGD4
(1.039) (P = 0.0275) at a level of P < 0.05. The transport
rate constants K1 and k3 (1/min), the receptor binding
potential (RBP) and the vascular fraction VB of both tra-
cers are presented in Figure 6. Kinetic data demonstrate
higher the values of VB for68Ga-BZH3 as compared
with68Ga-RGD4(0.0903 vs. 0.0574); furthermore, VB
was relatively low for both tracers, not exceeding 0.2.
Furthermore, the values of K1 and RBP were higher for
68Ga-BZH3 than the corresponding values for68Ga-
RGD4(0.3506 vs. 0.2728 and 0.0607 vs. 0.0442, respec-
tively). In addition, comparable k3 values without

Figure 4 3D fused PET-CT images for68Ga-RGD4(left) and68Ga-Bombesin (right). Besides the tumor region in the lower leg. The tracer
uptake in other sites covered heart (exhibit for68Ga-RGD4), liver, kidney, urinary bladder, and testicles (left for male rat).
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significant difference for both tracers were displayed in
Figure 6.
Discussion
PET with FDG is frequently used for oncological appli-
cation to assess tissue viability. However, owing to the
low FDG uptake in some tumor types, like in the neu-
roendocrine carcinomas, there is a need for new radio-
tracers. One idea is to study the expression of different
receptors in order to guide diagnostics and even more
therapy in that direction, e.g. using a radionuclide-based
therapy. NETs originate mostly from the gastroentero-
pancreatic tract and express specific receptors like
amine and peptide receptors (somatostatin, vasointest-
inal peptide receptors, bombesin, cholecystokinin, gas-
trin and/or substance P) [33]. Adams et al. reported the
comparison of different tracers in detecting malignant
NETs and revealed that increased FDG uptake was asso-
ciated with malignancy [34]. In nude mice bearing the
AR4-2J tumor, tumor uptake of both90Y and111In-
DOTATOC 4 h after injection was five times higher
than with111In-DTPA-octreotide [35]. We had reported
on68Ga-DOTATOC studies in patients with NETs and
an enhanced uptake in metastases of NETs [36].
Furthermore, we have shown that the global DOTA-
TOC uptake in NETs is mainly dependent on k1 (recep-
tor binding) and VB (fractional blood volume) and less
on the k3 (internalization).68Ga-DOTATOC was better
suited than18F-FDG for the diagnosis of metastatic
NETs. The68Ga-DOTATOC uptake was also used as a
parameter for a radionuclide therapy with90Y-DOTA-
TOC. Patients with lesions demonstrating an enhanced
68Ga-DOTATOC uptake (> 5.0 SUV) were selected for
radionuclide therapy [37].
Bombesin and the two mammalian bombesin-like pep-
tides, BB1 and BB2 regulate many biologic response
processes through activation of distinct receptor sub-
types, including modulation of smooth muscle contrac-
tion, secretion of neuropeptides and hormones, as well
as stimulation of cell growth [38,39]. Activation of
Figure 5 Box-Whiskers plots of the median SUV 55 to 60 min and FD for both tracers. *P values < 0.05. B,68Ga-BZH3; R,68Ga-RGD4.
Table 2 The value of statistically significant level P using
the Wilcoxon rank-sum test
P
VB
K1
k2
k3
k4 RBPSUV FD
0.05*0.05* 0.4624 0.80650.05* 0.0275*0.8065 0.0275*
*P values < 0.05 were considered significant.
Cheng et al. EJNMMI Research 2011, 1:34
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neuromedin B (BB1) receptors has been reported in var-
ious human cancers [39]. Experimental studies demon-
strated an enhanced bombesin receptor expression in
several human adult glioblastoma cell lines as well as in
two pediatric human glioblastoma cell lines [38]. We
reported on an enhanced68Ga-BZH3 uptake in a sub-
group of patients with gastrointestinal stromal tumors
[15], and quantitative68Ga-BZH3 studies were helpful
in patients with recurrent gliomas for tumor grading
and the differentiation between high- and low-grade
tumors [14]. In addition, other bombesin analogues
64Cu-,99mTc-,188Re-,177Lu-,90Y-, and111In have been
reported to be promising radiotracers for PET imaging
of many human cancers overexpressing the GRP recep-
tor such as breast cancer and prostate carcinoma
[6-13,40,41].
Integrins play a key role in angiogenesis and tumor
metastasis by mediating tumor cell invasion and move-
ment across blood vessel, whereas integrins expressed
on endothelial cells modulate cell migration and survival
during the angiogenic cascade. A common feature of
many integrins like aνb3 is that they bind to extracellu-
lar matrix proteins via the three amino acid sequence
arginine-glycine-aspartic acid(RGD)[42,43].
Radiolabeled RGD-peptides, the integrin aνb3-specific
tracers, have been developed for PET and SPECT ima-
ging. A mass of data suggested that aνb3 expression can
be quantified by radiolabeled RGD-peptides [44-46]. In
this study,68Ga-BZH3 and68Ga-RGD4were used as tra-
cers for PET to assess the receptor expression in AR42J
tumor-bearing nude rats by comparison.
Quantitative dynamic PET provides the possibility for
absolute tracer quantification and is superior to static
images, which are widely used, but do not provide infor-
mation on tracer kinetics. Furthermore, the use of a
two-compartment model is the superior approach for
the assessment of tracer kinetics, and is accepted for
research purposes [27]. Concerning the68Ga-BZH3
kinetics, k1 is a parameter that reflects the receptor
binding and k3 is a parameter that reflects the internali-
zation of the tracer. A lower receptor binding of68Ga-
BZH3 was reported in gliomas as compared with68Ga-
DOTATOC in meningiomas, but higher internalization,
were proved [47]. In the present study, the comparison
of the68Ga-BZH3 kinetics with the68Ga-RGD4kinetics
in the ARJ 42 tumor-bearing nude rats revealed higher
mean values of k1 for68Ga-BZH3 (median, 0.3506) as
compared with
68Ga-RGD4 (median 0.2728), and

Figure 6 Box-Whiskers plots of median values for both tracers for vB, K1, k3 and RBP. *P values < 0.05). B,68Ga-BZH3; R,68Ga-RGD4.
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comparable k3 values (median, 0.1177 vs. 0.1180).
According to these data, the tracers’ accumulation in
this neuroendocrine tumor cell line is primarily depends
on the receptor binding and less on the internalization.
Generally,68Ga-BZH3 uptake was lower than18F-FDG
[15]. Herein, we found68Ga-BZH3 uptake was higher
than that of68Ga-RGD4, and the values were relatively
comparable in comparison to that reported in gliomas
[14]. In particular, there were significant differences
between VB, K1, k4, RBP, and FD. The fractional blood
values VB of68Ga-BZH3 were higher than that of68Ga-
RGD4(median, 0.0903 vs. 0.0574), however for both tra-
cers they are low in comparison to those reported for
other tracers, like68Ga-DOTATOC and18F-FDG. This
is in accordance to previous published data, e.g. in mela-
noma patients and confirm the hypothesis that the abso-
lute value of VB depend on the applied tracer [48]. The
VB and RBP values for68Ga-BZH3 were more spread
out than those determined for68Ga-RGD4. A possible
explanation is that the tracer uptake of68Ga-RGD4was
generally lower than that of68Ga-BZH3.
Cancer is often characterized by chaotic, poorly regu-
lated growth. Recent studies have shown that fractal
geometry can be useful to describe the pathological
architecture of tumors and angiogenesis. Fractals can be
useful measures of pathologies of the vascular architec-
ture, the tumor border, and the cellular morphology
[49]. The FD is used to characterize the chaotic nature
of the tracer’s distribution in primary tumors and
metastases, based on the box counting procedure of
chaos theory, for the analysis of dynamic PET data. In
the present study, FD values for68Ga-BZH3 were ran-
ged from 1.066 to 1.150 (median, 1.142), higher than
that for68Ga-RGD4(median, 0.989), but both are lower
compared with those measured in malignancies with dif-
ferent tracers, such as68Ga-DOTATOC,18F-FDG,15O-
water, and
[48,50].
18F-DOPA (a median FD exceed 1.25)
Conclusion
In general, a high SUV indicates high receptor binding.
The preliminary results give evidence for a higher BZH3
uptake, which is related to higher bombesin and neuro-
medin B gene expression than that of aνb3 in neuroen-
docrine tumors.68Ga-BZH3 seems better for diagnosis
of NETs owing to higher values of global tracer uptake.
Further studies with a larger number of animals and in
patients are needed to confirm these preliminary results.
Author details
1Clinical Cooperation Unit Nuclear Medicine, German Cancer Research
Center, Heidelberg, Germany2Department of Radiopharmaceutical
Chemistry, German Cancer Research Center, Heidelberg, Germany3University
Hospital Munich, Department of Nuclear Medicine, Ludwig Maximilians-
University Munich, Munich, Germany
Authors’ contributions
CC participated in the whole study and wrote the manuscript. LP performed
the statistical analysis. MS, CW, and BW carried out the synthesis of tracers.
ADS and LGS did the design of the study and revised the whole manuscript.
UH gave financial support. All authors read and approved the final
manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 28 July 2011 Accepted: 13 December 2011
Published: 13 December 2011
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doi:10.1186/2191-219X-1-34
Cite this article as: Cheng et al.: Comparison between68Ga-bombesin
(68Ga-BZH3) and the cRGD tetramer68Ga-RGD4studies in an
experimental nude rat model with a neuroendocrine pancreatic tumor
cell line. EJNMMI Research 2011 1:34.
Cheng et al. EJNMMI Research 2011, 1:34
http://www.ejnmmires.com/content/1/1/34
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