Imaging CXCR4 Expression in Human Cancer Xenografts: Evaluation of Monocyclam Cu-64-AMD3465

Russell H Morgan Department of Radiology and Radiological Science, Johns Hopkins University, Baltimore, Maryland 21231, USA.
Journal of Nuclear Medicine (Impact Factor: 6.16). 06/2011; 52(6):986-93. DOI: 10.2967/jnumed.110.085613
Source: PubMed


The chemokine receptor 4 (CXCR4) is overexpressed in several cancers and metastases and as such presents an enticing target for molecular imaging of metastases and metastatic potential of the primary tumor. CXCR4-based imaging agents could also be useful for diagnosis, staging, and therapeutic monitoring. Here we evaluated a positron-emitting monocyclam analog, (64)Cu-{N-[1,4,8,11-tetraazacyclotetradecanyl-1,4-phenylenebis(methylene)]-2-(aminomethyl)pyridine} ((64)Cu-AMD3465), in subcutaneous U87 brain tumors and U87 tumors stably expressing CXCR4 (U87-stb-CXCR4) and in colon tumors (HT-29) using dynamic and whole-body PET supported by ex vivo biodistribution studies. Both dynamic and whole-body PET/CT studies show specific accumulation of radioactivity in U87-stb-CXCR4 tumors, with the percentage injected dose per gram reaching a maximum of 102.70 ± 20.80 at 60 min and tumor-to-muscle ratios reaching a maximum of 362.56 ± 153.51 at 90 min after injection of the radiotracer. Similar specificity was also observed in the HT-29 colon tumor model. Treatment with AMD3465 inhibited uptake of radioactivity by the tumors in both models. Our results show that (64)Cu-AMD3465 is capable of detecting lesions in a CXCR4-dependent fashion, with high target selectivity, and may offer a scaffold for the synthesis of clinically translatable agents.


Available from: Sridhar Nimmagadda
A study of the use of
Cu-AMD3465 as a PET imaging
agent has shown it capable of detecting lesions in a CXCR4-
dependent fashion with high tumor-to-muscle ratios.
In the PET/CT images shown here, CXCR4 expression
in subcutaneous U87 brain tumor xenografts is seen.
This positron-emitting monocyclam analog may serve as
a suitable scaffold for the synthesis of clinically translatable
imaging agents.
See page 989.
Volume 52 | Number 6 | June 2011
The Of cial Publication of
The Journal of Nuclear Medicine
June 2011 • Volume 52 • Pages 8411004
The Journal of Nuclear Medicine
Page 1
Imaging CXCR4 Expression in Human Cancer Xenografts:
Evaluation of Monocyclam
Ravindra A. De Silva, Kevin Peyre*, Mrudula Pullambhatla*, James J. Fox, Martin G. Pomper, and Sridhar Nimmagadda
Russell H. Morgan Department of Radiology and Radiological Science, Johns Hopkins University, Baltimore, Maryland
The chemokine receptor 4 (CXCR4) is overexpressed in several
cancers and metastases and as such presents an enticing
target for molecular imaging of metastases and metastatic
potential of the primary tumor. CXCR4-based imaging agents
could also be useful for diagnosis, staging, and therapeutic
monitoring. Here we evaluated a positron-emitting monocyclam
nylenebis(methylene)]-2-(aminomethyl)pyridine} (
in subcutaneous U87 brain tumors and U87 tumors stably
expressing CXCR4 (U87-stb-CXCR4) and in colon tumors
(HT-29) using dynamic and whole-body PET supported by ex
vivo biodistribution studies. Both dynamic and whole-body
PET/CT studies show specific accumulation of radioactivity in
U87-stb-CXCR4 tumors, with the percentage injected dose per
gram reaching a maximum of 102.70 6 20.80 at 60 min and
tumor-to-muscle ratios reaching a maximum of 362.56 6
153.51 at 90 min after injection of the radiotracer. Similar spe-
cificity was also observed in the HT-29 colon tumor model.
Treatment with AMD3465 inhibited uptake of radioactivity by
the tumors in both models. Our results show that
AMD3465 is capable of detecting lesions in a CXCR4-depend-
ent fashion, with high target selectivity, and may offer a scaffold
for the synthesis of clinically translatable agents.
Key Words: PET; tumor microenvironment; chemokine;
stem cells; molecular imaging; colon cancer; brain cancer;
J Nucl Med 2011; 52:986–993
DOI: 10.2967/jnumed.110.085613
Compelling evidence demonstrates that chemokine re-
ceptor 4 (CXCR4) expression plays an important role in
several diseases including HIV, cancer, and lupus (13).
Although CXCR4 was discovered as a coreceptor for
HIV-1 cell entry in the 1990s (4), recently much attention
has been paid to investigating its role in cancer progression
and metastasis. CXCR4 is overexpressed in more than 20
different human cancers (4,5); is required for tumor devel-
opment, growth, vascularization, and metastases (5,6); and
is considered a therapeutic target (7).
CXCR4 has been shown to be a prognostic factor
in several cancers, includ ing colorectal carcinoma (CRC).
CXCR4 is differentially expressed in CRC and is required
for the outgrowth of colon cancer metastasis (8,9). CXCR4
expression in primary CRC is associated with recurrence,
poor survival, and liver metastasis (8). Most recently, in
patients treated with the folinic acid, flu orouracil, and
oxaliplatin regimen, enhanced CXCR4 expression was
reported in resected liver metastases, suggesting that
CXCR4 may be contributing to drug resistance and i ndi-
c ating an expanding role of CXCR4 in cancer biology
In addi tion to the aforementioned biologic roles, several
comprehensive studies have demonstrated that CXCR4 is
necessary for cancer metastasis (11,12). Homing of cancer
cells to the common destinations of cancer metastas is is
mediated through the binding of CXCR4 to its cognate
ligand, CXCL12, which is secreted in tissues that eventu-
ally harbor metastases (6,13). Extensive immunohisto-
chemical studies have revealed that tumors with elevated
CXCR4 expression often have an aggressive phenotype and
that metastases often have elevated expression, com pared
with the primary tumor (1416). Inhibition of CXCR4–
CXCL12 signaling by antibodies, peptide analogs, small
molecules, or small interfering RNA knockdown has dem-
onstrated reduced metastatic burden in orthotopic and
metastatic models of various cancers (11,12,1722). Taken
together, those preclinic al and clinical observations demon-
strate that CXCR4 plays a critical role in metastasis. In
addition, the pleiotropic activity of CXCR4 and CXCL12
is critical for neural, vascular, and hematopoietic organo-
genesis, underscoring the need to stratify the patient pop-
ulation with high tumor CXCR4 levels. Accordingly,
development of imaging agents that can noninvasively
and repeatedly detect CXCR4 expression levels may prove
useful in managing cancer patients. CXCR4-targeted imag-
ing agents can be used to evaluate primary tumors for ele-
vated CXCR4 expression and therapeutic intervention, to
screen for secondary metastatic spread to both local and
distant sites, and for therapeutic monitoring.
Received Dec. 13, 2010; revision accepted Feb. 28, 2011.
For correspondence or reprints contact: Sridhar Nimmagadda, Johns
Hopkins Medical Institutions, 1550 Orleans St., CRB II, #4M07, Baltimore,
MD 21231.
*Contributed equally to this work.
COPYRIGHT ª 2011 by the Society of Nuclear Medicine, Inc.
Page 2
To image CXCR4 expression in tumor models, macro-
molecular agents such as
In- and
F-labeled pept ides
I-labeled monoclonal antibodies have been investi-
gated using either SPECT/CT or PET (2325). Others and
our own group reported the imaging of CXCR4 expression
using a low-molecular-weight agent,
Cu-AMD3100, in
control and tumor-bearing mice (26,27). Our studies with
Cu-AMD3100 in human cancer xenografts showed clear
detection of CXCR4 expression in an orthotopic breast
cancer model and in experimental lung metastases. Though
promising, the bicyclam AMD3100 has a relatively low
affinity (;651 6 37 nM) (28) and a structurally restricted
scaffold. In search of agents that are amenable to structural
modification, we pursued the monocyclam analog {N-
lene)]-2-(aminomethyl)pyridine} (AMD3465) to image
CXCR4 expression. Compared with AMD3100, AMD3465
has higher affinity, reduced size, and charge (29). The prop-
erties of AMD3465 as a CXCR4 inhibitor have been well
characterized (30). AMD3465 is reported to have a 50%
inhibitory concentration (IC50) of 41.7 6 1.2 nM and
12.07 6 2.02 nM for
I-CXCL12 binding and calcium
flux stimulated by stromal-derived factor-1a (29,30), re-
spectively. Also, AMD3465 was shown to be 10-fold more
effecti v e as a CXCR4 antagonist than the bicyclam AMD3100
(31). Because of those favorable qualities, we examined the
potential of AMD3465 as an imaging agent for CXCR4
expression. We used the ability of the cyclam to form
strong complexes with copper to develop
as an imaging agent for PET. Here, we report a detailed
evaluation of
Cu-AMD3465 in human subcutaneous brain
tumor xenografts stably expressing CXCR4 by whole-body
pseudodynamic imaging, whole-body static imaging, and
ex vivo biodistribution studies. We also show the poten-
tial of
Cu-AMD3465 to image graded levels of CXCR4
expression in a colon cancer model that has an intermediate
level of CXCR4, compared with that found in the brain
tumor model.
Cell Lines
All cell culture reagents were purchased from Invitrogen
unless otherwise specified. The human glioblastoma cell line
U87 and colorectal adenocarcinoma cell line HT-29 were pur-
chased from Am erican Type Culture Collection and cultured in
our laboratory in minimum essential medium and McCoy 5A
medium supplemented w ith 10% fetal bovine serum, 100 units
of penicillin per milliliter, and 100 mg of streptomycin per
milliliter, resp ectively. A U87 c e ll line st a bly tra nsfe cted w ith
human CD4 and CXCR4 (U 87-stb-CXCR4) was obtained from
the National Institutes of Hea lth AIDS Research Reference
Reagent Program (32) and cultured in Dulbecco’s modified
Eagle’s medium supplemented with 15% fetal bovine serum,
1 mg of puromycin per milliliter, 300 mg of G418 per milliliter,
100 units of penicillin per milliliter, and 100 m g of streptomycin
per milliliter. Cell lines were maintained in a humidified incu-
bator with 5% CO
Radiopharmaceutical Preparation
AMD3465 was a kind gift from Genzyme Corp., and
was obtained from the University of Wisconsin.
AMD3465 was radiolabeled with
using a standard
procedure, as described previously (26). Briefly, 200 mgof
AMD34656HCl was added to 370–740 MBq (10–20 mCi) of
concentrated in vacuo and adjusted to an approximate
pH of 5–5.5 with 0.1 M sodium acetate, and the pH-adjusted
reaction mixture was heated at 55–60C for 45 min.
AMD3465 was purified on a reversed-phase high-performance
liquid chromatography system (Varian) using a C-18 (Luna,
5 mm, 10 · 250 mm; Phenomenex) semipreparative column. A
mobile phase with 15% MeOH (with 0.1% trifluoroacetic acid)
and 85% H
O (with 0.1% trifluoroacetic acid) at a flow rate of
5 mL/min was used.
Cu-AMD3465 was collected between 16
and 18 min, dried, and diluted in saline for cell and animal studies.
Radioactive signal was detected using a single-channel radiation
detector (model 105S; Bioscan) with ultraviolet absorbance moni-
tored at 266 nm.
Determination of Partition Coefficient
The partition coefficient (log P) of
Cu-AMD3465 complex was
determined by adding 148–185 kBq (4–5 mCi) of the complex to a
solution containing 1 mL of water and 1 mL of octanol (n 5 3). The
resulting solution was then shaken well for 1 h at room temperature.
From each phase, 100-mLaliquotswere(n 5 3) counted separately.
The partition coefficient was calculated as a ratio between counts in
the octanol phase to counts in the water phase. An average log P
value was obtained from those ratios.
Flow Cytometry
Surface CXCR4 expression levels were analyzed using a
CXCR4 monoclonal antibody (clone 44716) conjugated to
phycoerythrin (R&D Systems) according to procedures described
previously (26).
Receptor Binding Assays
U87, U87-stb-CXCR4, and HT-29 cells seeded in 6-well plates
at 60%–80% confluence were used for receptor binding assays.
Cells were incubated with 37 kBq/mL (1 mCi/mL) of
AMD3465 in phosphate-buffered saline binding buffer (containing
5 mM MgCl
,1 mM CaCl
, 0.25% bovine serum albumin, pH 7.4)
for 30 min at 4C. After incubation, cells were washed quickly 4
times with 4C binding buffer, trypsinized using nonenzymatic
buffer, and cell-associated activity was determined in a g-spec-
trometer (1282 Compugamma CS; Pharmacia/LKB Nuclear, Inc.).
For internalization assays, cells were detached using non-
enzymatic buffer, and aliquots of 1 million cells per tube were
incubated with 37 kBq (1 mCi) of
Cu-AMD3465 per milliliter
for various times up to 4 h at 4Cor37C in the phosphate-
buffered saline binding buffer. Assuming minimal receptor endo-
cytosis at 4C, the internalization assay was performed only with
cells incubated at 37C. At 15-, 30-, 60-, and 240-min intervals,
the medium was removed and cells were washed once with bind-
ing buffer followed by a mild acidic buffer (50 mM glycine, 150
mM NaCl [pH 3.0]) at 4C for 5 min. Then the acidic buffer was
collected, and cells were washed twice with binding buffer. Pooled
washes (containing cell surface–bound
Cu-AMD3465) and cell
pellets (containing internalized
Cu-AMD3465) were counted in
an automated g-counter along with the standards. All of the radio-
activity values were converted into percentage of incubated dose
(%ID) per million cells. Experiments were performed in triplicate
PET OF CXCR4 EXPRESSION De Silva et al. 987
Page 3
and repeated 3 times. Data were fitted according to linear regres-
sion analysis using PRIZM software.
Animal Models
All experimental procedures using animals were conducted
according to protocols approved by the Johns Hopkins Animal
Care and Use Committee. Female nonobese diabetic (NOD)/severe
combined immune deficient (SCID) mice (age, 6–8 wk; weight, 25–
30 g) were purchased from the Johns Hopkins Immune Compro-
mised Animal Core. Mice were implanted subcutaneously with U87
and U87-stb-CXCR4 cells (4 · 10
cells/100 mL) in the upper left
and right anks, respectively. Similarly, HT-29 colon adenocar-
cinoma cells (4 · 10
cells/100 mL) were inoculated in the upper
right flanks of the mice. Animals were used for biodistribution
and PET/CT experiments when the tumor size reached 300–
400 mm
PET/CT and Analysis
Dynamic and whole-bo dy PE T and CT images were acquired
on an eXplore VISTA small-animal PET (GE Healthcare) and an
X-SPECT small SPECT/CT system (Gamma Medica Ideas),
respectively. For imaging studies, mice were induced with 3%
and maintained under 1.5% isoflurane (v/v). Pseud odynamic
imaging studies were performed on NOD/SCID mice bear ing
U87 and U87-stb-CXCR4 brain tumors. After intravenous
injection of
Cu-AMD3465 (range, 9.0–9.6 MBq; mean, 9.4
MBq), changes in radiotra cer accumulation were re corded over
the whole body using an imaging sequence consisting of 16
frames for a total of 70 min with variable dwell times (2 ·
60 s, 6 · 120 s, 4 · 240 s, and 4 · 600 s), as described previously
(33). After dynamic imaging, whole-body PET images (2 bed
positions, 15-min emission per bed position) were acquired at
1.5, 4, 8, and 24 h after injection of radiotrac er. For binding
specificity studies, a separate group of mice (n 5 2) was sub-
cutaneously administered w ith a blocking dose of AMD3465
(25 mg/kg) at 1 h b efore the injection of
Cu-AMD3465, and
another set of 2 mice was injected with
alone. In
colon cancer xenografts, whole-body images were acquired at
90 min after injection of
Cu-AMD3465 alone or with a block-
ing dose. After each PET scan, a CT scan was acquired in 512
projections for anatomic coregistration. PET emission data were
corrected for decay and d ead time and reconstructed using the
3-dimensional ordered-subse ts expectation maximization algo-
rithm. Data were analyzed based on regions of interest drawn
within the tumors or tissue. The percentage of injected dose per
gram (%ID/g) was c alculated based on a ca libration factor that
was determined using a known quantity of radioactivity. Time–
activity curves were calculated as an average from the region-of-
interest analyses of 4 mice. Data were analyzed using AMIDE
software (SourceForge), and volume-rendered images were gener-
ated using Amira 5.2.0 software (Visage Imaging Inc.).
Ex Vivo Biodistribution
NOD/SCID mice harboring either brain or colon xenografts
were injected intravenously with 740 kBq (20 mCi) of
AMD3465 in 200 mL of saline. At 30, 60, 90, 240, and 600 min
after injection, animals were sacrificed; blood, tumors, and
selected tissues were harvested and weighed; and the radioactivity
in the tissues was measured in an automated g-spectrometer. In the
case of colon cancer xenografts, biodistribution studies were per-
formed at 90 min after
Cu-AMD3465 injection.
To demonstrate the in vivo specificity of
Cu-AMD3465, a set
of mice received a blocking dose of AMD3465 (25 mg/kg) sub-
cutaneously at 1 h before the injection of radiotracer, and another
set of mice received
alone. At 90 min after injection
of radiotracers, ex vivo biodistribution studies were performed.
Aliquots of the injected dose were counted as reference standards
for the calculation of %ID/g values. A minimum of 4 animals per
time point was used.
HT-29 tumors from biodistribution experiments were used for
histologic examination. Sections of tumors were stained with
hematoxylin and eosin, and immunohistochemistry was performed
on tumor tissues as previously described using rabbit polyclonal
antibodies that recognize amino acids 328–338 of human CXCR4
(34), with minimum cross-reactivity to mouse CXCR4 (Imgenex)
Data Analysis
Statistical analysis was performed using PRIZM software. An
unpaired 2 tailed t test was used, and P values less than 0.05
for the comparison between tumors expressing high and tumors
expressing low CXCR4 uptake were considered to be significant.
FIGURE 1. In vitro characterization of CXCR4 expression. (A) Evaluation of surface CXCR4 expression in cancer cell lines used by flow
cytometry. (B) Uptake analysis of
Cu-AMD3465 in cancer cell lines. (C) Internalization of CXCR4 in U87-stb-CXCR4 cells: 4C (green),
37C total (red), 37C cell surface (orange), and 37C internalized (blue).
Page 4
Radiolabeling and Partition Coefficient
Radio–high-performance liquid chromatography assess-
ment showed the radiochemical purity of
to be greater than 98% (Supplemental Fig. 1; supplemental
materials are available online only at http://jnm.snmjour-, with specific radioactivity in the range of 6.0 6
3.1 GBq/mmol (162 6 84 mCi/mmol). The log P value for
the complex was 22.71 6 0.37.
Receptor Expression, In Vitro Radioligand Binding,
and Internalization
Flow cytometric analysis using CXCR4 antibody (clone
44716) revealed that 2%, 95%, and 30% of U87, U87-stb-
CXCR4, and HT-29 cells, respectively, were positive for
CXCR4 (Fig. 1A). The radioligand binding studies, sup-
ported by these data, show a gradual increase in the accu-
mulation of radioactivity on the order of U87-stb-CXCR4 .
HT-29 . U87 (Fig. 1B). The internalization assays were
done retrospectively to understand the retained radioactivity
uptake observed in the U87-stb-CXCR4 tumors. Over the
4-h incubation period, a continuous accumulation of radio-
activity was observed in cells incubated at 37C, with slopes
of 0.0360 6 0.0022 and 0.0492 6 0.0081 for internalized
and total fractions, respectively. For cells incubated at 4C, a
lower slope of 0.0215 6 0.0060 was observed (Fig. 1C).
The time–activity curves acquired in mice harboring U87
and U87-stb-CXCR4 subcutaneous brain tumor xenografts
over 70 min showed continuous
Cu-AMD3465 uptake in
U87-stb-CXCR4 tumors (Fig. 2). The %ID/g for U87-stb-
%ID/g values for U87-stb-CXCR4 tumors at 65 min were
nearly 3-fold greater than those of kidneys and liver and
nearly 18-fold greater than for the U87 tumors. The
whole-body PET/CT images acquired on U87 and U 87-
stb-CXCR4 brain xenografts at different times are shown
in Figure 3 and Supplemental Figure 2. The highest signal
intensity was observed in the U87-stb-CXCR4 tumors at
all time points. The best image contrast for U87-stb-
CXCR4 tumors was noticed at 90 min after injection.
Even after two
Cu half-lives and 24 h after injection
of the radiotracer, U87-stb-CXCR4 tumors were clearly
visible. Other organs with noticeable uptake were the liver
and kidneys. On the basis of these image contrast data, in
vivo speci ficity studies and im aging studies in the colon
tumor model were performed at 90 min after injection of
tracers. A 25 mg/kg blocking dose of AMD3465 resulted
FIGURE 2. Dynamic imaging of CXCR4 expression in subcuta-
neous U87 xenografts with
Cu-AMD3465. NOD/SCID mice bear-
ing U87 and U87-stb-CXCR4 glioblastoma xenografts on left and
right flanks, respectively, were given approximately 9.25 MBq (250
mCi) of
Cu-AMD3465 via tail vein injection, and whole-body pseu-
dodynamic imaging was performed for 70 min. Dynamic timeactiv-
ity curves are for various tissues: muscle (black), U87 (blue), kidney
(orange), liver (green), and U87-stb-CXCR4 (red). Data are mean 6
SD of 4 animals. Specific accumulation of radioactivity in U87-stb-
CXCR4 (red line) over U87 (blue line) is apparent.
FIGURE 3. PET/CT images of CXCR4
expression in subcutan eous bra in tumo r
xenografts with
Cu-AMD3465. NOD/
SCID mice bearing U87 and U87-
stb-CXCR4 glioblastoma xenografts on
left and right flanks, respectively, were
given approximately 9.25 MBq (250 mCi)
Cu-labeled radiotracers via tail
vein injection, and PET/CT images
were acquired. (A) Repre sentative trans-
axial PET, CT, and fused sections of
both tumors from a mouse injected with
(B) Representative volume-rendered whole-
body images of a
Cu-AMD3465 injected
mouse at 90 min (left), 4 h (middle), and 8 h
(right) after injection. All images were decay-
corrected and scaled to same maximum threshold value. B 5 bladder; K 5 kidney; L 5 liver; solid arrow 5 U87-stb-CXCR4 tumor; unfilled
arrow 5 U87 tumor.
PET OF CXCR4 EXPRESSION De Silva et al. 989
Page 5
in a significant r eduction of
Cu-AMD3465 uptake in
U87 and U87-stb-CXCR4 tumors, demonstrating the spe-
cificity of the radiotracer (Fig. 4A). Further evidence of
specificity was established by uniform uptake observed in
mice injected with only
ies of m ice w ith HT-29 tumors showed specific accumu-
lation of activity in the tumors (Fig. 5A). Also, the
blocking dose inhibited the radioactivity uptake in tumors,
further demonstrating the
Cu-AMD3465 specificity in
these tumor models (Fig. 5A). Supporting the CXCR4
expression levels observed in vit ro, the upta ke of
AMD3465 i n HT-29 tumors was interme diate betw een
that of U87 and U87-stb-CXCR4 tumors.
Ex Vivo Biodistribution
To validate the imaging studies and further quantify the
Cu-AMD3465 uptake, biodistribution studies were per-
formed at 30, 60, 90, 240, and 600 min in the brain tumor
model (Table 1). Those biodistribution studies showed the
highest uptake in the U87-stb-CXCR4 tumor at all time
points. Other tissues with noticeable uptake were the liver,
kidneys, and bone marrow. All other tissues had a low %ID/g,
in agreement with PET data. The tumor-to-muscle and tumor-
to-blood ratios for both tumors are shown in Table 2 and
support the best contrast observ ed in PET/CT at 90 min after
injection. The blocking dose of AMD3465 resulted in a
greater than 95% reduction in the tumor uptake of radioactiv-
bone marro w reduced roughly by half, suggesting that a sig-
nificant portion of that uptake was CXCR4-mediated. In mice
injected with 740 kBq (20 mCi) of
, relativ ely low
but uniform distrib ution of radioactivity was observed in sev-
eral tissues and both tumors.
Biodistribution studies in mice bearing HT-29 colon
tumors were performed at 90 min after injection (Fig. 5B).
The %ID/g for the HT-29 tumors was 5.62 6 0.90, and the
tumor-to-muscle and tumor-to-blood ratios were 24.78 6
2.38 and 8.05 6 0.60, respectively. No significant differ-
ences in radioactivity distribution were observed in other
tissues, compared with the brain tumor model. Representa-
tive images of sections stained with hem atoxylin and eosin
or CXCR4 are shown in Figure 5C.
We investigated a radiolabeled version of a known high-
affinity monocyclam inhibitor of CXCR4, AMD3465, as an
imaging agent for CXCR4 expression.
Cu-AMD3465 eval-
uation in U87 glioblastoma cells, U87 cells stably expressing
CXCR4, and a colon tumor expressing intermediate levels of
CXCR4 showed specific accumulation of radioactivity. The
specificity, target selectivity, and tumor-to-muscle ratios
observed suggest that
Cu-AMD3465, compared with other
known agents such as AMD3100, is a superior agent for PET
of CXCR4 expression and can delineate graded levels of
CXCR4 expression in tumors.
To determine the pharmacokinetics and establish the
specificity, we initially investigated
Cu-AMD3465 in sub-
cutaneous brain tumor models stably expressing CXCR4. In
vitro binding studies showed specific binding of radioactiv-
ity to U87-stb-CXCR4 cells, compared with the parental
U87 cells. Also, the in vivo dynamic and whole-body
PET/CT studies clearly demonstrated selective accumula-
tion of radioactivity in the U87-stb-CXCR4 tumors, starting
as early as 5 min after injection of the radiotracer. The
tumor-to-muscle ratios reached a maximum at 90 min after
injection of the radiotracer. The radioactivity uptake in liver
and kidneys reached a maximum within few minutes after
injection and remained constant up to 70 min. The radio-
tracer accumulation in the U87-stb-CX CR4 tumors was
evident even at 24 h. Retrospective in vitro analysis of
FIGURE 4. In vivo specificity of
Cu-AMD3465 in subcutaneous
brain tumor xenografts. NOD/SCID mice bearing U87 and U87-stb-
CXCR4 glioblastoma xenografts on left and right flanks, respec-
tively, were given approximately 9.25 MBq (250 mCi) of
radiotracers via tail vein injection, and PET/CT images were ac-
quired. (A) Representative volume-rendered whole-body images of
Cu-AMD3465, AMD3465 blocking dose (25 mg/kg) followed by
Cu-AMD3465, and
alone. All images were scaled to
same maximum threshold value. (B) Biodistribution analysis of
selected tissues from mice injected with 740 kBq (20 mCi) of radio-
tracers and sacrificed at 90 min after injection. All radioactivity val-
ues were converted into %ID/g. Biodistribution data are mean 6 SD
of 45 animals. B 5 bladder; K 5 kidney; L 5 liver; solid arrow 5
U87-stb-CXCR4 tumor; unfilled arrow 5 U87 tumor.
Page 6
Cu-AMD3465 uptake at 37C in U87-stb-CXCR4 cells
revealed that accumulation and retention of radioactivity
was due to receptor-mediated endocytosis and perhaps
binding of this radioactivity to cytosolic proteins. CXCR4-
mediated endocytosis has been previously described using
fluorescently labeled CXCL12 (35).
To validate further the utility of
Cu-AMD3465, we
pursued imaging and biodistribution studies in a colorectal
adenocarcinoma cell line, HT-29, that has 30% of its cells
expressing CXCR4 as determined by flow cytometry. Both
PET and biodistribution studies showed clear and specific
accumulation of radioactivity in tumors derived from those
cells. This radioactivity uptake in tumors was significantly
reduced in block ing studies, further validating the specific-
ity of
Cu-AMD3465. Furthermore, the %ID/g values of
HT-29 tumors were intermediate to U87 and U87-stb-
CXCR4 tumors, suggesting that variable levels of CXCR4
expression could be delineated using this imaging agent.
Previously, we investigated the bicyclam AM D3100 as a
PET agent for CXC R4 expression in the same glioblastoma
models, which allows for comparison between these 2
agents (26). The tumor-to-muscle and tumor-to-blood ratios
Cu-AMD3465 Ex Vivo Biodistribution Studies for Subcutaneous Brain Tumor Xenografts
Time (min)
Organ/tissue 30 60 90 240 600
Blood 1.93 6 0.30 0.90 6 0.08 0.73 6 0.23 0.61 6 0.09 1.20 6 0.36
Heart 1.19 6 0.16 1.00 6 0.20 0.90 6 0.25 0.93 6 0.06 0.72 6 0.07
Lung 4.48 6 0.56 3.70 6 0.99 2.78 6 0.47 3.39 6 0.85 2.10 6 0.13
Liver 32.08 6 2.03 35.06 6 7.77 36.15 6 1.79 33.65 6 3.13 27.27 6 2.57
Stomach 1.91 6 0.31 2.02 6 0.76 2.17 6 0.65 1.98 6 0.102 1.21 6 0.03
Spleen 6.23 6 1.17 5.68 6 1.75 4.28 6 1.00 4.88 6 0.42 2.64 6 0.12
Kidney 32.45 6 2.33 32.05 6 4.03 37.93 6 3.57 33.57 6 6.39 25.15 6 3.29
Small intestines 3.12 6 0.53 3.33 6 0.87 2.99 6 1.01 2.77 6 0.69 1.79 6 0.19
Muscle 0.75 6 0.52 0.37 6 0.12 0.30 6 0.13 0.23 6 0.03 0.19 6 0.02
Bladder 2.65 6 0.32 4.75 6 3.43 2.41 6 0.51 2.17 6 0.71 0.96 6 0.21
Pancreas 1.19 6 0.18 1.53 6 0.32 1.13 6 0.22 1.47 6 0.62 0.68 6 0.05
Bone 2.79 6 0.74 1.53 6 0.44 1.38 6 0.19 1.12 6 0.19 0.80 6 0.36
Bone marrow 6.39 6 0.00 10.22 6 4.97 9.18 6 2.04 8.10 6 3.38 9.42 6 3.88
U87 3.34 6 0.59 2.56 6 0.20 4.15 6 0.94 4.23 6 1.30 4.03 6 0.94
U87-stb-CXCR4 106.58 6 5.99* 102.70 6 20.80* 96.29 6 13.98* 50.95 6 6.38* 28.92 6 2.65*
*P , 0.0001, and comparative reference is U87 tumor uptake.
FIGURE 5. CXCR4 expression imaging
in colon cancer xenografts with
AMD3465. NOD/SCID mice harboring HT-
29 colon cancer xenografts in upper right
flank received either approximately 9.25
MBq (250 mCi) of
Cu-AMD3465 or
AMD3465 (25 mg/kg) blocking dose followed
Cu-AMD3465 via tail vein injection, and
whole-body images were acquired at 90 min
after injection. (A) Representative volume-
rendered whole-body images showing clear
and specific accumulation of radioactivity in
HT-29 tumors. (B) Biodistribution analysis of
selected tissues from mice injected with 740
kBq (20 mCi) of
Cu-AMD3465 and sacri-
ficed at 90 min after injection. All radioactivity
values were converted into %ID/g. Biodistri-
bution data are mean 6 SD of 6 animals. (C)
Representative microscopy images of 10-
mm-thick sections stained with hematoxylin
and eosin and CXCR4 obtained at ·10 mag-
nification from HT-29 tumors. A ·20 mag-
nification is also shown in boxed region.
H&E5 hematoxylin and eosin; L 5 liver;
solid arrow 5 HT-29 tumor.
PET OF CXCR4 EXPRESSION De Silva et al. 991
Page 7
Cu-AMD3465 at 90 min after injection are 7- to 8-
fold higher than those of
Cu-AMD3100, suggesting the
superiority of
Cu-AMD3465 as an imaging agent. Even
Cu-AMD3465 has improved affinity and kinetics,
compared with
Cu-AMD3100, considerable uptake in the
liver and kidneys was also observed. CXCR4 expression in
the liver and kidneys has been reported (34,36 ), and
reduced uptake has been observed in blocking studies in
these tissues, suggesting that some of the uptake seen is
receptor-mediated. Also, copper–cyclam complexes are
reported to be thermodynamically stable; however, several
in vitro studies have shown dissociation of copper from the
complex (37,38). Therefore, some of this accumulation
could be attributed to possible transchelation of
Cu from
Cu-AMD3465 to plasma proteins. Our previous studies
with bicyclam
Cu-AMD3100 have shown that copper
bound in the cyclam is stable for at least 4 h after injection
of the radiotrac er. These previous reports, combined with
the loss of binding observed in our blocking studies and
rather uniform uptake of radioactivity seen in
injected mice, suggest that the observed uptake is not
related to transchelation but is receptor-mediated. Further-
more, the radioactivity in the blood of mice injected wi th
Cu-AMD3465 was 4-fold less than the %ID/g values
in blood from the mice injected with
. If there
was significant transchelation, we would have observed
higher %ID/g values in blood in mice injected with
AMD3465; however, higher values were not observed. The
increase in %ID/g in blood at 600 min also suggests trans-
chelation of copper at later time points. Cross-bridged
cyclam-based inhibitors due to stable copper binding may
lead to improved copper-based CXCR4 imaging agents
(39). Also, possible metabolites such as
Cu-cyclam have
been shown to have low affinity for CXCR4 (40,41), sug-
gesting that the uptake observed in our studies is due to
We have synthesized and performed a detailed evaluation
Cu-AMD3465 as a PET agent to detect CXCR4
expression in tumor models with graded levels of CXCR4
expression. The results presented indicate that AMD3465
may serve as a suitable scaffold for the synthesis of clin-
ically translatable CXCR4-targeted imaging agents.
The costs of publication of this article were defrayed in
part by the payment of page charges. Therefore, and solely
to indicate this fact, this article is hereby marked “adver-
tisement” in accordance with 18 USC section 1734.
We thank the University of Wisconsin team for providing
; Gilbert Green, Jianhua Yu, David L. Huso,
Lauren Hoffman, and Christopher Endres for assistance
with imaging and image analysis; and Ronnie Mease,
Sangeeta Ray, and Catherine Foss for helpful discussions.
This work was partially supported by an anonymous family
fund translational research grant from the American Brain
Tumor Association (SN), the Maryland Stem Cell Research
Fund (SN), an Elsa U. Pardee Foundation grant (SN), and a
National Cancer Institute grant (U24 CA92871; MGP).
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Tumor-to-Tissue Ratios for
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Time (min)
Ratio 30 60 90 240 600
U87-stb-CXCR4 to U87 32.57 6 5.51 40.19 6 7.53 21.62 6 4.36 12.85 6 4.03 5.38 6 2.97
U87-stb-CXCR4 to muscle 193.82 6 108.55 297.78 6 93.83 362.56 6 153.51 222.95 6 27.62 151.00 6 29.91
U87 to muscle 5.88 6 2.93 7.31 6 1.59 17.53 6 7.64 18.91 6 7.25 42.04 6 35.98
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The Invited Perspective “The Real Cost of Theoretic Risk Avoidance: The Need to Challenge Unsubstantiated
Concerns About
I Therapy,” by Goldsmith (J Nucl Med. 2011;52:681–682) contains an error in the first sentence
of the sixth paragraph. According to title 10 of Code of Federal Regulations part 35.75(a), the originally stated
value of 500 mSv in that sentence shoul d have been 5 mSv. The corrected sentence appears below. The author
regrets the error.
After the 1997 regulatory modification allowing release of patients receiving more than 1,110 MBq of
I—provided the nuclear
practitioner has demonstrated to the licensing authority that patients have been instructed on reasonable isolation and that conditions are
such that no member of the public is likely to be exposed beyond 5 mSv—Grigsby et al. (2) distributed radiation-monitoring devices to
family members (adults, children, and pets) of patients receiving 3.7–5.5 GBq of
PET OF CXCR4 EXPRESSION De Silva et al. 993
Page 9
  • Source
    • "Another cyclam-containing CXCR4 ligand, AMD3465, was also used for imaging CXCR4 expression. De Silva et al. reported that [ 64 Cu]- AMD3465 was capable of detecting tumor lesions using dynamic and whole-body PET/CT in a CXCR4-dependent fashion with high target selectivity in both U87 brain tumor and HT-29 colon tumor animal models [48]. Anti-CXCR4 antibodies are commonly used for fluorescence microscopy imaging, but they also showed potential in SPECT/CT imaging in vivo. "
    [Show abstract] [Hide abstract] ABSTRACT: This review discusses the potential of CXCR4 chemokine receptor in the design of anticancer and antimetastatic drug delivery systems. The role of CXCR4 in cancer progression and metastasis is discussed in the context of the development of several types of drug delivery strategies. Overview of drug delivery systems targeted to cancers that overexpress CXCR4 is provided, together with the main types of CXCR4-binding ligands used in targeting applications. Drug delivery applications that take advantage of CXCR4 inhibition to achieve enhanced anticancer and antimetastatic activity of combination treatments are also discussed.
    Preview · Article · Jan 2016
  • Source
    • "Preclinical molecular imaging of CXCR4 is focused around SPECT, PET and optical imaging. Where the first two have shown potential for the noninvasive visualization of disease extent [132, 133], the last enables microscopic evaluation of receptor interactions and has demonstrated potential in imageguided surgery applications [134] . Studies regarding this receptor nicely illustrate that the efforts to optimize affinity and kinetics have paid off. "
    [Show abstract] [Hide abstract] ABSTRACT: In view of the trend towards personalized treatment strategies for (cancer) patients, there is an increasing need to noninvasively determine individual patient characteristics. Such information enables physicians to administer to patients accurate therapy with appropriate timing. For the noninvasive visualization of disease-related features, imaging biomarkers are expected to play a crucial role. Next to the chemical development of imaging probes, this requires preclinical studies in animal tumour models. These studies provide proof-of-concept of imaging biomarkers and help determine the pharmacokinetics and target specificity of relevant imaging probes, features that provide the fundamentals for translation to the clinic. In this review we describe biological processes derived from the "hallmarks of cancer" that may serve as imaging biomarkers for diagnostic, prognostic and treatment response monitoring that are currently being studied in the preclinical setting. A number of these biomarkers are also being used for the initial preclinical assessment of new intervention strategies. Uniquely, noninvasive imaging approaches allow longitudinal assessment of changes in biological processes, providing information on the safety, pharmacokinetic profiles and target specificity of new drugs, and on the antitumour effectiveness of therapeutic interventions. Preclinical biomarker imaging can help guide translation to optimize clinical biomarker imaging and personalize (combination) therapies.
    Full-text · Article · Feb 2015 · European journal of nuclear medicine and molecular imaging
  • Source
    • "Further developments yielded AMD3465, a monocyclam, with higher affinity for CXCR4 and smaller size and charge than earlier cyclams. When labelled with 64 Cu, AMD3465 shows superior target specificity within this family of molecules, good pharmacokinetics and high tumour uptake, but also high liver retention which is the main obstacle in its clinical development [149]. Current research in this field is focused on generating the optimal radioprobe. "
    [Show abstract] [Hide abstract] ABSTRACT: Tumour cells exhibit several properties that allow them to grow and divide. A number of these properties are detectable by nuclear imaging methods. We discuss crucial tumour properties that can be described by current radioprobe technologies, further discuss areas of emerging radioprobe development, and finally articulate need areas that our field should aspire to develop. The review focuses largely on positron emission tomography and draws upon the seminal ‘Hallmarks of Cancer’ review article by Hanahan and Weinberg in 2011 placing into context the present and future roles of radiotracer imaging in characterizing tumours.
    Full-text · Article · Feb 2015 · European journal of nuclear medicine and molecular imaging
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