Evaluation of 99mTc-3PRGD2 integrin receptor imaging in hepatocellular carcinoma tumour-bearing mice: comparison with 18F-FDG metabolic imaging
Evaluation of 99mTc-3PRGD2 integrin receptor imaging in hepatocellular carcinoma tumour-bearing mice: comparison with 18F-FDG metabolic imaging
Jieling Zheng 0
Weibing Miao 0
Chao Huang 0
Haoxue Lin 0
0 Department of Nuclear Medicine, The First Affiliated Hospital of Fujian Medical University , 20 Chazhong Road, Taijiang District, Fuzhou 350005 , China
Objective Our study was designed to explore the utility of 99mTc-HYNIC-PEG4-E[PEG4-c(RGDfK)]2 (99mTc3PRGD2) for the detection of hepatocellular carcinoma (HCC) and specifically to compare the diagnostic performance of 99mTc-3PRGD2 integrin receptor imaging and 2-18-fluoro-2-deoxy-D-glucose (18F-FDG) metabolic imaging in a nude mouse model. Methods 99mTc-3PRGD2 was synthesized using a HYNIC3PRGD2 lyophilized kit with 99mTcO4 labelling. The nude mouse animal model was established by subcutaneously injecting 5 9 107/ml HepG2 cells into the shoulder flank of each mouse. Biodistribution studies were performed at 0.5, 1, 2 and 4 h after intravenous administration of 0.37 MBq of 99mTc-3PRGD2. Immunohistochemistry was performed to evaluate the expression level of integrin avb3 in the HCC tissues. Dynamic imaging was performed using listmode after the administration of 55.5 MBq of 99mTc3PRGD2, to reconstruct the multiphase images and acquire the best initial scan time. At 8, 12, 16, 20 and 24 days after inoculation with HepG2 cells, 55.5 MBq of 99mTc3PRGD2 and 37 MBq of 18F-FDG were injected successively into the nude mouse model, subsequently, simultaneous SPECT/PET imaging was performed to calculate the tumour volume and tumour uptake of 99mTc-3PRGD2 and 18F-FDG. Results The biodistribution study first validated that the tumour uptake of 99mTc-3PRGD2 at the different time
99mTc-3PRGD2; Integrin avb3; 18F-FDG
points was higher than that of all the other organs tested in
the experiment, except for the kidney. Integrin avb3
expressed highly in early stage HCC and declined for
further necrosis of the tumour tissue. Subcutaneous
tumours were visualized clearly with excellent contrast
under 99mTc-3PRGD2 SPECT/CT imaging, and the
multiphase imaging comparison showed the tumours were
prominent at 0.5 h, suggesting that the best initial scan time
is 0.5 h post-injection. The comparison of the imaging
results of the two methods showed that 99mTc-3PRGD2
integrin receptor imaging was more sensitive than 18F-FDG
metabolic imaging for the detection of early stage HCC,
meanwhile the tumour uptake of 99mTc-3PRGD2 was
consistently higher than that of 18F-FDG. However, as
tumour necrosis further increased in HCC tissues, the
uptake of 18F-FDG was higher than that of 99mTc-3PRGD2.
Conclusion Our study demonstrated that 99mTc-3PRGD2 is
a valuable tumour molecular probe for the detection of
early stage HCC compared with 18F-FDG, meriting further
investigation of 99mTc-3PRGD2 as a novel SPECT tracer
for tumour imaging.
Hepatocellular carcinoma (HCC) is a primary cancer of the
liver with a significant impact worldwide due to its high
incidence and mortality. Early detection and diagnosis of
HCC can bring great benefit for the prognosis of patients.
Currently, the definitive diagnosis of HCC depends
primarily on conventional imaging modalities, such as
ultrasonography, magnetic resonance imaging or computed
tomography . However, these techniques are usually
based on anatomical, morphological or haemodynamic
alterations. The appearance of HCC at the early stage is
atypical, and thus, there is no obvious difference between
small HCC and preneoplastic nodules. Moreover, the types
of hepatic lesions are complicated, various forms of
inflammation, granulomatous lesions and benign tumours
can mimic the morphology and haemodynamics of early
HCC, resulting in the difficult differential diagnosis of
early HCC in non-cirrhotic patients. Therefore,
conventional imaging modalities still have some limitation in the
diagnosis of early HCC.
Hepatocarcinogenesis is considered a multi-step
process; changes firstly come from gene, molecule and
metabolic function, with subsequent anatomical and
morphological changes. Hence, acquiring cellular, molecular or
functional information on HCC tumours through functional
imaging techniques has significant clinical value for the
early detection and diagnosis of HCC. Nowadays,
2-18fluoro-2-deoxy-D-glucose (18F-FDG) PET/CT is the most
widely employed tumour-functional imaging modality
worldwide, however, the positive detection rate for HCC is
still not ideal, particularly in terms of the low specificity
and sensitivity for the detection of early stage HCC [2, 3].
Thus, a large number of other compounds have been
investigated as an alternative or complement to 18F-FDG,
most recently 11C-acetate [4, 5], 11C-choline , and
18Flabelled fluorothymidine [7, 8]; however, none of these
compounds have gained broad approval. Accordingly,
there is still a distinct need for better functional imaging
modalities available for patients with HCC.
Integrin avb3, a member of the integrin receptor family, is
highly expressed in both tumour cells and activated
endothelial cells and plays a pivotal role in tumour growth,
invasion and metastasis [9–11]. The tri-peptide sequence of
arginine-glycine-aspartic acid (RGD) is a cellular recognition
site that can selectively bind to integrin avb3 . A variety of
radiolabeled RGDs have been developed as SPECT and PET
radiotracers for tumour imaging and have attracted
considerable attention in recent decades. Our study was designed to
investigate the feasibility of
99mTc-HYNIC-PEG4-E[PEG4c(RGDfK)]2 (99mTc-3PRGD2) for the detection of HCC and
specifically to compare the diagnostic performance of
99mTc3PRGD2 integrin receptor imaging and 18F-FDG metabolic
imaging in HCC tumour-bearing mice.
Materials and methods
The HYNIC-3PRGD2 lyophilized kit was obtained from
the Medical Isotope Research Center of Peking University.
The synthesis of the labelling precursor and subsequent
99mTc-labelling were performed according to previously
described methods . The vial containing the above
product was heated at 100 C for 20 min and then
incubated at room temperature for 10 min. The resulting
solution was analysed using radioactive thin layer
chromatography to verify that the radiochemical purity
was [95%. 18F-FDG was supplied by the Atom High-tech
Company of China (Fuzhou, Fujian, China). Doses were
prepared by dissolving the radiotracer in saline.
This study was approved by our hospital’s Ethics Committee
for medical research. A total of 36 female athymic nu/nu
mice were purchased from the Medical Animal Center,
FuZhou General Hospital of Nanjing Military Command
(6week-old, average body weight 24.16 ± 2.28 g). The
human HCC HepG2 cell line was purchased from the
Institute of Cytobiology, Chinese Academy of Science. The
HepG2 cells were cultured in DMEM supplemented with
10% foetal bovine serum (FBS, HyClone) and 1% penicillin
and streptomycin solution (Gibco) at 37 C in a humidified
atmosphere of 5% CO2 in air. The cells were split or
harvested when they reached 90–100% confluency to maintain
exponential growth. Each mouse was implanted
subcutaneously with 5 9 107/ml HepG2 cells into the left shoulder
flank. The tumour volume was measured every other day
with vernier callipers and calculated using the following
formula: (length 9 width2)/2.
A total of 16 HepG2 hepatoma-bearing mice, with a
maximum tumour diameter of 1.0 cm, were randomly
divided into 4 groups, with 4 animals in each group. Each
mouse was injected intravenously with 0.37 MBq of
99mTc-3PRGD2 in 0.1 ml. At 0.5, 1, 2 and 4 h after
administration, the mice were sacrificed by humane
euthanasia, and the important organs (tumour, heart, liver,
spleen, lungs, kidneys, stomach, intestine, muscle and
blood) were excised, dried, weighed and counted using a
ccounter. The percentage of the injected dose per gram
(%ID/g) and the target/non-target (T/NT) ratios were
calculated to evaluate the biological distribution
characteristics of 99mTc-3PRGD2 in the nude mice.
In total, 12 mice were chosen randomly for HepG2 cell
inoculation at different time points. When the subcutaneous
tumours grew to various sizes (0.06–1.0 g), the mice were
sacrificed by humane euthanasia, and the tumours were
excised, fixed in 10% formalin, embedded in paraffin, cut
into slices (6-lm-thick) and dried. After dewaxing in
xylene and hydrating in gradient ethyl alcohol (from 100 to
80%), the sections were incubated with the mouse
antiintegrin avb3 monoclonal antibody (1:100, Abcam,
Cambridge, USA), followed by sequential incubation with
reagent A (reaction enhancer) and B (enzyme-conjugated
goat anti-mouse/rabbit IgG polymer) of an
immunohistochemical EliVision plus kit, then washed with phosphate
buffer solution (PBS) and stained with fresh
diaminobenzidine (DAB) solution. Finally, the sections were
visualized under an Olympus optical microscope (Olympus
Corporation, Tokyo, Japan). The appearance of brown
colouring in the cytoplasm or cytomembrane of the tumour
tissues was regarded as positive integrin avb3 expression.
Dynamic SPECT/CT imaging with 99mTc-3PRGD2
All imaging studies were performed using microSPECT/
PET/CT equipment (Versatile Emission Computed
Tomography System, Milabs, Utrecht, the Netherlands),
which enables simultaneous submillimeter imaging of
single-photon and positron-emitting radiolabeled
molecules. Four HepG2 hepatoma-bearing mice were injected
with 55.5 MBq of 99mTc-3PRGD2 in 0.1 ml saline via the
tail vein. At 20 min after administration, the mice were
quickly transferred into a shielded chamber connected to an
isoflurane anaesthesia unit. After the induction of
anaesthesia, the mice were immediately placed supine on the
scanning bed. The 99mTc-3PRGD2 imaging commenced
30 min after the tracer injection. First, rectangular scans in
the regions of interest (ROIs) from both the SPECT and CT
were selected by X-ray. CT scanning was performed using
the ‘‘normal’’ acquisition settings at 55 keV and 615 lA,
and then SPECT data were acquired from 30 min to 3 h
and 4 to 4.5 h post-injection (pi) in list-mode using the
following settings: 84 projections per frame over 30 min
and a 20% energy window centred on 140 keV.
Multiphase image reconstruction and data analysis
CT reconstruction was performed using a cone-beam
filtered back-projection algorithm (NRecon v1.6.3, Skyscan,
Belgium). SPECT image reconstruction of the list-mode
raw data was performed using a pixel-based
ordered-subsets expectation maximization algorithm (POSEM), with
the initial scan time set as 0.5, 1, 2 and 4 h pi (a 30-min
scanning time). The new SPECT data and CT data were
fused together using PMOD software (PMOD
Technologies, Zurich, Switzerland). To achieve a higher
signal-tonoise ratio, Gaussian smooth 3D (1.0 mm full-width at half
maximum) was used and the image colour gradation was
adjusted. For each SPECT/CT fusion image, the ROI
covering the entire tumour was generated automatically by
setting a maximum pixel value threshold of 30% or more,
and then the tumour volume and radioactivity counts were
calculated using PMOD. The tumour uptake of
99mTc3PRGD2 was expressed as the percentage of the injected
dose (%ID) and the percentage of the injected dose per unit
volume (%ID/cm3). The background ROI (0.01 cm3) was
drawn over the muscle of the contralateral forelimb, and
then the T/NT ratio of tumour to muscle was calculated for
the semi-quantitative analysis.
Simultaneous SPECT/PET Imaging
with the 99mTc/18F dual-isotope
The HepG2 hepatoma-bearing mice were reared at
26–27 C. Four mice were chosen for simultaneous
SPECT/PET imaging at 8, 12, 16, 20 and 24 days after
inoculation with HepG2 cells, and they were fasted but
allowed to drink water for 12 h before imaging. The
selected mice were injected intravenously with 55.5 MBq
of 99mTc-3PRGD2 and 37 MBq of 18F-FDG successively.
A 45-min simultaneous SPECT/PET acquisition was
obtained, beginning at 45 min after the tracer injection.
Throughout the scanning, the body temperature of the mice
was monitored using a constant heating and feedback
device. To achieve signal separation of the two tracers, the
energy peak was set to 140 keV for 99mTc-3PRGD2 and
511 keV for 18F-FDG. All photopeak windows were set to
a width of 20%. The SPECT and PET images were
reconstructed using POSEM. The tumour volume and
tumour uptakes of 99mTc-3PRGD2 and 18F-FDG (%ID and
%ID/cm3) were calculated using PMOD software.
The quantitative data are expressed as x s. The
statistical analyses were performed using one-way analysis of
variance (ANOVA) for multiple comparisons, least
significant difference (LSD) t test for the comparisons
between groups and regression analyses for the correlation
analysis with the SPSS 18.0 statistical software package.
The level of significance was set at P \ 0.05.
Biodistribution of 99mTc-3PRGD2
As shown in Fig. 1a, the tumour uptake of 99mTc-3PRGD2
at the different time points was 4.301 ± 0.313 %ID/g
(0.5 h), 6.902 ± 0.717 %ID/g (1 h), 5.045 ± 0.193 %ID/g
(2 h) and 2.099 ± 0.388 % ID/g (4 h), with a statistically
Fig. 1 a The biodistribution
data in selected organs of
HepG2 hepatoma-bearing mice
obtained at 0.5, 1, 2 and 4 h
after injection of 0.37 MBq of
99mTc-3PRGD2 (x ± s, n = 4).
b Comparison of the T/NT
ratios of tumour to muscle and
tumour to liver in HepG2
hepatoma-bearing mice at 0.5,
1, 2 and 4 h after injection of
0.37 MBq of 99mTc-3PRGD2
(x ± s, n = 4)
significant difference between each group
(F = 78.864,P \ 0.05). 99mTc-3PRGD2 was primarily
excreted through the urinary system as evidenced by the
higher renal uptake (11.842 ± 1.304, 10.034 ± 1.051,
8.683 ± 0.437, 4.225 ± 0.332 %ID/g at 0.5, 1, 2 and 4 h
pi, respectively). Moderate accumulation was also found in
the intestine and liver, illustrating that 99mTc-3PRGD2 was
partially metabolized by the liver or intestine. Other organs
with relatively low radioactivity were the spleen, lung,
stomach, blood and muscle. Figure 1b shows the T/NT
ratios of tumour to muscle and tumour to liver, indicating
that the highest T/NT ratios were obtained at 2 h pi
(9.57 ± 2.37 and 2.56 ± 0.91, respectively).
Integrin avb3 expression in HCC tissues
HCC cells have large and atypical nuclei and mitosis in
haematoxylin-eosin (HE)-stained slices.
Immunohistochemistry showed that integrin avb3 positive expression,
as visualized by brown staining, was observed
predominantly in the cytoplasm and cytomembrane, as well as the
vascular epithelial cells (Fig. 2). The expression level of
avb3 in HCC cells varied among the different tumour sizes
(0.06–1.0 g). When the tumour weight was less than 0.23 g
without tumour necrosis, the overall expression level of
avb3 was relatively high. When the tumour weight was
between 0.35 and 1.0 g, the expression level of integrin
avb3 gradually declined in line with the necrosis of the
Dynamic SPECT/CT imaging results
The subcutaneous tumours were clearly visualized under
multiphase SPECT/CT imaging, with excellent contrast at
0.5, 1, 2 and 4 h pi (Fig. 3). The accumulation of
radioactivity in the subcutaneous tumours was most obvious when
the initial scan time was 0.5 h pi, and some radionuclide
remained until 4 h pi. Prominent renal uptake of
99mTc3PRGD2 was observed at all time points examined, whereas
abdomen uptake was moderate and radioactivity
accumulation in the lung, mediastinum and muscle was relatively
low. Furthermore, radioactivity distribution in all body tissue
types decreased over time. These results were consistent with
that of the biodistribution study.
The tumour uptake was 4.61 ± 1.17 %ID/cm3 (0.5 h),
3.61 ± 0.87 %ID/cm3 (1 h), 3.02 ± 0.75 %ID/cm3 (2 h)
and 2.63 ± 0.71 %ID/cm3 (4 h), suggesting that the
highest tumour uptake occurred when the initial scan time
Fig. 2 Representative microscopic images of the tumour slices from
the different sized xenografted HepG2 tumours after
haematoxylineosin (HE) staining and immunohistochemical staining (0.09 and
0.35 g). a HE staining showed the HCC cells and tumour blood
vessels without tumour necrosis. b Immunohistochemistry showing
the positive expression of integrin avb3 was visualized as brown
staining. The results showed that avb3 was predominantly expressed
in the cytoplasm and cytomembrane, as well as the vascular epithelial
cells (arrows). c HE staining showing the central visual field was
characterized by cell necrosis. d Immunohistochemistry showing that
avb3 staining was negative in the necrotic region. The images were
obtained by an Olympus optical microscope (10 9 20 magnification)
Fig. 3 The traverse views
(a) and coronal views (b) of
multiphase SPECT/CT images
after the injection of 55.5 MBq
of 99mTc-3PRGD2 in HepG2
hepatoma-bearing mice, with
the initial scan time set as 0.5, 1,
2 and 4 h pi (a 30-min scanning
was 0.5 h pi. The T/NT ratios of tumour to muscle were
3.39 ± 1.33 (0.5 h), 3.17 ± 1.33 (1 h), 3.66 ± 0.92 (2 h)
and 2.86 ± 0.49 (4 h). Although the highest T/NT ratio
was found when the initial scan time was 2 h pi, there were
no statistically significant differences among the four
groups (P [ 0.05). Both the direct visualization and
quantitative analysis indicated that the best initial scan time
was 0.5 h pi.
We assessed the performance differences between
99mTc3PRGD2 integrin receptor imaging and 18F-FDG metabolic
imaging during the growth of tumour with the
microSPECT/PET/CT system (Fig. 4). 99mTc-3PRGD2 SPECT/
CT could sensitively detect subcutaneous tumours at
8 days after inoculation with HepG2 cells. The tumours
were clearly visualized at 8, 12, and 16 days after
inoculation with HepG2 cells for the distinct concentration and
homogenous distribution of radionuclides, and the tumour
volume gradually increased over time. In contrast, there
was only minimal uptake in the subcutaneous tumours
under 18F-FDG PET/CT imaging at 8, 12, and 16 days after
inoculation with HepG2 cells. From day 20 to day 24, as
tumour necrosis further increased, both the 99mTc-3PRGD2
SPECT/CT fusion images and 18F-FDG PET/CT fusion
images showed uneven tracer distribution in the HCC
tissues. Notably, the uptake of 18F-FDG into the brown
adipose tissue located in the neck and back or multiple muscle
tissues was too high, thereby affecting image quality and
forcing some measures including warming, fasting and
anaesthesia to be performed in advance.
The tumour uptake of 99mTc-3PRGD2 and 18F-FDG at the
different imaging time points is shown in Table 1. Except
Fig. 4 The three-dimensional
views of simultaneous SPECT/
PET images obtained 45 min
after the injection of 55.5 MBq
of 99mTc-3PRGD2 and 37 MBq
of 18F-FDG in 99mTc-window
(a) and 18F-widow (b), with a
45-min scanning time. These
images were acquired at 8, 12,
16, 20 and 24 days after HepG2
for the uptake measurements on day 20 (P \ 0.05), there
were no statistically significant differences among the
different groups, which were divided according to the
imaging day (t = 1.62, P \ 0.05). A regression analysis
was used to assess the relationship between the tumour
volume and tumour uptake of 99mTc-3PRGD2 and 18F-FDG
(Fig. 5). The relationship between the tumour volume
and %ID of 99mTc-3PRGD2 was modelled as quadratic
polynomial, with an R2 value of 0.85. The conversion
formula was y = -17.81x2 ? 11.16x-0.38, indicating that
the uptake of 99mTc-3PRGD2 increased to its peak as the
tumour volume increased and then declined due to necrosis
(day 24). The relationship between the tumour volume
and %ID of 18F-FDG was also modelled as quadratic
polynomial, with an R2 value of 0.96. The conversion
formula was y = -6.61x2 ? 7.68x-0.18, illustrating that
the tumour uptake of 18F-FDG was consistently less than
that of 99mTc-3PRGD2 from day 8 to day 16. When all the
tumour volume was greater than 0.30 cm3 from day 20 to
day 24, the uptake of 18F-FDG, in turn, was higher than
that of 99mTc-3PRGD2 during this time period.
Among the various forms of radioactive medications,
99mTc-3PRGD2 stands out for its superior avb3-targeting
ability, biodistribution and pharmacokinetics [13–15]. It
has been shown to be a promising tracer for the diagnosis,
staging, and treatment evaluation of thoracic malignancies
Fig. 5 The blue line represents
the relationship between the
tumour volume (cm3) and
tumour uptake of
99mTc3PRGD2 (%ID) at 8, 12, 16, 20
and 24 days after HepG2 cell
inoculation. The green line
represents the relationship
between the tumour volume
(cm3) and tumour uptake of
18FFDG (%ID) at 8, 12, 16, 20 and
24 days after HepG2 cell
Table 1 Comparison of tumor uptakes of 99mTc-3PRGD2 and 18F-FDG at 8, 12, 16, 20 and 24 days after inoculation with HepG2 cells (%ID,
x s, n = 4)
such as lung cancer, breast cancer and metastasis [16–19].
However, few studies have investigated the use of
99mTc3PRGD2 for the diagnosis of HCC, especially no studies
have compared the efficacy of 99mTc-3PRGD2 integrin
receptor imaging with 18F-FDG metabolic imaging.
Integrin avb3 express lowly in normal liver tissues including
hepatocytes, stellate cells, Kupffer cells, and other
nonparenchymal cells . Studies have reported that the
expression of integrin avb3 is significantly higher in HCC
tissues than in adjacent normal hepatic tissue . In this
study, we demonstrated that HCC could be detected using
SPECT/CT with 99mTc-3PRGD2 as the radiotracer.
To further estimate the usefulness of 99mTc-3PRGD2 in
the non-invasive monitoring of HCC tumours, 18F-FDG
was chosen as the positive control. The imaging results
indicated that 99mTc-3PRGD2 SPECT/CT has an advantage
over 18F-FDG PET/CT for HCC imaging in two aspects.
First, 99mTc-3PRGD2 SPECT/CT could distinctly and
sensitively detect the HCC tissues at 8 days after HepG2
cell inoculation, a time point at which the smallest tumour
volume was only 0.041 cm3 but the uptake of
99mTc3PRGD2 reached 10.67 %ID/cm3 at this point. In contrast,
the tumours were unclear under 18F-FDG images so the
imaging could not be used to accurately detect early stage
HCC. Furthermore, the tumour uptake of 99mTc-3PRGD2
was significantly higher than that of 18F-FDG from day 8 to
day 16. This finding suggests that 99mTc-3PRGD2 has the
potential to be used as a SPECT tracer for early detection
of HCC over 18F-FDG. Second, it is well known that
18FFDG PET/CT imaging is affected by many factors, such as
glucose levels, insulin, and temperature. Muscle or brown
adipose tissue can also competitively uptake 18F-FDG
[22, 23]. In contrast, 99mTc-3PRGD2 integrin receptor
imaging shows less interference from these factors,
resulting in the acquisition of clearer images. Moreover,
99mTc-3PRGD2 can be more widely applied to improve the
screening rates for early HCC detection because it is more
cost-effective, feasible and practical as a SPECT tracer.
We analysed the possible reasons for the different
imaging results with 99mTc-3PRGD2 and 18F-FDG comes
from two completely different biological processes they are
targeting. In this study, we found that avb3 expression in
the xenografted HepG2 tumours was high in the early stage
and showed an increasing trend until the tumours became
necrotic. Given that 99mTc-3PRGD2 is a target-specific
radiotracer whose biodistribution is determined by its
receptor, the high expression level of avb3 in early HCC
contributes to the high tumour uptake of 99mTc-3PRGD2;
therefore, 99mTc-3PRGD2 imaging is able to sensitively
detect early HCC while 18F-FDG imaging is used to
evaluate glucose metabolism, the main mechanism of
18FFDG uptake in malignant tumours largely depends on the
presence of facilitated glucose transporters, especially type
1 (Glut 1) and a rate-limiting glycolytic enzyme, especially
hexokinase (HK) type II. However, HCCs have different
glucose-regulating mechanisms and enzyme expression
patterns, and the low expression of Glut 1 and the existence
of dephosphorylase are the main reasons for the low
18FFDG uptake [24–26]. The uptake of 18F-FDG in HCC is
also related to the degree of differentiation and lesion size,
with well-differentiated HCC exhibiting a high rate of
gluconeogenesis compared with that in normal liver
tissues, resulting in similar uptake of 18F-FDG . In
particular, early stage HCC tumours, which are well
differentiated and small in size, show low radioactivity
concentrations under 18F-FDG PET/CT imaging.
Except 99mTc-3PRGD2 SPECT/CT was more sensitive
than 18F-FDG PET/CT for the detection of early HCC, it is
worth noting that HCC uptake of 18F-FDG was higher than
that of 99mTc-3PRGD2 in advanced stage HCC, indicating
that the diagnostic efficiency of 18F-FDG PET/CT might be
better than 99mTc-3PRGD2 SPECT/CT for imaging
advanced HCC. On one hand, the tumour uptake of
99mTc3PRGD2 may have decreased due to the concomitant
maturity of blood vessels, tumour necrosis, and larger
interstitial space that accompanied the rapid growth of the
tumour . On the other hand, cancer cell growth is
heavily dependent on glucose metabolism as a major
energy substrate, particularly poorly differentiated
advanced HCC. The association of neoplastic growth with
increased aerobic glycolysis is known as the Warburg
effect, which might help explain why the uptake of
18FFDG increased in advanced stage HCC . Therefore,
combining 99mTc-3PRGD2 SPECT/CT and 18F-FDG PET/
CT imaging could aide in the detection of HCC by
compensating for the deficiency of 18F-FDG in the detection of
early stage HCC and 99mTc-3PRGD2 in the diagnosis of
advanced HCC. Moreover, multimodality imaging using
multiple tracers can also provide complementary
information on tumour-associated functions with excellent
sensitivity and high selectivity; thus, multimodality
imaging might improve the tumour diagnosis, differential
diagnosis and therapeutic monitoring of HCC compared
with those of a single radiological modality alone.
In the complex pathogenesis of hepatic carcinoma,
angiogenesis is one of the crucial events, relying on the
migration and invasion of vascular endothelial cells, which
is regulated by various cell adhesion receptors including
the integrin family . Among the 24 members of
integrin family, avb3 is the most widely studied because its
expression is an important factor in determining the
invasiveness and metastatic potential of malignancy . In our
study, we demonstrated that avb3 was highly expressed on
the activated endothelial cells of HCC tissues. Currently,
sorafenib chemotherapy, an anti-angiogenic therapy, is the
first line therapy for HCC patients who are not eligible for
surgical removal or liver transplantation. However, there is
still no appropriate imaging modality to monitor the
efficacy of anti-angiogenic therapy. Research has shown that
99mTc-3PRGD2 can be applied to guide anti-angiogenic
therapy by visualizing and quantifying the expression level
of avb3 [18, 31]. Thus, 99mTc-3PRGD2 imaging has the
potential to select suitable patients who could benefit from
sorafenib chemotherapy and supply an objective basis for
its curative effect.
It should be noted that our results are preliminary and
obtained using a very limited number of animals; therefore,
insufficient data may have caused errors in our analysis.
Further studies with a substantial number of animals or a
comparable translational human clinical trial are still
needed. Moreover, most HCCs arise in the setting of chronic
hepatitis induced by HCV or HBV infection. However,
current animal models cannot mimic the inflammatory
microenvironment that leads to the pathogenesis of HCC
and the pathogenic sequence of human HCC that starts
with fibrosis and cirrhosis prior to the development of
HCC. Several studies have reported that 99mTc-3PRGD2
can assess activation of hepatic stellate cells and diagnose
liver fibrosis [32, 33]. The uptake in normal hepatic,
hepatitis and liver fibrosis tissues may decrease the T/NT ratio
and influence the detection of HCC. Finally, the relation
between the pathologic grade and tumour uptake of
99mTc3PRGD2 was not assessed in the present study; therefore,
we cannot verify whether various pathologic types of liver
cancer impact 99mTc-3PRGD2 SPECT/CT imaging.
In conclusion, 99mTc-3PRGD2 is more sensitive for the
detection of early stage HCC than 18F-FDG, but in advanced
HCC, the diagnostic performance of 18F-FDG might be
better than that of 99mTc-3PRGD2 because the tumour uptake
of 99mTc-3PRGD2 significantly declined due to tumour
necrosis. Combined 99mTc-3PRGD2 SPECT/CT and
18FFDG PET/CT imaging would be helpful for the imaging
diagnosis of HCC. We believe that 99mTc-3PRGD2 is worth
further investing as a promising radiotracer for the diagnosis
of HCC and guidance of the anti-angiogenic therapy.
Acknowledgements The authors thank the Medical Isotopes
Research Center of Peking University for supplying the
HYNIC3PRGD2 lyophilized kit. This work was financially supported by the
Fujian Provincial Natural Science Foundation (2015J01459) and the
Fujian Provincial Medical Innovate Foundation (2015-CX-21).
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://creative
commons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appropriate
credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
1. Vilarinho S , Taddei T. Therapeutic strategies for hepatocellular carcinoma: new advances and challenges . Curr Treat Options Gastroenterol . 2015 ; 13 : 219 - 34 .
2. Khan MA , Combs CS , Brunt EM , Lowe VJ , Wolverson MK , Solomon H , et al. Positron emission tomography scanning in the evaluation of hepatocellular carcinoma . J Hepatol . 2000 ; 32 : 792 - 7 .
3. Delbeke D , Martin WH , Sandler MP , Chapman WC , Wright JK Jr, Pinson CW. Evaluation of benign vs malignant hepatic lesions with positron emission tomography . Arch Surg . 1998 ; 133 : 510 - 5 .
4. Territo PR , Maluccio M , Riley AA , Brian P , et al. Evaluation of 11C-Acetate and 18 F-FDG PET/CT in mouse multidrug resistance gene-2 deficient mouse model of hepatocellular carcinoma . BMC Med Imaging . 2015 ; 15 : 15 .
5. Hwang KH , Choi DJ , Lee SY , Lee MK , Choe W. Evaluation of patients with hepatocellular carcinomas using [11C]acetate and [18F]FDG PET/CT: a preliminary study . Appl Radiat Isot . 2009 ; 67 : 1195 - 8 .
6. Yamamoto Y , Nishiyama Y , Kameyama R , Okano K , Kashiwagi H , Deguchi A , et al. Detection of hepatocellular carcinoma using 11C-choline PET: comparison with 18F-FDG PET . J Nucl Med . 2008 ; 49 : 1245 - 8 .
7. Talbot JN , Gutman F , Fartoux L , Grange JD , Ganne N , Kerrou K , et al. PET/ CT in patients with hepatocellular carcinoma using [18F]fluorocholine: preliminary comparison with [18F]FDG PET/ CT . Eur J Nucl Med Mol Imaging . 2006 ; 33 : 1285 - 9 .
8. Eckel F , Herrmann K , Schmidt S , Hillerer C , Wieder HA , Krause BJ , et al. Imaging of proliferation in hepatocellular carcinoma with the in vivo marker 18F-fluorothymidine . J Nucl Med . 2009 ; 50 : 1441 - 7 .
9. Hodivala-Dilke K. avb3 integrin and angiogenesis: a moody integrin in a changing environment . Curr Opin Cell Biol . 2008 ; 20 : 514 - 9 .
10. Cai W , Chen X. Anti-angiogenic cancer therapy based on integrin avb3 antagonism . Anticancer Agents Med Chem . 2006 ; 6 : 407 - 28 .
11. Brooks PC , Clark RA , Cheresh DA . Requirement of vascular integrin avb3 for angiogenesis . Science . 1994 ; 264 : 569 - 71 .
12. Ruoslahti E , Pierschbacher MD. New perspectives in cell adhesion: RGD and integrins. Science . 1987 ; 238 : 491 - 7 .
13. Jia B , Liu Z , Zhu Z , Shi J , Jin X , Zhao H , et al. blood clearance kinetics, biodistribution, and radiation dosimetry of a kit-formulated integrin avb3-selective radiotracer 99mTc-3PRGD2 in nonhuman primates . Mol Imaging Biol . 2011 ; 13 : 730 - 6 .
14. Wang L , Shi J , Kim YS , Zhai S , Jia B , Zhao H , et al. Improving tumor-targeting capability and pharmacokinetics of 99mTc-labeled cyclic RGD dimers with PEG4 linkers . Mol Pharm . 2009 ; 6 : 231 - 45 .
15. Zhou Y , Kim YS , Chakraborty S , Shi J , Gao H , Liu S. 99mTclabeled cyclic RGD peptides for noninvasive monitoring of tumor integrin avb3 expression . Mol Imaging . 2011 ; 10 : 386 - 97 .
16. Zhou Y , Shao G , Liu S. Monitoring breast tumor lung metastasis by U-SPECT-II/CT with an integrin avb3-targeted radiotracer 99mTc-3P-RGD2 . Theranostics. 2012 ; 2 : 577 - 88 .
17. Miao W , Zheng S , Dai H , Wang F , Jin X , Zhu Z , et al. Comparison of 99mTc-3PRGD2 integrin receptor imaging with 99mTcMDP bone scan in diagnosis of bone metastasis in patients with lung cancer: a multicenter study . PLoS One . 2014 ; 9 : e111221 .
18. Ma Q , Min K , Wang T , Chen B , Wen Q , Wang F , et al. 99mTc3PRGD2 SPECT/CT predicts the outcome of advanced nonsquamous non-small cell lung cancer receiving chemoradiotherapy plus bevacizumab . Ann Nucl Med . 2015 ; 29 : 519 - 27 .
19. Chen Q , Xie Q , Zhao M , Chen B , Gao S , Zhang H , et al. Diagnostic value of 99mTc-3PRGD2 scintimammography for differentiation of malignant from benign breast lesions: comparison of visual and semi-quantitative analysis . Hell J Nucl Med . 2015 ; 18 : 193 - 8 .
20. Li F , Song Z , Li Q , Wu J , Wang J , Xie C , et al. Molecular imaging of hepatic stellate cell activity by visualization of hepatic integrin avb3 expression with SPECT in rat . Hepatology . 2011 ; 54 : 1020 - 30 .
21. Jin Y , Chen JN , Feng ZY , Zhang ZG , Fan WZ , Wang Y , et al. OPN and avb3 expression are predictors of disease severity and worse prognosis in hepatocellular carcinoma . Plos One . 2014 ; 9 : e87930 .
22. Yeung HWD , Grewal RK , Gonen M , Scho¨der H, Larson SM . Patterns of 18F-FDG uptake in adipose tissue and muscle: a potential source of false-positive for PET . J Nucl Med . 2003 ; 44 : 1789 - 96 .
23. Christensen CR , Clark PB , Morton KA . Reversal of hypermetabolic brown adipose tissue in18F-FDG PET imaging . Clin Nucl Med . 2006 ; 31 : 193 - 6 .
24. Lee JD , Yang WI , Park YN , Kim KS , Choi JS , Yun M , et al. Different glucose uptake and glycolytic mechanisms between hepatocellular carcinoma and intrahepatic mass-forming cholangiocarcinoma with increased 18F-FDG uptake . J nucl Med . 2005 ; 46 : 1753 - 9 .
25. Mamede M , Higashi T , Kitaichi M , Ishizu K , Ishimori T , Nakamoto Y , et al. [18F] FDG uptake and PCNA, glut-1, and hexokinase-II expressions in cancers and inflammatory lesions of the lung . Neoplasia . 2005 ; 7 : 369 - 79 .
26. Ahn KJ , Hwang HS , Park JH , Bang SH , Kang Won Jun , Yun Mijin , et al. Evaluation of the role of hexokinase type II in cellular proliferation and apoptosis using human hepatocellular carcinoma cell lines . J Nucl Med . 2009 ; 50 : 1525 - 32 .
27. Torizuka T , Tamaki N , Inokuma T , Magata Y , Sasayama S , Yonekura Y , et al. In vivo assessment of glucose metabolism in hepatocellular carcinoma with FDG-PET . J Nucl Med . 1995 ; 36 : 1811 - 7 .
28. Shao G , Zhou Y , Wang F , Liu S. Monitoring glioma growth and tumor necrosis with the U-SPECT-II/CT scanner by targeting integrin avb3 . Mol Imaging . 2013 ; 12 : 39 - 48 .
29. Warburg O. On the origin of cancer cells . Science . 1956 ; 123 : 309 - 14 .
30. Liu S. Radiolabeled cyclic RGD peptide bioconjugates as radiotracers targeting multiple integrins . Bioconjug Chem . 2015 ; 26 : 1413 - 38 .
31. Ji B , Chen B , Wang T , Song Y , Chen M , Ji T , et al. 99mTc3PRGD2 SPECT to monitor early response to neoadjuvant chemotherapy in stage II and III breast cancer . Eur J Nucl Med Mol Imaging . 2015 ; 42 : 1362 - 70 .
32. Zhang X , Xin J , Shi Y , Xu W , Yu S , Yang Z , et al. Assessing activation of hepatic stellate cells by 99mTc-3PRGD2 scintigraphy targeting integrin avb3: a feasibility study . Nucl Med Biol . 2015 ; 42 : 250 - 5 .
33. Yu X , Wu Y , Liu H , Gao L , Sun X , Zhang C , et al. Small-animal SPECT/CT of the progression and recovery of rat liver fibrosis by using an integrin avb3-targeting radiotracer . Radiology . 2016 ; 279 : 502 - 12 .