TECHNICAL ADVANCEOpen Access
Imaging of thyroid tumor angiogenesis with
microbubbles targeted to vascular endothelial
growth factor receptor type 2 in mice
Marcello Mancini1,2*, Adelaide Greco3,5, Giuliana Salvatore4, Raffaele Liuzzi1, Gennaro Di Maro6, Emilia Vergara3,
Gennaro Chiappetta7, Rosa Pasquinelli7, Arturo Brunetti3,5and Marco Salvatore3
Background: To evaluate whether Contrast Enhanced Ultrasund (CEUS) with microbubbles (MBs) targeted to
VEGFR-2 is able to characterize in vivo the VEGFR-2 expression in the tumor vasculature of a mouse model of
thyroid cancer (Tg-TRK-T1).
Methods: Animal protocol was approved by Institutional committee on Laboratory Animal Care. Contrast-enhanced
ultrasound imaging with MBs targeted with an anti-VEGFR-2 monoclonal antibody (UCAVEGFR-2) and isotype control
antibody (UCAIgG) was performed in 7 mice with thyroid carcinoma, 5 mice with hyperplasia or benign thyroid
nodules and 4 mice with normal thyroid. After ultrasonography, the tumor samples were harvested for histological
examination and VEGFR-2 expression was tested by immunohistochemistry. Data were reported as median and
range. Paired non parametric Wilcoxon’s test and ANOVA of Kruskal-Wallis were used. The correlation between the
contrast signal and the VEGFR-2 expression was assessed by the Spearman coefficient.
Results: The Video intensity difference (VID) caused by backscatter of the retained UCAVEGFR-2was significantly
higher in mice harboring thyroid tumors compared to mice with normal thyroids (P < 0.01) and to mice harboring
benign nodules (P < 0.01). No statistically significant differences of VIDwere observed in the group of mice carrying
benign nodules compared to mice with normal thyroids. Moreover in thyroid tumors VIDof retained VEGFR-2-targeted
UCA was significantly higher than that of control UCAIgG(P <0.05). Results of immunohistochemical analysis confirmed
VEGFR-2 overexpression. The magnitude of the molecular ultrasonographic signal from a VEGFR-2-targeted UCA
retained by tissue correlates with VEGFR-2 expression determined by immunohistochemistry (rho 0.793, P=0.0003).
Conclusions: We demonstrated that CEUS with UCAVEGFR-2might be used for in vivo non invasive detection
and quantification of VEGFR-2 expression in thyroid cancer in mice, and to differentiate benign from malignant
Keywords: Thyroid, Transgenic, High resolution ultrasound, Cancer, Contrast agent
Angiogenesis is a critical determinant of tumor growth
and invasion [1,2] and successful application of novel ther-
apies that target tumor vasculature will require selection
of susceptible tumors and precise evaluation of early treat-
ment response. Vascular endothelial growth factor and its
main receptor vascular endothelial growth factor recep-
tor 2 (VEGFR-2), are overexpressed on tumor vascular
endothelial cells and have been identified as targets for
antiangiogenic drugs [3-10].
Papillary thyroid carcinoma (PTC) is the most common
malignancy of the thyroid gland. At the molecular level
PTC is characterized by genetic alterations of components
of the mitogen-activated protein kinase (MAPK) pathway
. These include structural chromosome rearrangements
affecting NTRK1 (TRK-T1) tyrosine kinase receptor
that undergo in-frame recombination with various partner
* Correspondence: firstname.lastname@example.org
1Institute of Biostructure and Bioimaging, Italian National Research Council
(CNR), Naples, Italy
2SDN Foundation IRCCS, Naples, Italy
Full list of author information is available at the end of the article
© 2013 Mancini et al.; licensee BioMed Central Ltd. 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.
Mancini et al. BMC Medical Imaging 2013, 13:31
genes . Specifically, the TRK-T1 oncogene results
from a paracentric inversion of chromosome 1q25 that
fuses the 5′ end of the TPR (Translocated Promoter
Region) to the 3′ end of NTRK1 genes generating the
constitutively active and oncogenic kinase NTRK1 .
Transgenic mice featuring the thyroid-specific expres-
sion of TRK-T1 under the transcriptional control of the
thyroid-specific bovine thyroglobulin (Tg) promoter were
generated previously . Twenty-three% of TRK-T1 mice
of age ≤ 7 months and 78% of mice > 7 months developed
thyroid nodules characterized by malignant features, such
as the proliferation of follicular epithelial cells containing
scant cytoplasm, mitotic figures and papillae with fibro-
vascular stalks [13,14].
In papillary thyroid carcinoma, increased VEGFR-2
expression correlates with an increased cancer cell pro-
liferation assessed by Ki-67 index, with increased thy-
roid tumor size [15,16] and with poor prognosis [16-19].
The thyroid cancer cells of primary tumors taken from
patients with metastases had an higher VEGFR-2 ex-
pression compared to cells taken from primary tumors
of patients without metastases [15,16]. These observa-
tions have been suggested to be clinically useful in
identifying patients who are more prone to develop
Recently, tumor angiogenesis imaging in vivo has been
noninvasively explored using contrast enhanced ultra-
sound (CEUS) with microbubbles (MBs) targeted to αvβ3
integrin, endoglin, and VEGFR2 [20-24]. This technique
is rapidly emerging as a noninvasive and quantitative
molecular imaging modality that combines the advan-
tages of high spatial resolution, real-time imaging, and
lack of ionizing radiation and may be particularly advan-
tageous in clinical oncology because VEGFR-2 has been
implicated as marker of metastatic potential and poor
prognosis in certain tumors [25-27].
Microbubbles are gas-filled echogenic US contrast agents
that can be targeted to specific molecular markers by
means of the attachment of appropriate ligands to the
surface of the MBs. A specific characteristic of MBs is
their relatively large size, which prevents them from
leaking into the extravascular space. This property can
be exploited for imaging by targeting the MBs to disease
processes reflected on the vascular endothelial cells
lining the luminal surface of capillaries and vessels, such
as inflammation and angiogenesis. When these func-
tionalized MBs are injected intravenously, they distrib-
ute throughout the whole body and attach at tissue sites
expressing the targeted molecular marker, leading to a
local increase of the US imaging signal. This approach
allows the exclusive visualization of molecular markers
of angiogenesis expressed on tumor vascular endothelial
cells, have a potential clinical translation in future
and should improve the ability to detect, diagnose
stage, select appropriate treatments, and determine prog-
nosis in patients with thyroid pathologies.
To our knowledge, no study has addressed the poten-
tial of targeted CEUS imaging for assessment of thyroid
tumor angiogenesis in vivo by using MBs targeted to
This study aimed to investigate whether targeted CEUS
allows noninvasive assessment of VEGFR-2 expression
on tumor vascular endothelium in Tg-TRK-T1 mice, a
murine model of thyroid cancer. We also investigated
whether the evaluation of expression levels of VEGFR2
in vivo can differentiate benign from malignant nodules
of the thyroid.
Animal studies were performed in accordance with
National Institutes of Health (NIH) recommendations
and Animal Research Advisory Committee (ARAC) pro-
cedure  and the approval of the Italian Institutional
animal research committee (Institutional Animal and Care
Committee of the University of Naples “Federico II” and
the Italian Ministry of Health). All animal procedures
in this study were conducted by a veterinarian and
conformed to all regulations protecting animals used
for research purposes, including national guidelines
[D.L. 27 Gennaio 1992, 116 Suppl. G.U 40 18 Febbraio
1992. Direttiva CEE n.609/86] as well as the protocols
recommended by Workamn et al. .
Tg-TRK-T1 transgenic mice have been previously
described . From 2010 to 2011, thyroid Ultrasound
was performed in 16 Tg-TRK-T1 transgenic mice model
of thyroid carcinogenesis . Body weight range of
animals was 29–32 gr, equally distributed among male
(n=9) and female (n=7). Mice were examined every six
months and were sacrificed immediately after the last
ultrasound scanning. At the time of the necroscopy, the
age range of mice was 12–15 months.
High frequency ultrasound with targeted contrast
A Vevo 770 microimaging system (Visualsonics, Toronto,
Ontario, Canada) with a single element probe, center fre-
quency of 40-MHz was used for all the examinations. The
transducer has an active face of 3 mm, a lateral resolution
of 68.2 μm, axial resolution of 38.5 μm, focal length of
6 mm, mechanical index 0.14, transmit power 50%, and a
dynamic range 52 dB [29,30]. Precise and repeatable con-
trol over the position of the two-dimensional image plane
was obtained with a rail system (Vevo Integrated Rail
System II; Visualsonics). Mice were anesthetized using
1.5–2% isoflurane vaporized in oxygen (2Lt/min) on a
heated stage, with constant monitoring of their body
temperature, using physiological monitoring platform .
Mancini et al. BMC Medical Imaging 2013, 13:31
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Cite this article as: Mancini et al.: Imaging of thyroid tumor
angiogenesis with microbubbles targeted to vascular endothelial
growth factor receptor type 2 in mice. BMC Medical Imaging 2013 13:31.
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