Key words
breast - ultrasound - angiogenesis - animal investigations
The present work was originally published in European Radiology [1]
[2] and as a PhD thesis at the RWTH Aachen University [3] and was conducted at the Institute for Experimental Molecular Imaging at the RWTH
Aachen University.
Among women, breast cancer is the most frequently diagnosed cancer worldwide. Accurate
and sensitive imaging techniques are needed to help decrease the mortality rate by
early detection of breast cancer. Contrast-enhanced ultrasound with non-targeted microbubbles
in patients was shown to possibly help to predict prognosis and to noninvasively detect
highly aggressive breast cancers [4]. This technique is called functional ultrasound. Due to their size of 2 – 5 µm,
the microbubbles stay strictly intravascular and the relative blood volume (rBV) of
a tumor can be determined, reflecting its angiogenic activity und thus, the tumor’s
aggressiveness. A further technique for the characterization of the breast cancer’s
aggressiveness may be molecular ultrasound imaging. The microbubbles, now targeted
to tumor-endothelial angiogenic markers, bind to their specific target upon injection
and a relative quantification of marker expression within the tumor endothelium can
be performed noninvasively. In the present work two types of microbubbles were used
[1]
[2]. For molecular ultrasound this was BR55. A heterodimer selectively binding to human
and murine vascular endothelial growth factor receptor type 2 (VEGFR-2) was conjugated
to DSPE-PEG-2000-NH2 which was then incorporated into the microbubbles’ lipid shell.
A detailed description can be found in [5]. VEGFR-2 is one of the most characterized tumor-angiogenic markers. For functional
ultrasound BR38 was applied, consisting of non-targeted, PEGylated microbubbles, i. e.
microbubbles with a Polyethylenglycol (PEG) brush. Their lipid shell contains DPPE-MPEG
5000 that contributes to a long persistence of the microbubbles in the blood system
[6]. Both, BR38 and BR55 were designed for clinical application.
In the first study the circulating characteristics of BR55 and BR38 microbubbles in
healthy mice and their ability to characterize the angiogenic activity of differently
aggressive human breast adenocarcinomas were evaluated [1]. BR38 showed a long blood half-life of more than ten minutes and a relatively slow
uptake by the liver. For clinical use the long circulation time of BR38 might be beneficial
because it allows an intensive examination of one or even several different organs.
The VEGFR-2 targeted BR55 microbubbles had a relatively short blood half-life of about
4 min. An early accumulation of these microbubbles was observed in the liver which
led to their rapid clearance from the blood. The fast elimination of freely circulating
microbubbles is strongly desired in order to minimize background signals, as BR55
is designed to assess VEGFR-2 expression.
In highly aggressive, estrogen-independent MDA-MB-231 and less aggressive, estrogen-dependent
MCF-7 breast cancer xenografts, both BR55 and BR38 proved to be very well suited for
distinguishing and characterizing the different angiogenic activity. Although the
more aggressive breast tumors showed a significantly higher rBV compared to the less
aggressive model determined by functional ultrasound using BR38, the differences in
the angiogenic activity between both tumor models were more distinct with the molecular
information obtained with BR55 molecular ultrasound imaging ([Fig. 1]). All data was validated by quantitative immunofluorescence.
Fig. 1 Ultrasound images showing s. c. breast cancer xenografts before a) and after injection of BR55 microbubbles. There is a higher peak enhancement in more
aggressive MDA-MB-231 compared to less aggressive MCF-7 tumours approximately 1 minute
after injection b) due to circulating microbubbles. Ten minutes after injection c) contrast enhancement is still higher in MDA-MB-231 and is mostly due to receptor
bound BR55. Reproduced from: Jessica Bzyl, Wiltrud Lederle, Anne Rix, et al. (2011)
Molecular and functional ultrasound imaging in differently aggressive breast cancer
xenografts using two novel ultrasound contrast agents (BR55 and BR38). Eur Radiol
21(9): 1988 – 95.
Abb. 1 Ultraschallbilder von Brustkrebsmodellen vor a) und nach Injektion von BR55 Mikrobläschen. Etwa eine Minute nach Injektion b) war das Peak Enhancement in den aggressiveren MDA-MB-231 höher als in den weniger
aggressiven MCF-7-Tumoren. Zehn Minuten nach Injektion c) ist die Kontrastverstärkung in MDA-MB-231 immer noch höher und resultiert aus rezeptorgebundenen
BR55. Entnommen aus: Jessica Bzyl, Wiltrud Lederle, Anne Rix, et al. (2011) Molecular
and functional ultrasound imaging in differently aggressive breast cancer xenografts
using two novel ultrasound contrast agents (BR55 and BR38). Eur Radiol 21(9): 1988 – 95.
In the second study 3-dimensional molecular and functional ultrasound imaging comprising
the whole of small developing MCF-7 tumors of only 2 mm in diameter compared to larger
tumors was performed [2]. Reliable imaging of the VEGFR-2 expression with BR55 microbubbles was possible
for all tumor sizes ([Fig. 2]). However, due to the particularly high VEGFR-2 expression in the smallest tumors,
molecular ultrasound may detect micro-tumors even more sensitively than larger tumors.
These results indicate that molecular ultrasound imaging may improve the detection
of small invasive breast carcinomas, which are known to overexpress VEGFR2 on the
endothelium [7]. In contrast, the degree of vascularization assessed by functional ultrasound with
BR38 was more or less constant in all tumor sizes. This phenomenon was elucidated
by immunofluorescence analysis which strongly indicated vessel maturation. Vessel
maturation is characterized by more stabilized vessels due to a close association
of vessel supporting cells with the endothelium, like α-SMA-expressing pericytes.
Furthermore, vessel maturation is characterized by the down-regulation of VEGFR-2
expression. In consequence, maturing vessels have an enlarged lumen resulting in a
higher blood volume. Significantly enlarged and significantly more α-SMA positive
vessels in larger tumors were observed. In addition, the number of vessels was decreased
in the larger tumors compared to the 2 mm tumors, thus explaining the constant rBV
for all tumor sizes measured by functional ultrasound.
Fig. 2 Representative images of A) a 4 mm³ and B) a 34 mm³ MCF-7 tumor (arrowheads). On the contrast-enhanced image signals of BR55
microbubbles can be seen as a overlay (white arrows show representative signals).
In the 4 mm³ tumors significantly more signals of BR55 microbubbles were detected
compared with all other tumor sizes. Reproduced from: Jessica Bzyl, Moritz Palmowski,
Anne Rix, et al. (2013) The high angiogenic activity in very early breast cancer enables
reliable imaging with VEGFR2-targeted microbubbles (BR55). Eur Radiol 23(2): 468 – 75.
Abb. 2 Repräsentative Bilder A) eines 4 mm³ und B) eines 34 mm³ großen MCF-7-Tumors (Pfeilspitzen). In dem kontrastverstärkten Bild
sind die Signale der BR55 Mikrobläschen dargestellt (weiße Pfeile deuten auf repräsentative
Signale). In den 4 mm³ großen Tumoren konnten signifikant mehr BR55-Signale detektiert
werden als in Tumoren aller anderen Größen. Entnommen aus: Jessica Bzyl, Moritz Palmowski,
Anne Rix, et al. (2013) The high angiogenic activity in very early breast cancer enables
reliable imaging with VEGFR2-targeted microbubbles (BR55). Eur Radiol 23(2): 468 – 75.
In conclusion, these two studies show how beneficial molecular ultrasound imaging
with clinically applicable microbubbles might be in regards to a noninvasive characterization
of breast cancer aggressiveness and an early detection of small breast lesions.
