Longitudinal MRI Evaluation of Intracranial Development
and Vascular Characteristics of Breast Cancer Brain
Metastases in a Mouse Model
Heling Zhou1, Min Chen2, Dawen Zhao1*
1Department of Radiology, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America, 2Clinical Sciences, University of Texas Southwestern
Medical Center, Dallas, Texas, United States of America
Longitudinal MRI was applied to monitor intracranial initiation and development of brain metastases and assess tumor
vascular volume and permeability in a mouse model of breast cancer brain metastases. Using a 9.4T system, high resolution
anatomic MRI and dynamic susceptibility contrast (DSC) perfusion MRI were acquired at different time points after an
intracardiac injection of brain-tropic breast cancer MDA-MB231BR-EGFP cells. Three weeks post injection, multifocal brain
metastases were first observed with hyperintensity on T2-weighted images, but isointensity on T1-weighted post contrast
images, indicating that blood-tumor-barrier (BTB) at early stage of brain metastases was impermeable. Follow-up MRI
revealed intracranial tumor growth and increased number of metastases that distributed throughout the whole brain. At
the last scan on week 5, T1-weighted post contrast images detected BTB disruption in 160 (34%) of a total of 464 brain
metastases. Enhancement in some of the metastases was only seen in partial regions of the tumor, suggesting intratumoral
heterogeneity of BTB disruption. DSC MRI measurements of relative cerebral blood volume (rCBV) showed that rCBV of brain
metastases was significantly lower (mean =0.8960.03) than that of contralateral normal brain (mean =1.0060.03;
p,0.005). Intriguingly, longitudinal measurements revealed that rCBV of individual metastases at early stage was similar to,
but became significantly lower than that of contralateral normal brain with tumor growth (p,0.05). The rCBV data were
concordant with histological analysis of microvascular density (MVD). Moreover, comprehensive analysis suggested no
significant correlation among tumor size, rCBV and BTB permeability. In conclusion, longitudinal MRI provides non-invasive
in vivo assessments of spatial and temporal development of brain metastases and their vascular volume and permeability.
The characteristic rCBV of brain metastases may have a diagnostic value.
Citation: Zhou H, Chen M, Zhao D (2013) Longitudinal MRI Evaluation of Intracranial Development and Vascular Characteristics of Breast Cancer Brain Metastases
in a Mouse Model. PLoS ONE 8(4): e62238. doi:10.1371/journal.pone.0062238
Editor: Alexander Annala, City of Hope, United States of America
Received January 11, 2013; Accepted March 19, 2013; Published April 29, 2013
Copyright: ? 2013 Zhou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by the DOD IDEA Awards W81XWH-08-1-0583 and W81XWH-12-1-0317. MRI experiments were performed in the
Advanced Imaging Research Center, an NIH BTRP # P41-RR02584 facility, and ultrasound-guided intracardiac injection was performed with VisualSonics Vevo 770
under 1S10RR02564801. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Dawen.Zhao@UTSouthwestern.edu
Brain metastasis is the most common intracranial malignancy
in adults. The prognosis is extremely poor, with a median
survival of 4–6 months even with aggressive treatment. Breast
cancer is one of the three major primary cancers with a high
morbidity of brain metastasis (15–25%) [1–3]. Benefited from the
routine mammography tests and improved therapeutic options,
mortality of breast cancer has been decreasing in the past decade
. Advances in chemotherapy and immunotherapy played an
important role in treating primary breast cancer as well as its
systemic metastasis. However, the incidence of brain metastasis
seems to have increased over the past decade, especially within
patients undergoing these systematic therapies [5–8]. In part, this
is due to the fact that most chemotherapeutic agents that show
efficacy against systemic disease have poor penetration of blood-
brain barrier (BBB). Brain metastases containing an intact BBB
are hereby inaccessible to the therapeutics and remain untreated
Current understandings for vascular development in brain
metastases are largely based on invasive histological studies on
animal models [12,13]. Several brain-tropic cancer lines derived
from primary melanoma, lung or breast cancer are capable of
developing brain metastases upon intracardiac or intracarotid
injection [14–17]. Using this model, several studies by others have
applied molecular tracers to evaluate BTB permeability by
measuring their uptake in brain metastases versus normal brain
tissues on ex vivo brain sections. These studies have shown that
BTB is intact at earlier stage of brain metastases, but becomes
disruptive while the metastases growing larger [12,13]. However,
histological studies normally require a large number of mice that
are killed at different time points after tumor implantation. More
importantly, information about temporal development in individ-
ual lesions is lacking from histological studies.
In vivo imaging promises greater efficiency since each animal
serves as its own control and multiple time points can be examined
sequentially. Not only intra- and inter-tumoral heterogeneity but
also temporal comparison in the same individual lesions can be
studied with longitudinal imaging. MRI that has a superb spatial
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resolution is the most widely used imaging modality for brain
tumors of clinical patients. MRI at 1.5 or 3 T has previously been
applied by others to study the intracardiac model of brain
metastasis of breast cancer MDA-MB231Br (231Br) in mice or
rats. Successful detection of multifocal brain metastases and
alteration of BTB permeability based on the leakage of MR
contrast agents has been reported in their studies [18,19].
Tumor blood perfusion is another main factor that may affect
efficient delivery of chemotherapeutics to brain metastasis. There
are several noninvasive imaging modalities available for vascular
perfusion assessment. Dynamic susceptibility contrast (DSC)
perfusion MRI is one of the most applied MR imaging techniques
for brain perfusion measurement [20,21]. A series of T2*-weighted
images is acquired to capture the rapid passage of gadolinium
contrast agent post i.v. injection. The perfusion parameters
extracted from decreased signal intensity during the first pass of
the contrast are used to calculate cerebral blood volume (rCBV)
and flow (rCBF) in a disease site relative to the contralateral
healthy brain region. DSC perfusion MRI has been widely used
for noninvasive assessment of tumor vascularity in both preclinical
and clinical settings [22–24]. However, there are currently no
studies that have assessed vascular perfusion and their changes
with intracranial development of brain metastases. The charac-
teristics of the 231Br model containing multifocal brain lesions
that are widespread throughout the whole mouse brain present a
technical challenge. To facilitate intertumoral rCBV comparison
within and between individual animals, in contrast to the widely-
used single normal reference, we have developed a novel approach
that utilizes multiple normal references contralateral to the tumor
In this study, we have applied a high field 9.4 T MRI system to
monitor longitudinal development of brain metastases based on
T2-weighted images after intracardiac inoculation of breast cancer
MDA-MB231Br cells. Along with intracranial tumor growth,
changes in BTB and tumor vascular volume, rCBV were
evaluated by longitudinal T1-weighted post contrast images and
DSC perfusion-weighted MRI, respectively. Comprehensive
analysis was performed to study correlations between tumor
volume, BTB disruption and rCBV in individual metastases.
Materials and Methods
Brain-tropic human breast cancer MDA-MB-231/BR-GFP cell
line (231-BR) was previously described [14,15]. The 231-BR cells
(kindly provided by Dr. Steeg, NCI) were incubated in Dulbecco’s
modified Eagle’s medium (DMEM) with 10% FBS, 1% L-
Glutamine and 1% penicillin-streptomycin at 37uC with 5%
CO2. Once 80% confluence was reached, the cells were harvested,
and suspended in serum-free medium.
Breast Cancer Brain Metastasis Model
All animal procedures were approved by the Institutional
Animal Care and Use Committee of University of Texas
Southwestern Medical Center. Female nude mice (n
BALB/c nu/nu, 6–8 weeks old; NCI, Frederick, MD) were
anesthetized with inhalation of 2% isoflurane. 26105231-BR cells
(in 100 ml of serum free medium) were injected directly into the left
ventricle of a mouse heart under the imaging guidance of a small
animal ultrasound (Vevo 770, VisualSonics; Toronto, Canada).
Longitudinal MRI Studies
MRI monitoring of the initiation and development of
MRI was initiated two weeks after tumor
implantation and repeated once a week for up to three weeks.
Animals were sedated with 3% isoflurane and maintained under
general anesthesia (1.5% isoflurane). Animal body temperature
and respiration were monitored and maintained constant
throughout the experiment. MR measurements were performed
using a 9.4 T horizontal bore magnet with a Varian INOVA
Unity system (Palo Alto, CA). A tail vein of mouse was
catheterized using a 27 G butterfly for Gd-DTPA (MagnevistH;
Bayer HealthCare, Wayne, NJ) contrast agent administration.
High resolution multi-slice (14 slices with 1 mm-thick, no gap) T1-
and T2-weighted coronal images, covering from the frontal lobe to
the posterior fossa, were acquired with the following parameters:
T1-weighted images: spin echo multiple slice (SEMS), TR/TE
=400 ms/20 ms, matrix: 2566256, FOV 20620 mm, resolution:
78678 mm2in plane. T2-weighted images: fast spin echo multiple
slice (FSEMS) sequences, TR/TE =2500 ms/48 ms, 8 echo
trains, matrix: 2566256, FOV 20620 mm, resolution: 78678
mm2in plane. We determined tumor size on T2-weighted images
by manually outlining the enhancing portion of the mass on each
image by using MatLab (Mathworks, Natick, MA) programs
written by us. With high spatial resolution, hyperintense lesions
can be identified as small as 310 mm in diameter. Since the
metastases in this study were very small and development of
necrosis and edema was minimal, the hyperintense lesion on T2-
weighted images truly represented the tumor mass. Given most of
the tumor diameters were smaller than the slice thickness (1 mm),
the tumor size was presented as in plane area rather than the
Once brain lesions were identified on T2-weighted
images, DSC MRI was performed on 4 of the slices containing
most of the metastases. A series of gradient echo multiple slice
(GEMS) T2*-weighted images over 5 mins was acquired before
and after a bolus injection of Gd-DTPA contrast (0.1 mmol/kg
body weight) via a tail vein. DSC MRI parameters: TR/TE
=27 ms/4 ms, FA =20u, matrix: 64664, FOV 20620 mm,
number of slices =4 with 1 mm thick. As illustrated in Fig. 1, raw
data of signal intensity were extracted from the image series on a
voxel-by-voxel basis and plotted into a time course curve. DR2*
was calculated using equation:
where St is the signal intensity (SI) of each time point, S0is the
mean SI of baseline, and TE is the echo time. First-pass
pharmacokinetic modeling (FPPM) fitting  was applied to
DR2*curve to detect the starting and the ending points of the
bolus. The equation used for this step is as follows:
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Figure 1. Dynamic susceptibility contrast (DSC) MRI of rCBV. A. A series of T2*-weighted images of a mouse brain was acquired before and
after a bolus injection of the contrast agent, Gd-DTPA via a tail vein (arrow). Immediately after the injection, significant loss in signal intensity was
observed, which gradually recovered to the baseline level 5 mins later. B. The flowchart illustrates the data process of DSC MRI to generate rCBV map.
Raw data of DSC MRI signal versus time curve was first plotted, depicting the first pass of the contrast agent via the brain as the dip on the curve. The
DR2*was then calculated from the signal time course. FPPM was applied to determine the general trend of DR2*and a three-segment baseline was
generated. Finally, Gamma-variate fitting was used to correct DR2*, and the area under the bolus was calculated, which is proportional to CBV. C. An
anatomic T2-weighted image was obtained from a normal mouse brain. A color-coded rCBV map generated from DSC MRI was overlaid on the T2-
weighted image showing symmetric distribution of rCBV between the two hemispheres (D).
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And the bolus shape function is defined as:
Baseline correction was separated into three parts. Pre-bolus
baseline was set to zero. Then, a linear fit was applied from this
point to the end of the data to obtain post bolus baseline, and
connection of these two segments served as the under-bolus
baseline. Subtraction of DR2*with baseline obtained in previous
step provided a corrected DR2*. CBV was calculated from the
gamma-variate fitting applied to the corrected curve and the area
under the fitted curve was proportional to CBV . Gamma
fitting was obtained using the following equation:
ROIs of brain metastases were drawn based on the high
resolution T2-weighted images. The corresponding contralateral
normal ROIs were selected symmetrically to the tumor ROIs
according to the distinct landmarks in brain, such as ventricles,
hippocampus and the interfaces of different anatomical structures.
In cases where the contralateral sites appeared abnormal,
neighboring regions in the same anatomical structure were
selected instead. The mean CBV value of all the normal ROIs
in each animal served as the reference. rCBV for both individual
metastases and the normal brain was determined by normalizing
the CBV to the reference CBV. Analysis was performed on a
home written MATLAB program. A color-coded rCBV map was
obtained and overlaid on the corresponding T2-weighted image.
Brain Tumor Barrier (BTB) permeability.
spin echo multislice images were immediately acquired after DSC
MRI, about 5 mins post contrast injection to evaluate BTB
permeability. A non-permeable metastasis is isointense, while a
permeable metastasis appears hyperintense to surrounding brain
tissue on T1-weighted post contrast image.
Histological and Immunohistochemical Studies
Animals were sacrificed immediately after the last MR follow-up
and the brain was dissected and frozen for preparation of
cryosections. H&E staining was performed on coronal brain
sections (10 mm), while their adjacent sections were used for
immunohistological staining. Vascular endothelium was stained
using a rat anti-mouse CD31 antibody (Serotec Inc., Raleigh, NC)
followed by Cy3-labeled goat anti-rat IgG (Jackson Immunor-
esearch Laboratory, West Grove, PA). Fluorescence images of
tumor cells (green, GFP) and vasculature (red) were captured on
the same microscopic field using a Coolsnap digital camera
mounted on a Nikon microscope and analyzed with MetaVue
software (Universal Imaging Corporation). Microvascular density
(MVD) was calculated as follows: mean number of red vessels/
mean area of green tumors. A total of twelve metastases and their
contralateral normal brain tissues in 3 tumor-bearing mouse
brains were evaluated.
Statistical analysis was conducted using R (R Development
Core Team, 2012), a language and environment for statistical
computing. A natural log transformation was applied to all the
rCBV values to achieve normal distribution before Student’s t-test
was performed for significance analysis. The statistical analysis of
correlation was based on Regression and Bivariate plots (SAS Inst.
Inc., Cary, NC). Two sample paired Student’s t-test was
performed to evaluate the significant difference of rCBV or
MVD of tumor and its contralateral reference tissue. After
dividing tumors into two groups based on the permeability status,
comparison of rCBV or tumor size between these two groups was
evaluated using unpaired t-test. Paired t-test was performed for
longtitudinal studies to assess the changes of individual lesions over
Ultrasound imaging-guided left ventricular injection of brain-
tropic breast cancer 231BR cells ensured the accuracy so that
every animal in this study developed brain metastases, as
compared to a successful rate of 50% with the manual injection
used in our previous study. Longitudinal MRI monitoring was
initiated 2 weeks post injection and repeated once a week for up to
3 more weeks (Fig. 2). With the high spatial resolution of T2-
weighted images, metastatic lesions, appearing as hyperintense,
became visible in mouse brain 3 or 4 weeks post injection; the
minimum detectable tumor was ,310 mm in diameter, whereas all
of these early stage metastases were isointense on T1-weighted post
contrast images (Figs. 2, 3, 4, 5). Follow-up scans revealed
increased tumor size and appearance of new lesions (Figs. 2A and
5A), which correlated well with H&E staining (Fig. 2B). A total of
464 metastases, with a size ranging from 0.09 mm2to 1.7 mm2, in
9 mice were depicted on T2-weighted images of the last MR
follow-up at week 5. These metastases distribute throughout the
mouse brain with a higher incidence in the cerebral cortex (49%),
whereas the least number in the thalamus, midbrain or cerebellum
(5%; Fig. 2C). Of these metastases, 160 (34%) lesions were
enhanced on T1-weighted post contrast images, indicating a
locally disrupted BTB (Figs. 2A, 3A, 4 and 5A). However,
enhancement in some of the metastases was only seen in partial
regions of the tumor (Fig. 3A), suggesting intratumoral heteroge-
neity of BTB disruption.
Dynamic susceptibility contrast (DSC) MRI, based on a bolus
injection of contrast agent Gd-DTPA into a tail vein, was applied
for rCBV measurement (Fig. 1A). The first pass of the contrast via
brain vasculature was clearly depicted as the dip region in the
signal intensity time cure (Fig. 1B). A color-coded rCBV map of a
normal mouse brain, projected on the T2-weighted image, showed
the high level of symmetry between hemispheres and significant
higher rCBV in the cortical regions (Fig. 1C and D). For brain
metastases-bearing mice (n =9), 212 of the 464 metastases,
identified at the last scan, were subject to rCBV measurements and
compared with their contralateral normal brain. As shown in
Fig. 3, the metastatic lesions were found to have significantly lower
rCBV, compared to their contralateral normal brain (mean
=0.8960.03 (s.e.) vs. 1.0060.03 (s.e.), p,0.005; Fig. 3C). It is
important to notice marked heterogeneity in rCBV between
individual metastases, ranging from 0.16 to 2.84 (Figs. 3, 4, 5).
This is also true for rCBV in normal brain, as demonstrated in
Fig. 1. Thus, it is necessary to compare rCBV between a pair of a
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specific metastases and its contralateral normal brain. Further
analysis showed no significant correlation between rCBV and
tumor size (R2,0.02; Fig. 4A). When these metastases were
separated into the permeable (n =70) and non-permeable (n
=142) group, determined upon whether it was enhanced by T1
contrast agent, the data showed that neither rCBV (mean
=0.8860.04 vs. 0.9060.03; p.0.1; Fig. 4B) nor tumor size (mean
=0.4960.11 mm2vs. 0.4760.14 mm2; p.0.1; Fig. 4C) signifi-
cantly differed between the two groups.
Figure 2. Longitudinal MRI monitoring of the initiation and development of intracranial brain metastases. A. MRI scans of the whole
mouse brain were initiated 3 weeks after intracardiac injection of MDA-MB231Br cells and repeated once a week for 2 weeks. Four consecutive
coronal MRI sections of a representative mouse brain showed no apparent intracranial lesions on T2-weighted images. However, follow-up images at
week 4 identified multiple lesions with hyper-intensity on T2-weighted images (arrowhead), but none of them was enhanced on T1-weighted post
contrast images. An increased number of lesions (arrowheads) appeared on the images at week 5, only a few of which (arrowheads) were enhanced
post Gd-DTPA. B. Corresponding histological sections of H&E staining showed a good correlation with MRI. C. MRI evaluation of a total of 464
metastases in 9 mice brains indicated that these metastases distributed through the whole mouse brain with a higher incidence in the brain cortex
(49%). Note: OB: Olfactory bulb; BG: Basal ganglia; Hippo: Hippocampus.
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Longitudinal MRI studies allowed in vivo non-invasive evalua-
tion of tumor growth and changes in BTB permeability and rCBV
of individual brain metastases. Thirty two metastases that were
identified from 5 of the 9 mouse brains in the scans of week 3 were
followed a week later. As shown in a representative mouse brain in
Fig. 5, five small hyperintense metastases first appeared on T2-
weighted images at week 3. All the 5 lesions grew larger, along
with many other new lesions becoming visible in the following
week’s scan. All the 5 lesions showed no enhancement on T1-
weighted contrast images at week 3, only one of them became
enhanced at week 4. DSC MRI found that rCBV values decreased
in 4 of the 5 metastases (Fig. 5B). For the total of 32 metastases,
rCBV values were initially similar to those of their contralateral
normal brain (mean =1.0560.05 vs. 0.9660.04), but decreased
significantly and became significantly lower than those of their
contralateral normal brain in the late scan (mean =0.8860.06 vs.
1.0060.06, p,0.05; Fig. 5C).
Figure 3. Significantly lower rCBV in brain metastases than contralateral normal brain. A. Four weeks after intracardiac injection of 231Br
cells, T2-weighted MRI revealed multiple high signal intensity lesions (arrowheads) on four consecutive coronal sections of a representative mouse
brain. Only a few of the lesions (arrowheads) were enhanced on T1-weighted post contrast images, one (blue arrowhead in the MRI section 3) of
which showed partial enhancement, indicating intratumoral heterogeneity of BTB disruption. rCBV maps of the four sections were generated and
overlaid on the T2-weighted images. B. The rCBV values of the metastatic lesions and their contralateral normal brain were obtained and summarized
in the table. Note the color presented in the table coincides with the color of arrowhead on each of the MR images. Most of metastatic lesions had
lower rCBV values than their contralateral counterparts of normal brain. C. Statistical analysis of rCBV in a total of 212 lesions of 9 animals obtained
from the last follow-up MRI showed significantly lower rCBV of the metastatic tumors with a mean value of 0.8960.03 (s.e.), compared to the
contralateral normal brain (mean =1.0060.03; p,0.005).
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Immunohistochmical staining of vascular endothelium (CD31)
showed that MVD was 6696201/mm2within the metastatic
lesions (n =12), which was significantly lower than that of the
contralateral normal brain (9656177/mm2; p,0.05; Fig. 6).
Moreover, in contrast to the normal brain comprising of regularly-
shaped micro-vessels, the irregular and dilated vessels were often
seen for tumor vessels (Fig. 6).
In the present study, we have demonstrated the utility of
longitudinal MRI to evaluate intracranial growth and vascularity
of breast cancer brain metastases in a mouse model. Using the
9.4 T MRI, high resolution T2-weighted images enabled the
detection of multifocal tumor initiation at a diameter of as small as
310 mm. Longitudinal monitoring of BTB permeability based on
T1-weighted contrast enhanced images revealed that BTB in early-
stage (week 2 or 3) brain metastases were exclusively impermeable;
even at the late stage (week 4 or 5), T1contrast enhancement was
only found in a small proportion (34%) of brain metastases,
indicating that the BTB is still intact in the majority of the
metastases (Figs. 2, 3 and 5). This observation is in good
agreement with a recent MRI study of the 231BR brain metastases
mouse model. In that study, Percy et al observed no contrast
enhancement for brain metastases by day 20, while 28% of the
metastases by day 30 appeared hyperintense on T1-weighted post
contrast images . The MRI data are consistent with
histological studies conducted previously by others. Zhang and
colleagues administered fluorescent dye, sodium fluorescein
systemically into the mice bearing brain metastases to study BBB
permeability. The microscopic observations on brain sections
showed differential permeability of the dye among the metastases,
of which lesions smaller than 0.2 mm2had intact BBB, while larger
metastases were leaky because of tumor angiogenesis and/or
central necrosis . Although our data of the current study
indicated that the mean tumor size of permeable metastases was
larger than that of the non-permeable ones, there was no
significant difference in tumor size between the two groups
(Fig. 4C). Similarly, lack of correlation between tumor size and
BBB disruption was reported in a recent study by Lockman et al.,
suggesting other factors may also be involved in this dynamic
On many occasions, enhancement on T1-contrast images was
only seen in partial regions of the tumor (Fig. 3C), implicating
inhomogeneous disruption of BTB. This finding concurs with a
recent study of biodistribution of anti-cancer drugs in brain
metastases . Lockman and colleagues assessed the uptake of
radio-labeled paclitaxel or doxorubicin in brain metastases of
Figure 4. Lack of correlation between rCBV, tumor size and permeability of brain metastases. Based on T1-weighted contrast enhanced
MRI, the 212 metastases studied by DSC MRI were separated into the permeable (enhanced, n =70) and non-permeable (not enhanced, n =142)
group. A. A plot of rCBV versus individual tumor size showed no correlation in either the permeable (filled; R2=0.01) or non-permeable group
(empty; R2,0.02). B. The rCBV values of the permeable lesions (median =0.84, ranging from 0.34 to 2.10) were not significantly different from those
of the non-permeable ones (median =0.82, ranging from 0.16 to 2.84; p.0.1). C. Further comparison found no significant difference in tumor size
between the permeable (mean =0.4960.11 mm2) and the non-permeable (mean =0.4760.14 mm2; p =0.1) metastases.
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MDA-MB231Br-Her2 mouse model. Heterogeneous intratumoral
distribution of the chemotherapeutics was clearly visualized on ex
vivo brain sections by phosphorescence. Even in the most
permeable metastases, the drug concentrations were far below
that in visceral metastases . Taken together, all these data
support the notion that systemic anti-cancer therapeutics has
limited utility in treating brain metastases.
Despite its small molecular weight (MW , 500), the hydrophilic
MRI contrast, Gd-DTPA is found not to penetrate across the
intact BBB and is thus suitable for BBB permeability study. Many
strategies to overcome this barrier have been exploited to facilitate
the delivery of effective anti-cancer therapeutics into brain tumors,
i.e, hemispherical opening of the BBB using high-concentration
intra-carotid injections of mannitol or temporary disruption of
localized BBB by high-intensity focused ultrasound [26,27]. Thus,
it is critical to develop a means to enable non-invasive evaluation
of BBB damage after exposure to intervention in order to predict
drug delivery. Indeed, Treat et al recently reported a linear
correlation between T1 signal intensity post Gd-DTPA and
doxorubicin concentrations in brain regions after BBB damage
induced by sonication .
Perfusion MRI has been widely used to provide important
diagnostic and prognostic information on pathological conditions.
Arterial spin labeling (ASL) MRI, utilizing magnetically tagged
arterial blood as endogenous contrast has proven feasible in
quantitative measurements of rCBF and rCBV in clinical studies
[29,30]. However, the low sensitivity and poor signal to noise ratio
(SNR) of ASL perfusion MRI limits its application to mouse brain.
Dynamic contrast susceptibility (DSC) MRI acquired after
infusion of MRI contrast agents is another technique to measure
rCBV. DSC MRI has been widely applied to study microvascu-
lature and hemodynamics in brain tumors. Recent studies have
correlated DSC MRI of rCBV with histological grade and degree
of neovascularization in human glioma [23,31,32]. In a preclinical
mouse glioma study, a positive correlation between rCBV and
tumor microvascular density (MVD) was found . However,
Figure 5. Longitudinal MRI study of changes in BTB permeability and rCBV of brain metastases. A. Longitudinal MRI of a representative
mouse brain was initiated 2 weeks after intracardiac injection of 231Br cells. At week 3, five small metastases (arrowhead) were identified on four
consecutive T2-weighted coronal images. At week 4, many more lesions appeared on T2-weighted coronal images, while all the 5 lesions seen on
week 3 were found to increase in size (arrowhead). Changes in BTB permeability and rCBV were then evaluated for these five lesions. There was
initially no contrast enhancement seen in the five tumors at week 3, indicating an intact BTB. All the tumors except one (yellow arrowhead) still kept
BTB intact at week 4. rCBV maps were created and rCBV values of the tumors were presented in the table (B). C. A total of 32 lesions in 5 animals were
seen on both scans of weeks 3 and 4. rCBV of brain metastases (solid) was initially similar to that of contralateral normal brain (open; mean
=1.0560.05 (se) vs. 0.9660.04), but decreased significantly (p,0.05) and became significantly lower as compared to their contralateral normal brain
in the late scan (mean =0.8860.06 vs. 1.0060.06; p,0.05).
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little is known about rCBV abnormality of brain metastases, in
particular, its variation among individual brain metastases as well
as its alteration with tumor development.
Our data showed marked heterogeneity of rCBV for both
normal brain and metastatic lesions (Figs. 1 and 4). To acquire an
rCBV value of a lesion, a region of normal brain needs to be
chosen to serve as a reference. Previously published clinical or
preclinical works on vascular perfusion of a solitary brain tumor
have utilized either a region of white matters or the normal brain
contralateral to the tumor as a single reference . Tthe
intracardiac brain metastasis model used in this study developed
multiple intracranial lesions. As shown in Fig. 2, these multifocal
metastases distributed well throughout the whole mouse brain. It is
difficult to define a specific region of normal brain that can be used
as a common reference for each individual case. Moreover, given
the marked heterogeneity in vascular perfusion of normal brain,
we applied multiple normal regions that locate contralaterally to
the individual metastases as the references. The mean CBV value
of all the normal ROIs in each animal was used to serve as the
reference for both metastatic lesions and normal brain regions. In
cases where the contralateral sites appeared abnormal, neighbor-
ing regions in the same anatomical structure were selected instead.
As shown in Fig. 1D, using this approach, the reliable rCBV map
was generated in a normal mouse brain. We believe that this
approach can significantly minimize the individual variation and
facilitate a comparison of rCBV between individual animals.
DSC MRI measurement of rCBV revealed significantly lower
rCBV of brain metastases than that of contralateral normal brain
(p,0.005). The rCBV data were in good agreement with
histological findings that MVD within the metastases was
significantly lower than that of contralateral normal brain. Fidler’s
group has documented in their published studies that the MVD
within the metastases was 20 times lower that in the surrounding
normal brain . Unlike the robust angiogenesis observed in
primary high grade glioma, vessel sprouting, the characteristic of
angiogenesis, is rarely seen in brain metastases. Instead, dilation of
Figure 6. Immunohistochemical study of microvascular density (MVD) in brain metastases. A. Anti-CD31 staining was performed on a
brain section bearing metastases. A cortical lesion (, 600 mm in diameter) was depicted with green fluorescence (GFP). Microvessels (red) within the
lesion appeared less dense, as compared to abundant fine vessels in the contralateral normal brain tissues (B). Some of the tumor vessels were
irregular in shape and larger in diameter (arrow). C. Quantitative data of MVD showed a significantly lower MVD in brain metastases versus
contralateral normal brain (mean =6696201/mm2vs. 9656177/mm2; p,0.05).
Longitudinal MRI of Brain Metastases
PLOS ONE | www.plosone.org9April 2013 | Volume 8 | Issue 4 | e62238
blood vessel lumen, as shown in fig. 6, and reported elsewhere in
both the experimental brain metastases and surgical specimens of
human lung cancer brain metastases, is considered as a result of
the division of endothelial cells [12,33]. The characteristic rCBV
may have a diagnostic value for brain metastases to differentiate
them from those highly vascularized and perfused malignant
Longitudinal MRI allows rCBV of individual brain metastases
to be examined over time. Intriguingly, our data showed that
rCBV of individual metastases at early stage was similar to, but
became significantly lower than that of their healthy counterparts
at the late stage of tumor development (Fig. 5C; p,0.05). By using
multiphoton laser scanning microscopy and a mouse cranial
window model, Kienast et al. followed in real time brain
metastases formation from lung cancer and melanoma in mouse
brain. After extravasation, the metastatic cells grow along the
preexisting normal brain vessels . In another study, Kusters
et al. showed that a melanoma brain metastasis could grow up to
3 mm through co-opting preexisting blood vessels without
induction of an angiogenic switch . All these data indicate
that there is no angiogenic compensation for the tissue volume
increase at the lesion site, resulting in a lower MVD and rCBV, as
compared to normal brain. For the pooled data, however, we
found that rCBV was not correlated with either the size or
permeability of metastasis (Fig. 4A and B), implicating that the
intertumoral heterogeneity of rCBV may result from regional
variation in vascularity in normal brain where metastases locate.
In summary, we have applied 9.4 T MRI to study brain
metastases formation after intracardiac injection of breast cancer
MDA-MB231Br-GFP cells into mice. High resolution T2-weight-
ed MRI enables the detection of multifocal metastases at early
stage. MRI contrast, Gd-DTPA based T1-weighted contrast
enhanced MRI and T2*-weighted DSC MRI allow non-invasive
characterization of vascular permeability and blood volume during
intracranial development of brain metastases. Significant lower
BTB permeability and vascular volume in brain metastases than
normal brain underscore the urgent need to develop brain
permeable drugs or a means to alter the BTB permeability in
order to achieve therapeutic concentrations of anti-cancer
We are grateful to Drs. Diane Palmieria and Patricia Steeg (CCR, NCI) for
providing breast cancer MDA-MB231Br cells, Dr. Glyn Johnson (New
York University School of Medicine) for valuable discussions, and Dr.
Mason and Mr. Jason Reneau for technical and collegial support.
Designed the software used in analysis: HZ MC. Obtained permission for
use of cell line: DZ. Conceived and designed the experiments: DZ.
Performed the experiments: HZ DZ. Analyzed the data: HZ MC DZ.
Contributed reagents/materials/analysis tools: HZ MC DZ. Wrote the
paper: HZ MC DZ.
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