Real-Time Visualization and Quantitation of Vascular
Permeability In Vivo: Implications for Drug Delivery
Desmond B. S. Pink1, Wendy Schulte1, Missag H. Parseghian2, Andries Zijlstra1,3, John D. Lewis1,4*
1Innovascreen, Inc., Halifax, Nova Scotia, Canada, 2Stonsa Biopharm Inc., Carlsbad, California, United States of America, 3Department of Pathology, Vanderbilt University,
Nashville, Tennesee, United States of America, 4Department of Oncology, University of Alberta, Edmonton, Alberta, Canada
The leaky, heterogeneous vasculature of human tumors prevents the even distribution of systemic drugs within cancer
tissues. However, techniques for studying vascular delivery systems in vivo often require complex mammalian models and
time-consuming, surgical protocols. The developing chicken embryo is a well-established model for human cancer that is
easily accessible for tumor imaging. To assess this model for the in vivo analysis of tumor permeability, human tumors were
grown on the chorioallantoic membrane (CAM), a thin vascular membrane which overlays the growing chick embryo. The
real-time movement of small fluorescent dextrans through the tumor vasculature and surrounding tissues were used to
measure vascular leak within tumor xenografts. Dextran extravasation within tumor sites was selectively enhanced an
interleukin-2 (IL-2) peptide fragment or vascular endothelial growth factor (VEGF). VEGF treatment increased vascular leak in
the tumor core relative to surrounding normal tissue and increased doxorubicin uptake in human tumor xenografts. This
new system easily visualizes vascular permeability changes in vivo and suggests that vascular permeability may be
manipulated to improve chemotherapeutic targeting to tumors.
Citation: Pink DBS, Schulte W, Parseghian MH, Zijlstra A, Lewis JD (2012) Real-Time Visualization and Quantitation of Vascular Permeability In Vivo: Implications
for Drug Delivery. PLoS ONE 7(3): e33760. doi:10.1371/journal.pone.0033760
Editor: Eric J. Bernhard, National Cancer Institute, United States of America
Received October 18, 2011; Accepted February 16, 2012; Published March 29, 2012
Copyright: ? 2012 Pink 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 study was supported by Canadian Cancer Society Research Institute grant #700537 to Dr. Lewis and National Institutes of Health/National Cancer
Institute grant #CA120711-01A1 to Dr. Zijlstra. Dr. Pink was supported by National Research Council-Industrial Research Assistance Program #636054. 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 read the journal’s policy and have the following conflicts: Dr. Lewis and Dr. Zijlstra are employees of Innovascreen Inc.
and hold stock in Innovascreen Inc. Dr. Pink and Dr. Schulte are employees of Innovascreen Inc. Dr. Parseghian is an employee of Stonsa Biopharm Inc. This does
not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: email@example.com
Tumors develop a chaotic vascular network characterized by
variable blood pressure and vascular permeability that inhibits
effective drug delivery . Many areas within tumors contain
irregular blood vessels that are leaky and allow influx of circulating
blood components. Sporadic high cell density within the tumor
prevents normal tissue drainage . This promotes the accumu-
lation of cellular and blood proteins in the interstitial space,
leading to high interstitial oncotic pressure, which inhibits the
extravasation of systemic drugs . Ultimately the distribution of
systemically circulating drugs in tumors can be unpredictable and
irregular since it depends heavily on the passive extravasation of
the drug from the vasculature into target tissues [2,4,5,6].
By transiently altering tumor blood vessel physiology during
systemic anti-cancer treatment, tissue perfusion and drainage can
be enhanced, thereby relieving interstitial hypertension [7,8].
Prolonged treatment with anti-angiogenic drugs, such as Sunitinib
or DC101, normalizes blood flow through the remaining stabilized
vasculature. These treatments can improve tumor micro-hemo-
dynamics and effectively lower the interstitial pressure. Conse-
quently, the efficacy of concomitantly or subsequently adminis-
tered drugs is enhanced due to improved vascular delivery
[7,9,10]. Similarly, treatment of hepatic tumors with interferon-b
(IFN-b) induces tumor vessel maturation and tissue perfusion,
which improves delivery of additional therapeutics . Altering
oncogenic signaling in tumors can also be used to change their
blood-flow dynamics . Specifically, inhibition of the PI3K
pathway increases tumor perfusion and simultaneously enhances
doxorubicin delivery . These findings indicate that the
strategic use of adjuvants to transiently modify tumor blood flow
and hemodynamics can facilitate drug delivery to cancer sites.
Normalizing blood flow promotes drug delivery by reducing the
interstitial pressure that counteracts diffusion. However, normal-
izing agents can also reduce vascular permeability. Vascular
permeability greatly influences the extravasation of drugs associ-
ated with carriers, including liposomes, micelles or other
nanoparticles [14,15,16]. Recent advances to manipulate vascular
permeability exemplify how adjuvant therapy might facilitate the
targeting of future and existing anti-cancer therapies to tumor
tissues [6,17,18]. Unfortunately, the lack of accurate means to
quantify vascular permeability is a significant hurdle to predicting
its direct influence on drug localization and uptake in vivo.
Classically, vascular permeability has been measured using the
Miles Assay . This assay determines the leakage of a visible dye
from the vasculature into the surrounding tissue spectrophoto-
metrically, with the relative vascular permeability determined as
the ratio of extravasated versus intravascular dye. This assay has
several limitations, however, that preclude its use in many cases. It
is limited to the analysis of a single time-point, which must be
selected empirically from pilot experiments. Furthermore, due to
the wide range of experimental approaches described in the
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literature, results are subject to a high degree of variability and
their repeatability must be considered. Variability can be
mitigated somewhat by using large tissue volumes. Consequently,
these experiments are generally performed in rodent models with
large group sizes , which is both expensive and time-
consuming. As the Miles assay is limited to the determination of
average permeability over an entire tissue, localized differences in
vascular permeability, particularly within tumors, cannot be
detected. A dynamic measure of vascular permeability would
allow for the assessment of the impact of regional and temporal
changes in vascular permeability on drug distribution within solid
Here, we present an integrated method to visualize and quantify
the real-time dynamics of dextrans in a shell-less chick chorioal-
lantoic (CAM) model. Regional and temporal differences in vessel
permeability within the tumor microenvironment are captured at
high resolution using an intravital imaging approach. The use of
dextrans of different molecular weights allows for the concurrent
evaluation of vascular permeability and vascular structural
integrity. The dynamics of anti-cancer drugs as they move through
the vasculature and into tumor tissues can be mimicked with
dextrans . Dextrans of various molecular weights can mimic
the diffusion of various sized macromolecules including macro-
molecular drug carriers (,70 kDa) and antibodies (,150 kDa)
into the tumor interstitial space. Large dextrans of ,2000 kDa,
are sequestered within the lumen of the tumor vasculature .
This work builds upon earlier observations in the shell-less chicken
embryo model, which examined microvascular perselectivity
during normal angiogenesis in the early stages of CAM
development . These authors demonstrated a rapid reduction
in microvascular permeability to FITC-dextrans of varying sizes
(20–150 kDa) between days 4.5–5.5 of the normal 21-day
gestation. They also demonstrated that dextran size correlated
with permeability (dextran-20.dextran-40.dextran-70.dextran-
150 kDa). Furthermore, while these authors report tumor
permeability values for 70 kDa and 150 kDa dextrans, they did
not examine it in the shell-less or ex ovo chick model. The leakage
of small versus large molecular weight dextrans from the
vasculature in this model may provide a high-resolution measure
of vascular permeability predictive of drug localization in vivo.
The CAM is a thin, respiratory tissue for the developing chick
embryo characterized by a dense, highly organized network of
blood vessels [22,23]. The physiological responses of the CAM are
consistent with those of mammalian tissues [24,25] and it has
provided a physiologically relevant setting for angiogenesis
research for more than a century [26,27,28,29,30,31]. The
commercial availability of fertilized eggs, the ease of embryo
culture, and the robustness of the CAM model facilitate large,
statistically powerful studies and make it suitable for high
throughput approaches. The CAM is not fully immunocompetent
in the early embryo , and it supports the growth of human and
murine tumor xenografts [33,34,35]. In addition, in the ex ovo
model, the CAM is directly accessible for experimental manipu-
lation and imaging. Paired with a fluorescence microscopy
platform, this model is well-suited for analyzing drug-induced
changes in vascular permeability in tumor xenografts and their
We demonstrate, using this intravital imaging approach, that
vascular permeability can be manipulated to modulate the
extravasation of small molecules into the local tumor microenvi-
ronment. Treatment with vascular endothelial growth factor
(VEGF) or a permeability enhancing peptide (PEP) fragment of
IL-2  either locally or systemically results in a temporary
enhancement of vascular permeability that can be precisely
monitored over time. We show that this transient increase in
vascular permeability can be exploited to significantly enhance the
accumulation of a chemotherapeutic drug within the tumor.
In vivo detection of vascular leak
A modified Miles assay  was adapted for the CAM model.
Chicken embryos (day 15) were injected intravenously with
phosphate buffered saline (PBS) recombinant human VEGF121
(40 ng, Peprotech) or PEP (0.1 nM, Peregrine Pharmaceuticals) in
50 mL volumes. For local applications, reagents were applied to
the CAM via a small hole in a sterilized glass coverslip (18 mm
diameter). Embryos were then incubated for 2 hours at which time
100 mL of 0.5% Evan’s Blue, 5% BSA in PBS was injected and
embryos were further incubated for 60 minutes. After incubation,
the embryos were perfused with saline. The tissue underlying the
coverslip was removed after the treatment period and blotted dry,
weighed, homogenized and incubated in 200 mL of 100%
formamide to release the extravasated dye. Tissue samples were
homogenized for 30 sec and then incubated for 48 hr at 38uC.
The samples were centrifuged (14000 g for 10 minutes) and
175 mL of supernatant quantified spectrophotometrically against a
formamide blank at 620 nm. Vascular permeability index was
calculated as dye concentration in treated tissue sections/dye
concentration in matched vehicle (PBS) treated samples. For rat
studies, Evans Blue dye solution (10 ml/kg body weight, 0.5%
Evans blue (w/v) in endotoxin-free PBS) was injected intrave-
nously. Ten minutes after injection, each rat (n=5) was injected
intradermally with 25 mL of PBS into the left ear and 0.15nmoles
of reagent (,25 mL) into the right ear. Thirty minutes later, rats
were anesthetized, their ears photographed, and then perfused
with 100 mL PBS thru a ventricular infusion to remove free
intravascular dye. The ears were removed, the area of extrava-
sation cut out with a biopsy punch (8 mm wide), and then the
tissue was weighed and subsequently placed into 1 mL of
formamide for elution of the Evans Blue dye at 60uC over the
course of 48 hours. The amount of extravasated dye was then
determined spectrophotometrically as described above. The
absolute amount of dye was determined using a standard curve.
Cell Lines and Tumor Xenografts
Epidermoid carcinoma (HEp3) or breast cancer (MDA-MB435)
cells expressing green fluorescent protein (GFP) were maintained
as described previously . For imaging studies involving
xenograft tumors, day 10 chicken embryos had 0.1–0.56106
tumor cells in serum free media applied directly to a section of the
CAM surface that had been lightly abraded with a piece of filter
paper. For embryos being prepared for intravital imaging,
sterilized coverslips were applied on top of the tumor 24 hr post
tumor cell application.
Fertilized Dekalb White chicken eggs were received from Cox
Bros Poultry Farm (Maitland, NS) and incubated in a humidified
chamber at 38uC. At day 4, embryos were removed from their
shells using a Dremel tool with a cutting wheel and maintained
under shell-less conditions, in a covered dish in a humidified air
incubator at 38uC and 60% humidity as previously described
[38,39]. On day 10 of development, chicken embryos were
injected with 50 mg of fluorescein isothiocyanate (FITC)-dextran (2
MDa) (Ex/Em 494 nm/521 nm), tetramethyl rhodamine isothio-
cyanate (TRITC)-dextran (158 kDa) (Ex/Em 550 nm/573 nm) or
doxorubicin hydrochloride (Sigma) (Ex/Em 470 nm/556 nm)
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using a glass microinjection needle into a small venule in the
CAM. Injected volumes were maintained at 50 mL. The natural
fluorescence of doxorubicin was captured using the same filters
(Ex: BP 550/25 (HE), Em: BP 605/70 (HE)) used for TRITC
signal capture. Immediately after injection of the fluorescent
reagents, real-time imaging of the CAM was performed using a
previously described chick-embryo-imaging unit [39,40].
Image capture and processing
Real-time imaging of vascular leak was performed using an
upright epifluorescence microscope with a motorized Z stage
(AxioImager Z1, Carl Zeiss, Thornwood, NY) controlled by
Volocity software (Improvision, Lexington, MA). A four dimen-
sional image series was collected by capturing a mosaic of 3D
image stacks at distinct time-points from regions of interest within
the CAM. Specifically, a 150 nm image stack mosaic was captured
with a 15 nm step size every 15 min for 3–6 hours (13–25 frames).
From this raw data at each time point, the image stack was
cropped to a 100 nm stack containing the in-focus images of the
tumor. This was then flattened into a maximum intensity
projection for the majority of the analyses using Volocity software
(Improvision, Lexington, MA).
Time 0 was defined as the time of the first image capture,
5 minutes after injection of the fluorescent dextran mixture. The
captured images were corrected for drift and rotation using the
Stackreg plugin (Biomedical Imaging Group, http://bigwww.epfl.
ch/) of ImageJ (NIH, Rasband, W.S., ImageJ, National Institutes
of Health, Bethesda, Maryland, USA, http://rsb.info.nih.gov/ij/,
1997–2004). To generate the time-dependent changes in fluores-
cence localization in the CAM, the time 0 image stack was
subtracted from the subsequent time-points using the Image
Calculator function within Image J, hence time 0 intensity was set
at 0. For the generation of surface plots, end point images were
processed using the Interactive 3D Surface Plot plug-in for ImageJ
(Internationale Medieninformatik, Berlin, Germany, http://
rsbweb.nih.gov/ij/plugins/surface-plot-3d.html). Relative intensi-
ties in the surface plots were qualified using a spectrum LUT
normalized from standard 255 levels to 100 levels for ease of
interpretation. The pseudo colored spectrum LUT is based on 255
shades of grey in which a Value 0=black and a Value
The Miles assay predictably measures vascular
permeability changes in the CAM
To validate the shell-less chicken embryo as a suitable model for
vascular leak analysis, we performed an adapted Miles assay to
assess the impact of permeability enhancing factors VEGF and
PEP. PEP is a 37 amino acid peptide fragment of IL-2 that
possesses the vasopermeability activity of intact IL-2 but lacks its
cytokine activity . A dose response curve for PEP indicated
that maximal dye leakage from vessels resulted from 0.1 nM PEP
treatment (data not shown), and this concentration was used in the
subsequent experiments. A VEGF concentration of 200 nM was
selected for experiments, since this concentration induces
significant vascular leakage in rodent models [41,42,43]. VEGF,
PEP or PBS control was injected into a CAM vein distal to the site
of analysis or applied topically to a defined area of the CAM and
the embryo was incubated for 2 hours. This was followed by a
systemic injection of 0.5% Evan’s Blue, 5% BSA in PBS and
embryos were further incubated for 60 minutes before processing.
The relative vascular leak in PBS, VEGF or PEP-treated vessels
was determined in CAMs of day 15 chicken embryos (n$15 in all
cases). Embryos treated with PBS only showed no visible leakage
of Evan’s Blue dye (Figure 1A, left panel). Leakage of dye was
visibly increased in the CAM following injection of VEGF
relative to control (Figure 1A, right panel). Systemic injections of
VEGF or PEP induced significant increases in CAM vascular
permeability, * p,0.05, compared to PBS vehicle controls
(Figure 1B). When these agents were applied locally to the
surface of the CAM, a significant vascular leak was observed for
VEGF, p,0.05 but not PEP (Figure 1B). This may be due to a
reduced ability of PEP to diffuse into the tissue. Following a five
day growth period of human tumor xenografts MDA-MB435 or
HEp3 on the CAM surface (n.22 in all cases), vascular
permeability was measured following systemic injection of either
PBS or PEP (0.1 nM). The presence of Hep3 tumors, but not
MDA-MB435 tumors, resulted in significant vascular permeabil-
ity when compared to CAM with no tumor xenografts. Injection
of PEP increased vascular permeability in the CAM as expected.
However, PEP injection significantly amplified vascular perme-
ability in the CAM and in CAMs with HEp3 xenografts
(Figure 1C). These findings were consistent with the increase in
vascular leak observed in rat models following the administration
of PEP (Figure S1). We conclude that the chicken embryo model
responds predictably to the systemic administration of perme-
ability factors VEGF and PEP or to in situ factors secreted by a
tumor xenograft and the resulting changes in vascular leak are
quantifiable using the Miles assay.
Changes in vascular permeability can be visualized using
The ex ovo chicken embryo is an emerging platform for intravital
imaging of angiogenesis and the tumor microenvironment .
We have previously demonstrated that dye-labeled dextrans are
not particularly useful for the long-term visualization of vascula-
ture in vivo, as they leak progressively into the interstitium [44,45].
As indicators of changes in vascular permeability and tumor
perfusion, however, dextrans are potentially very useful, since they
mimic endogenous proteins by extravasating from vessels
predictably based on size . To assess this, a 158 kDa
TRITC-dextran was selected for its similar size to immunoglob-
ulins, which passively extravasate through blood vessel walls .
Based on the published pharmacokinetics of fluorescently labeled
dextrans , we selected a particle size that should extravasate
from the vasculature at a slow but measurable rate, that might be
influenced by vascular permeability factors. Intravital imaging
following systemic injection of VEGF or PEP revealed a significant
induction of vascular leak, manifested by a decrease in TRITC
fluorescence in the vessels over time, and a simultaneous increase
of signal in the surrounding tissues (Figure 2A). Extravasation of
the 158 kDa TRITC-dextran was detectable within 30 minutes
after PEP or VEGF treatment (Figure 2B and Video S1).
Significant levels of vascular leak were not detected in PBS-
treated controls over 3 hours of imaging (Figure 2B). This basic
approach allows for real-time visualization of the vascular network
and the dynamic measurement of temporal changes in vascular
Measuring the structural integrity and permeability of
The tumor vasculature is heterogeneous, consisting of irregu-
larly formed vessels that are both leaky and often fenestrated .
The extravasation of plasma proteins would be expected to occur
via enhanced vascular permeability and leak through discontin-
uous vessel walls . In order to distinguish between these two
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phenomena, we visualized co-injected dextrans of 2000 kDa or
158 kDa simultaneously. The 2000 kDa fluorescent dextran was
selected to mimic large molecular weight blood components, such
as LDL (2500–3500 kDa) and VLDL (10–806104kDa), which
are largely retained by structurally intact vasculature . We
hypothesized that structurally intact vasculature would retain the
2000 kDa FITC-dextran, and thus it could be used to define the
functional vessel framework in a given region of interest.
Furthermore, we surmised that the change in the ratio of the
158 kDa dextran to the 2000 kDa dextran could provide a
quantitative measure of vascular permeability changes over time at
the tumor site. To this end, 158 kDa TRITC-dextran and
2000 kDa FITC-dextran were systemically co-injected into CAMs
bearing human tumor xenografts with an average weight of 25–
50 mg and a diameter range of 3–8 mm. At the tumor site,
significant vascular leak of the smaller 158 kDa TRITC dextran
was detected after 45 minutes, and after 180 minutes was 3.5-fold
greater in the tumor than in normal tissue distal to the tumor site.
Vascular leak of either the large or small dextrans was nominal in
the absence of a tumor. The increased vascular leak seen in tumor-
bearing CAMs was greatest in regions immediately surrounding or
within the tumor (see Videos S2 and S3). By imaging in real time
the extravasation of these two dextran populations, it is possible to
simultaneously monitor both structural and functional aspects of
Figure 1. The Miles assay measures vascular permeability changes in the CAM. A. Bright field images of CAM vasculature following
injection of Evan’s blue dye subsequent to the systemic administration of PBS (left panel) or VEGF (right panel). Arrows indicate areas of visible
vascular leak. B. When VEGF or PEP is injected intravenously distal to the site of analysis, a significant level of vascular permeability is observed in the
CAM (left). Topically administered VEGF but not PEP induces a significant level of vascular permeability (right). C. Vascular permeability changes in
the CAM were evaluated in the presence of human tumor xenografts. Increased vascular permeability was observed at the tumor site, particularly in
HEp3 tumors. Systemically administered PEP (0.1 nM) further increases vascular permeability. Data are presented as Mean +/2 SEM, n.15 for each
group. * indicates statistical significance, p,0.05, ** p,0.01.
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the vasculature, and to precisely evaluate regional differences in
vascular permeability (Figure 3). Comparison of stitched images
(30–40 frames) from the tumor versus normal tissue shows
significantly more leak in the necrotic tumor core versus the
non-tumor tissue after 60 minutes (2-way ANOVA, p,0.05 and
Bonferroni post-tests for each time point, p,0.05). Necrotic core
vascular leak of the 158 kDa TRITC-dextran was more than 6-
fold greater than leak in tissue distant to the tumor after
180 minutes. Comparison across tissue in the tumor shows
significantly more leak was detected in the necrotic core versus
the tumor after 120 minutes (2-way ANOVA, p,0.05 and
Bonferroni post-tests for each time point, P,0.05). Indeed,
significantly increased extravasation of the 158 kDa dextran was
seen at the tumor core compared to the entire tumor or to normal
Figure 2. Intravital imaging assesses real-time changes in vascular permeability induced by VEGF and PEP. A. A series of
representative images from intravital imaging experiments is shown. An accumulation of fluorescence outside the vasculature over time is seen in
those embryos treated with VEGF or PEP compared to PBS. B. Images were captured and quantified every 15 minutes over a period of 3 hours to
evaluate the extent of vascular leak. Vascular leak values were generated by subtracting time 0 values from subsequent time points. Asterisks indicate
significant leak of dextran from the vasculature (2-way ANOVA, p,0.05 followed by Bonferroni post-tests, (p,0.05)) comparing either PBS vs VEGF, or
PBS vs PEP at each time point.
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vasculature distal from the tumor (Figure 3C). Because the tumor
xenografts in this assay develop on top of the CAM, there was
some concern that the environment may be atypically oxygen rich.
We found that this was unlikely to be the case, as tumors grown in
the CAM underneath a glass coverslip had equivalent vascular
leak levels as those exposed to the air.
Increased vascular permeability enhances drug delivery
to tumor sites
the vasoactiveagents VEGF and PEP enhance vascularpermeability,
we hypothesized that the delivery of chemotherapeutic drugs to
tumor sites could be improved using these agents. To test this, we
Figure 3. Assessment of regional permeability and vascular integrity in human tumors. A. Representative fluorescence micrographs from
two human HEp3 tumors displaying peri-tumoral (i) and tumor core (ii) vascular leak are shown with the 2000 kDa FITC-dextran (green) and 158 kDa
TRITC-dextran (red). The normalized images were generated by subtracting the 0 hour image from the 3 hour image, and represent the net vascular
leak. Tumor induced vascular leak is localized primarily to the tumor and especially to the central, necrotic core of the tumor. B. Areas utilized for
regional vascular leak analyses are delineated. The solid circle represents an area of non-tumor tissue; the dashed circle denotes tumor and the
dotted line indicates the avascular necrotic core. C. Quantitation of leak of large (green) and small (red) dextrans is shown for non-tumor tissue, the
entire tumor and the core of the tumor. The relative leak of both dextrans was normalized to time zero; n=6 for each analysis. Two-way ANOVA,
(p,0.05) followed by Bonferroni post-tests, (p,0.05) was used to assess significant leak of the TRITC-dextran of either tumor versus non-tumour
tissue, and necrotic core versus non-tumour tissue at each timepoint. Timepoints that demonstrated significance are indicated by an asterisk.
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measured the delivery of chemotherapeutic agents to tumor sites in
real time using intravital imaging in the presence and absence of
VEGF (Figure 4A–C). Doxorubicin was detected by its natural
fluorescence using intravitalimaging.Whendoxorubicinwasinjected
systemically into embryos bearing HEp3-GFP tumor xenografts, its
uptake into tumors in the absence of VEGF increases over
60 minutes, reaching maximum levels after 2 hours (Figure 4D).
When 200 nM VEGF was co-administered with doxorubicin,
doxorubicin uptake by tumor tissues was enhanced over 46within
to control levels (Figure 4E). At 60 minutes, doxorubicin uptake in
the presence of VEGF was approximately twice that of the controls.
Doxorubicin uptake into normal tissues was also increased by co-
administration of VEGF, but to a lesser extent (40%) than at the
tumor site. Thus, the delivery of doxorubicin to tumor xenografts was
significantly and selectively enhanced by the transient systemic
administration of VEGF.
Here, we present a novel approach to visualize and quantitate
hemodynamics and vascular leak in tumors. In contrast to
traditional, endpoint analysis methods, real-time intravital imaging
is sensitive to changes in both permeability and vessel integrity,
and can effectively track rapid and dynamic changes in vascular
permeability. We demonstrate that vascular permeability is
increased in xenograft tumors compared to distal normal tissues,
and that it can be further enhanced by VEGF and the PEP
fragment of IL-2. Utilizing standard epifluorescence microscopy,
we could also monitor localization of the chemotherapeutic,
doxorubicin, which is a naturally fluorescent DNA intercalating
agent, using this approach. We show that the uptake of
systemically administered doxorubicin in xenograft tumors is
enhanced by co-administered VEGF, suggesting that transiently
increasing the vascular leak in tumors using adjuvant therapies can
improve the uptake of chemotherapy at the tumor site.
As an alternative to the model we present here, vascular
dynamics can be visualized in rodent models with the use of
surgically placed skin flaps. Skin flaps have been used to estimate
the vascular leakage of florescent-labeled particles under various
conditions in tumors in rats  hamsters , and mice [9,20,50]
and to measure vessel regeneration during wound healing .
Imaging through skin flaps can predict drug localization  and
the influence of treatments on hemodynamics and vascular
Figure 4. Increasing vascular permeability enhances the accumulation of doxorubicin into the tumor. Doxorubicin was injected
intravenously subsequent to administration of PBS or VEGF and its uptake at the tumor site in real time was estimated using its natural fluorescence.
A–B. Representative images of doxorubicin uptake over time, tumors (green) and doxorubicin uptake (red) are shown. C. Heat map of doxorubicin
uptake after 3 hours in control and VEGF-treated tumors. D. Graph showing relative uptake of doxorubicin in the tumor in the presence or absence
of systemic VEGF treatment. E. Graph showing relative uptake of doxorubicin in the normal tissues distal to the tumor in the presence or absence of
systemic VEGF treatment. N=4 per treatment; data were analyzed by 2 way ANOVA.
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permeability [9,48,49,50]. However, imaging protocols in rodents
can be complicated. Creating the necessary skin flaps requires
microsurgical implantation of a frame in anesthetized animals to
provide a viewable imaging area. This nontrivial procedure can
complicate vascular dynamics and permeability around the
viewing area by inducing inflammation. By comparison, the chick
model described here is relatively easy to maintain while the thin,
vascular and transparent nature of the CAM is amenable to
imaging without surgical intervention. Despite the chick CAM’s
simplicity, our biological findings are consistent with those
reported in more complex models. Therefore, this work further
validates the chick CAM’s use as a tumor model and suggests on
its potential use for semi-high throughput imaging and screening
analysis that should facilitate and compliment the use of more
complex mammalian models.
The chick CAM responds predictably to permeabilization
factors and supports the growth of human tumor xenografts. While
increased vascular permeability induced by VEGF and PEP
occurred within minutes, imaging time courses of up to 72 hours,
can be accommodated in the ex ovo chicken embryo model .
Although fluorescent dextrans were used in the methods described
here, the CAM model will likely accommodate alternate molecules
to further expand its utility, such as labeled immunoglobulins or
LDL. These considerations along with the conservation of the key
chicken and human angiogenesis factors make it a useful model to
understand angiogenesis [52,53], drug targeting [54,55], response
to therapeutics  and vascular permeability.
Selective modification of the tumor vasculature is emerging as
a powerful means to enhance drug delivery and ultimately
efficacy. Common vasoactive agents used in oncology follow two
principle approaches; perturbation of the tumor vasculature by
vascular disrupting agents (VDAs) or normalization of the tumor
vasculature by anti-angiogenic agents. For example, the tubulin-
binding agent, combretastatin-serine (AVE8062) is a small
molecular weight VDA that causes a rapid and extensive
shutdown of established tumor vasculature. Prior dosing with
AVE8062 can therapeutically synergize with docetaxel, oxalipla-
tin or cisplatin [56,57]. Therapies to normalize the tumor
vasculature, as described by Jain , suggest that following
disruption of the immature vessels, the mature tumor vasculature
becomes strengthened and hence more susceptible to drug
therapy. Current strategies typically include the use of VEGF
inhibitors such as bevacizumab or anti-VEGF antibody, which
has shown benefits in animal models and patients [7,59]. Tong
and coworkers showed decreased interstitial hypertension caused
by targeting VEGF produced a morphologically and functionally
‘‘normalized’’ vascular network resulting in pressure gradients
favoring extravasation and hence improving drug penetration in
Increasing the uptake of co-administered chemotherapies by
overcoming the high interstitial fluid pressures in the tumor
microenvironment has previously been accomplished through
inhibition of the PDGF receptor with imatinib [60,61],
remodeling of the extracellular matrix using collagenase and
hyaluronidase [8,62], vascular normalization using anti-VEGF
antibodies  and targeted vasopermeation using PEP  (for
review, see Cairns et al., 2006 ). While the overall goal is the
same, evidence suggests that similar strategies can have markedly
different consequences. Tong et al.  suggest that drug uptake
is improved at the tumor site during vascular normalization
because a pressure gradient is briefly formed across the vessel
walls in tumors. This gradient dissipates rapidly, however,
against the high interstitial fluid pressure in the tumor. They
suggest that this short time window should be sufficient to
improve drug uptake. This contrasts with observations by Khawli
et al. , who administer PEP immunoconjugates two hours
prior to chemotherapy to achieve an optimal increase in drug
uptake at the tumor site. Our data indicate that VEGF and PEP
rapidly increase vascular leak, with measurable increases in
dextran efflux over controls that are apparent within 15 minutes
and continue to increase for 3 hours. Interestingly, VEGF
treatment resulted in a different dynamic in doxorubicin uptake,
with an initial spike in doxorubicin accumulation in tumor tissue
that was maintained at a steady level throughout the 3 hours of
analysis. The subtle difference in dextran versus doxorubicin
accumulation may result from size differences between the
dextran and doxorubicin molecules. The larger dextran molecule
likely requires a greater change in permeability and thus
responds more slowly than the smaller doxorubicin molecule.
Clearly, understanding the unique features of tumor blood
dynamics and vascular leak will help tease out these subtle, but
consequential affects to appropriately focus chemotherapeutic
Given the dynamic interplay of vascular signaling factors and
their individual roles in the modulation of vascular permeability, it
is difficult to predict the impact of adjuvant permeability
enhancement agents on drug uptake at the tumor site. The
approach and the model presented here offer a powerful tool to
investigate mechanisms of vasopermeability in vivo and to screen
the most appropriate strategies for improving drug uptake.
permeability. Injection of PEP (0.15 nmoles) into the ears of rats
(n=5) induces significant levels of vascular permeability similar to
CAM data in Figure 1. As an internal control, PBS was injected
into the corresponding left ear. The ratio of vascular leakage seen
for the reagent ear (right ear) divided by the value for the PBS ear
(left ear) was graphed as a VL index. Data are presented as Mean
+/2 SEM. * indicates statistical significance, p,0.05.
Comparison to rodent ear model of vascular
in vascular permeability. Representative intravital imaging
experiments representing the extravasation of 158 kDa dextran
are shown for PBS, VEGF and PEP-treated embryos over 3 hours.
Top panels represent the raw imaging data; bottom panels are
normalized to the first time point to denote leaked dextran.
Intravital imaging assesses real-time changes
HEp3 tumor. Intravital imaging experiments of 158 kDa (red)
and 2000 kDa (green) dextran extravasation over time are shown
for human epidermoid carcinoma (HEp3) tumors established in
the CAM. Raw and normalized data are shown.
Intravital imaging of vascular permeability in
158 kDa (red) and 2000 kDa (green) dextran extravasation over
time are shown for human breast carcinoma (MDA-MB435)
tumors established in the CAM. Raw and normalized data are
Intravital imaging of vascular permeability in
We thank Kristin Kain for her valuable editorial input, and Dr. Longen
Zhou for his assistance with the rodent model.
Real-Time Measurement of Vascular Permeability
PLoS ONE | www.plosone.org8 March 2012 | Volume 7 | Issue 3 | e33760
Conceived and designed the experiments: DP AZ JL. Performed the
experiments: DP WS. Analyzed the data: DP AZ JL. Contributed
reagents/materials/analysis tools: MP. Wrote the paper: DP MP AZ JL.
1. Minchinton AI, Tannock IF (2006) Drug penetration in solid tumours. Nat Rev
Cancer 6: 583–592.
Jang SH, Wientjes MG, Lu D, Au JL (2003) Drug delivery and transport to solid
tumors. Pharm Res 20: 1337–1350.
Stohrer M, Boucher Y, Stangassinger M, Jain RK (2000) Oncotic pressure in
solid tumors is elevated. Cancer Res 60: 4251–4255.
Jain RK (1989) Delivery of novel therapeutic agents in tumors: physiological
barriers and strategies. J Natl Cancer Inst 81: 570–576.
Campbell RB (2006) Tumor physiology and delivery of nanopharmaceuticals.
Anticancer Agents Med Chem 6: 503–512.
Fukumura D, Jain RK (2007) Tumor microvasculature and microenvironment:
targets for anti-angiogenesis and normalization. Microvasc Res 74: 72–84.
Tong RT, Boucher Y, Kozin SV, Winkler F, Hicklin DJ, et al. (2004) Vascular
normalization by vascular endothelial growth factor receptor 2 blockade induces
a pressure gradient across the vasculature and improves drug penetration in
tumors. Cancer Res 64: 3731–3736.
Eikenes L, Tari M, Tufto I, Bruland OS, de Lange Davies C (2005)
Hyaluronidase induces a transcapillary pressure gradient and improves the
distribution and uptake of liposomal doxorubicin (Caelyx) in human osteosar-
coma xenografts. Br J Cancer 93: 81–88.
Czabanka M, Vinci M, Heppner F, Ullrich A, Vajkoczy P (2009) Effects of
sunitinib on tumor hemodynamics and delivery of chemotherapy. Int J Cancer
10. Jain RK, Tong RT, Munn LL (2007) Effect of vascular normalization by
antiangiogenic therapy on interstitial hypertension, peritumor edema, and
lymphatic metastasis: insights from a mathematical model. Cancer Res 67:
11. Dickson PV, Hamner JB, Streck CJ, Ng CY, McCarville MB, et al. (2007)
Continuous delivery of IFN-beta promotes sustained maturation of intratumoral
vasculature. Mol Cancer Res 5: 531–542.
12. Qayum N, Muschel RJ, Im JH, Balathasan L, Koch CJ, et al. (2009) Tumor
vascular changes mediated by inhibition of oncogenic signaling. Cancer Res 69:
13. Qayum N, Im J, Stratford MR, Bernhard EJ, McKenna WG, et al. (2011)
Modulation of the Tumor Microvasculature by Phosphoinositide-3 Kinase
Inhibition Increases Doxorubicin Delivery In Vivo. Clin Cancer Res.
14. Wu NZ, Da D, Rudoll TL, Needham D, Whorton AR, et al. (1993) Increased
microvascular permeability contributes to preferential accumulation of Stealth
liposomes in tumor tissue. Cancer Res 53: 3765–3770.
15. Yuan F, Dellian M, Fukumura D, Leunig M, Berk DA, et al. (1995) Vascular
permeability in a human tumor xenograft: molecular size dependence and cutoff
size. Cancer Res 55: 3752–3756.
16. Mikhail AS, Allen C (2009) Block copolymer micelles for delivery of cancer
therapy: transport at the whole body, tissue and cellular levels. J Control Release
17. Tailor TD, Hanna G, Yarmolenko PS, Dreher MR, Betof AS, et al. (2010)
Effect of pazopanib on tumor microenvironment and liposome delivery. Mol
Cancer Ther 9: 1798–1808.
18. Fukumura D, Duda DG, Munn LL, Jain RK (2010) Tumor microvasculature
and microenvironment: novel insights through intravital imaging in pre-clinical
models. Microcirculation 17: 206–225.
19. Miles AA, Miles EM (1952) Vascular reactions to histamine, histamine-liberator
and leukotaxine in the skin of guinea-pigs. J Physiol 118: 228–257.
20. Dreher MR, Liu W, Michelich CR, Dewhirst MW, Yuan F, et al. (2006) Tumor
vascular permeability, accumulation, and penetration of macromolecular drug
carriers. J Natl Cancer Inst 98: 335–344.
21. Rizzo V, Kim D, Duran WN, DeFouw DO (1995) Ontogeny of microvascular
permeability to macromolecules in the chick chorioallantoic membrane during
normal angiogenesis. Microvasc Res 49: 49–63.
22. Ausprunk DH, Knighton DR, Folkman J (1974) Differentiation of vascular
endothelium in the chick chorioallantois: a structural and autoradiographic
study. Dev Biol 38: 237–248.
23. Leeson TS, Leeson CR (1963) The Chorio-Allantois of the Chick. Light and
Electron Microscopic Observations at Various Times of Incubation. J Anat 97:
24. Valdes TI, Klueh U, Kreutzer D, Moussy F (2003) Ex ova chick chorioallantoic
membrane as a novel in vivo model for testing biosensors. J Biomed Mater Res A
25. Valdes TI, Kreutzer D, Moussy F (2002) The chick chorioallantoic membrane
as a novel in vivo model for the testing of biomaterials. J Biomed Mater Res 62:
26. Cruz A, DeFouw LM, DeFouw DO (2000) Restrictive endothelial barrier
function during normal angiogenesis in vivo: partial dependence on tyrosine
dephosphorylation of beta-catenin. Microvasc Res 59: 195–203.
27. Cruz A, Rizzo V, De Fouw DO (1997) Microvessels of the chick chorioallantoic
membrane uniformly restrict albumin extravasation during angiogenesis and
endothelial cytodifferentiation. Tissue Cell 29: 277–281.
28. DeFouw LM, DeFouw DO (2000) Vascular endothelial growth factor fails to
acutely modulate endothelial permeability during early angiogenesis in the chick
chorioallantoic membrane. Microvasc Res 60: 212–221.
29. DeFouw LM, DeFouw DO (2000) Differentiation of endothelial barrier function
during normal angiogenesis requires homotypic VE-cadherin adhesion. Tissue
Cell 32: 238–242.
30. Rizzo V, Steinfeld R, Kyriakides C, DeFouw DO (1993) The microvascular unit
of the 6-day chick chorioallantoic membrane: a fluorescent confocal microscopic
and ultrastructural morphometric analysis of endothelial permselectivity.
Microvasc Res 46: 320–332.
31. Rizzo V, DeFouw DO (1997) Microvascular permselectivity in the chick
chorioallantoic membrane during endothelial cell senescence. Int J Microcirc
Clin Exp 17: 75–79.
32. Vargas A, Zeisser-Labouebe M, Lange N, Gurny R, Delie F (2007) The chick
embryo and its chorioallantoic membrane (CAM) for the in vivo evaluation of
drug delivery systems. Adv Drug Deliv Rev 59: 1162–1176.
33. Chambers AF, Wilson SM, Tuck AB, Denhardt GH, Cairncross JG (1990)
Comparison of metastatic properties of a variety of mouse, rat, and human cells
in assays in nude mice and chick embryos. In Vivo 4: 215–219.
34. Strojnik T, Kavalar R, Barone TA, Plunkett RJ (2010) Experimental model and
immunohistochemical comparison of U87 human glioblastoma cell xenografts
on the chicken chorioallantoic membrane and in rat brains. Anticancer Res 30:
35. Palmer TD, Lewis J, Zijlstra A (2011) Quantitative Analysis of Cancer
Metastasis using an Avian Embryo Model. J Vis Exp.
36. Epstein AL, Mizokami MM, Li J, Hu P, Khawli LA (2003) Identification of a
protein fragment of interleukin 2 responsible for vasopermeability. J Natl Cancer
Inst 95: 741–749.
37. Zijlstra A, Mellor R, Panzarella G, Aimes RT, Hooper JD, et al. (2002) A
quantitative analysis of rate-limiting steps in the metastatic cascade using
human-specific real-time polymerase chain reaction. Cancer Res 62:
38. Seandel M, Noack-Kunnmann K, Zhu D, Aimes RT, Quigley JP (2001) Growth
factor-induced angiogenesis in vivo requires specific cleavage of fibrillar type I
collagen. Blood 97: 2323–2332.
39. Leong HS, Steinmetz NF, Ablack A, Destito G, Zijlstra A, et al. (2010) Intravital
imaging of embryonic and tumor neovasculature using viral nanoparticles. Nat
Protoc 5: 1406–1417.
40. Zijlstra A, Lewis J, Degryse B, Stuhlmann H, Quigley JP (2008) The inhibition
of tumor cell intravasation and subsequent metastasis via regulation of in vivo
tumor cell motility by the tetraspanin CD151. Cancer Cell 13: 221–234.
41. Stacker SA, Vitali A, Caesar C, Domagala T, Groenen LC, et al. (1999) A
mutant form of vascular endothelial growth factor (VEGF) that lacks VEGF
receptor-2 activation retains the ability to induce vascular permeability. J Biol
Chem 274: 34884–34892.
42. Murohara T, Horowitz JR, Silver M, Tsurumi Y, Chen D, et al. (1998) Vascular
endothelial growth factor/vascular permeability factor enhances vascular
permeability via nitric oxide and prostacyclin. Circulation 97: 99–107.
43. Brkovic A, Sirois MG (2007) Vascular permeability induced by VEGF family
members in vivo: role of endogenous PAF and NO synthesis. J Cell Biochem
44. Lewis JD, Destito G, Zijlstra A, Gonzalez MJ, Quigley JP, et al. (2006) Viral
nanoparticles as tools for intravital vascular imaging. Nat Med 12: 354–360.
45. Cho C, Ablack A, Leong HS, Zijlstra A, Lewis J (2011) Evaluation of
Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging. . J Vis
46. Dvorak HF, Weaver VM, Tlsty TD, Bergers G (2011) Tumor microenviron-
ment and progression. J Surg Oncol 103: 468–474.
47. Maeda H, Fang J, Inutsuka T, Kitamoto Y (2003) Vascular permeability
enhancement in solid tumor: various factors, mechanisms involved and its
implications. Int Immunopharmacol 3: 319–328.
48. Reyes-Aldasoro CC, Wilson I, Prise VE, Barber PR, Ameer-Beg M, et al. (2008)
Estimation of apparent tumor vascular permeability from multiphoton
fluorescence microscopic images of P22 rat sarcomas in vivo. Microcirculation
49. Strieth S, Eichhorn ME, Werner A, Sauer B, Teifel M, et al. (2008) Paclitaxel
encapsulated in cationic liposomes increases tumor microvessel leakiness and
improves therapeutic efficacy in combination with Cisplatin. Clin Cancer Res
50. Czabanka M, Parmaksiz G, Bayerl SH, Nieminen M, Trachsel E, et al. (2011)
Microvascular biodistribution of L19-SIP in angiogenesis targeting strategies.
Eur J Cancer 47: 1276–1284.
Real-Time Measurement of Vascular Permeability
PLoS ONE | www.plosone.org9 March 2012 | Volume 7 | Issue 3 | e33760
51. Machado MJ, Mitchell CA (2011) Temporal changes in microvessel leakiness Download full-text
during wound healing discriminated by in vivo fluorescence recovery after
photobleaching. J Physiol 589: 4681–4696.
52. Ribatti D (2008) Chick embryo chorioallantoic membrane as a useful tool to
study angiogenesis. Int Rev Cell Mol Biol 270: 181–224.
53. Staton CA, Reed MW, Brown NJ (2009) A critical analysis of current in vitro
and in vivo angiogenesis assays. Int J Exp Pathol 90: 195–221.
54. Tartis MS, McCallan J, Lum AF, LaBell R, Stieger SM, et al. (2006)
Therapeutic effects of paclitaxel-containing ultrasound contrast agents. Ultra-
sound Med Biol 32: 1771–1780.
55. Saw CL, Heng PW, Liew CV (2008) Chick chorioallantoic membrane as an in
situ biological membrane for pharmaceutical formulation development: a
review. Drug Dev Ind Pharm 34: 1168–1177.
56. Kim TJ, Ravoori M, Landen CN, Kamat AA, Han LY, et al. (2007) Antitumor
and antivascular effects of AVE8062 in ovarian carcinoma. Cancer Res 67:
57. Delmonte A, Sessa C (2009) AVE8062: a new combretastatin derivative vascular
disrupting agent. Expert Opin Investig Drugs 18: 1541–1548.
58. Jain RK (2005) Normalization of tumor vasculature: an emerging concept in
antiangiogenic therapy. Science 307: 58–62.
59. Presta LG, Chen H, O’Connor SJ, Chisholm V, Meng YG, et al. (1997)
Humanization of an anti-vascular endothelial growth factor monoclonal
antibody for the therapy of solid tumors and other disorders. Cancer Res 57:
60. Pietras K, Ostman A, Sjoquist M, Buchdunger E, Reed RK, et al. (2001)
Inhibition of platelet-derived growth factor receptors reduces interstitial
hypertension and increases transcapillary transport in tumors. Cancer Res 61:
61. Pietras K (2004) Increasing tumor uptake of anticancer drugs with imatinib.
Semin Oncol 31: 18–23.
62. Eikenes L, Bruland OS, Brekken C, Davies Cde L (2004) Collagenase increases
the transcapillary pressure gradient and improves the uptake and distribution of
monoclonal antibodies in human osteosarcoma xenografts. Cancer Res 64:
63. Khawli LA, Hu P, Epstein AL (2005) NHS76/PEP2, a fully human
vasopermeability-enhancing agent to increase the uptake and efficacy of cancer
chemotherapy. Clin Cancer Res 11: 3084–3093.
64. Cairns R, Papandreou I, Denko N (2006) Overcoming physiologic barriers to
cancer treatment by molecularly targeting the tumor microenvironment. Mol
Cancer Res 4: 61–70.
Real-Time Measurement of Vascular Permeability
PLoS ONE | www.plosone.org10 March 2012 | Volume 7 | Issue 3 | e33760