Article

Longitudinal Imaging Studies of Tumor Microenvironment in Mice Treated with the mTOR Inhibitor Rapamycin

Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.
PLoS ONE (Impact Factor: 3.23). 11/2012; 7(11):e49456. DOI: 10.1371/journal.pone.0049456
Source: PubMed
ABSTRACT
Rapamycin is an allosteric inhibitor of mammalian target of rapamycin, and inhibits tumor growth and angiogenesis. Recent studies suggested a possibility that rapamycin renormalizes aberrant tumor vasculature and improves tumor oxygenation. The longitudinal effects of rapamycin on angiogenesis and tumor oxygenation were evaluated in murine squamous cell carcinoma (SCCVII) by electron paramagnetic resonance imaging (EPRI) and magnetic resonance imaging (MRI) to identify an optimal time after rapamycin treatment for enhanced tumor radioresponse. Rapamycin treatment was initiated on SCCVII solid tumors 8 days after implantation (500-750 mm(3)) and measurements of tumor pO(2) and blood volume were conducted from day 8 to 14 by EPRI/MRI. Microvessel density was evaluated over the same time period by immunohistochemical analysis. Tumor blood volume as measured by MRI significantly decreased 2 days after rapamycin treatment. Tumor pO(2) levels modestly but significantly increased 2 days after rapamycin treatment; whereas, it decreased in non-treated control tumors. Furthermore, the fraction of hypoxic area (pixels with pO(2)<10 mm Hg) in the tumor region decreased 2 days after rapamycin treatments. Immunohistochemical analysis of tumor microvessel density and pericyte coverage revealed that microvessel density decreased 2 days after rapamycin treatment, but pericyte coverage did not change, similar to what was seen with anti-angiogenic agents such as sunitinib which cause vascular renormalization. Collectively, EPRI/MRI co-imaging can provide non-invasive evidence of rapamycin-induced vascular renormalization and resultant transient increase in tumor oxygenation. Improved oxygenation by rapamycin treatment provides a temporal window for anti-cancer therapies to realize enhanced response to radiotherapy.

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Available from: J. Silvio Gutkind
Longitudinal Imaging Studies of Tumor
Microenvironment in Mice Treated with the mTOR
Inhibitor Rapamycin
Keita Saito
1
, Shingo Matsumoto
1
, Hironobu Yasui
1,2
, Nallathamby Devasahayam
1
,
Sankaran Subramanian
1
, Jeeva P. Munasinghe
3
, Vyomesh Patel
4
, J. Silvio Gutkind
4
, James B. Mitchell
1
,
Murali C. Krishna
1
*
1 Radiation Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America, 2 Laboratory
of Radiation Biology, Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan, 3 National Institute
of Neurological Disorder and Stroke, National Institutes of Health, Bethesda, Maryland, United States of America, 4 National Institute of Dental and Craniofacial Research,
National Institutes of Health, Bethesda, Maryland, United States of America
Abstract
Rapamycin is an allosteric inhibitor of mammalian target of rapamycin, and inhibits tumor growth and angiogenesis. Recent
studies suggested a possibility that rapamycin renormalizes aberrant tumor vasculature and improves tumor oxygenation.
The longitudinal effects of rapamycin on angiogenesis and tumor oxygenation were evaluated in murine squamous cell
carcinoma (SCCVII) by electron paramagnetic resonance imaging (EPRI) and magnetic resonance imaging (MRI) to identify
an optimal time after rapamycin treatment for enhanced tumor radioresponse. Rapamycin treatment was initiated on
SCCVII solid tumors 8 days after implantation (500–750 mm
3
) and measurements of tumor pO
2
and blood volume were
conducted from day 8 to 14 by EPRI/MRI. Microvessel density was evaluated over the same time period by
immunohistochemical analysis. Tumor blood volume as measured by MRI significantly decreased 2 days after rapamycin
treatment. Tumor pO
2
levels modestly but significantly increased 2 days after rapamycin treatment; whereas, it decreased in
non-treated control tumors. Furthermore, the fraction of hypoxic area (pixels with pO
2
,10 mm Hg) in the tumor region
decreased 2 days after rapamycin treatments. Immunohistochemical analysis of tumor microvessel density and pericyte
coverage revealed that microvessel density decreased 2 days after rapamycin treatment, but pericyte coverage did not
change, similar to what was seen with anti-angiogenic agents such as sunitinib which cause vascular renormalization.
Collectively, EPRI/MRI co-imaging can provide non-invasive evidence of rapamycin-induced vascular renormalization and
resultant transient increase in tumor oxygenation. Improved oxygenation by rapamycin treatment provides a temporal
window for anti-cancer therapies to realize enhanced response to radiotherapy.
Citation: Saito K, Matsumoto S, Yasui H, Devasahayam N, Subramanian S, et al. (2012) Longitudinal Imaging Studies of Tumor Microenvironment in Mice Treated
with the mTOR Inhibitor Rapamycin. PLoS ONE 7(11): e49456. doi:10.1371/journal.pone.0049456
Editor: Kwan Man, The University of Hong Kong, Hong Kong
Received June 19, 2012; Accepted October 9, 2012; Published November 20, 2012
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This work was supported by the Intramural Research Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health. 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: murali@helix.nih.gov
Introduction
Multiple genetic and epigenetic events are known to result in the
dysregulation of several signaling pathways that have an impact on
neoplastic disease progression, such as squamous cell carcinomas
(SCC) [1,2]. One such pathway, the phosphatidylinositol 3-kinase
(PI3K)-Akt pathway is frequently activated in many cancers, and
controls cellular metabolism, growth, and proliferation [3–6]. The
mammalian target of rapamycin (mTOR) is an atypical serine/
threonine kinase, which acts downstream of PI3K/Akt and,
therefore has become an attractive therapeutic target [7–10]. It
follows that inhibitors of mTOR, such as rapamycin and its
derivatives are currently being evaluated for molecular targeted
therapy of neoplastic diseases [9].
The inhibition of mTOR with its specific allosteric inhibitor,
rapamycin, provokes a rapid death of squamous xenografts,
resulting in tumor regression [11]. The molecular basis of this is
currently an active area of research [12]. For example, a recent
study using a reverse-pharmacology approach, which involved the
expression of a rapamycin-insensitive form of mTOR in squamous
cancer cells, showed that cancer cells are the primary targets of
rapamycin in vivo, and that mTOR controls the expression of
hypoxia-inducible factor-1a (HIF-1a), a key transcription factor
that orchestrates the cellular response to hypoxic stress, including
the regulation of the expression of angiogenic factors, thus
providing a likely mechanism by which rapamycin exerts its
tumor suppressive and antiangiogenic effects [13]. Blocking
mTOR pathway in SCC tumors was also shown to prevent
accumulation of HIF-1a resulting in inhibition of processes
involved in glucose metabolism as well as decrease in pro-
angiogenic factors such as vascular endothelial growth factor
(VEGF) [13].
Recent studies using magnetic resonance imaging (MRI)
showed that treatment with mTOR inhibitors results in strong
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Page 1
antiangiogenic and anti-vascular effects in solid tumors [12].
Although there are distinctions between the effects of mTOR
inhibitors and antiangiogenic agents on tumor vasculature, it was
suggested that rapamycin induced antiangiogenic effects also
mediate vascular re-normalization as in the case of conventional
antiangiogenic agents [14]. Since vascular normalization improves
tumor oxygenation as well as delivery of therapeutic drugs [15–
19], examining whether such a process occurs in the case of
mTOR inhibitors may explain the efficacy of rapamycin’s
radiosensitizing effects [20]. If such a temporal change of tumor
oxygenation can be identified for rapamycin by using a non-
invasive pO
2
mapping technique such as by electron paramagnetic
resonance imaging (EPRI) it becomes then possible to appropri-
ately schedule the two modalities for better therapeutic outcomes.
Electron paramagnetic resonance (EPR) is a spectroscopic
technique similar to nuclear magnetic resonance. EPR detects
paramagnetic species that have unpaired electrons such as
transition metal complexes and free radicals. With the recent
availability of triarylmethyl radical probes (TAM) as in vivo
compatible paramagnetic tracers, EPRI is now being explored for
mapping tissue oxygen in live animals [21–24]. The fundamental
basis for EPRI in monitoring tissue oxygen using TAM stems from
the paramagnetic nature of molecular oxygen arising from its two
unpaired electrons. The collisional interaction between TAM and
dissolved paramagnetic oxygen leads to a broadening of the
spectral line width of TAM. The EPR spectral broadening of
TAM is linear with oxygen concentration, providing quantitative
capability of EPR in determining tissue pO
2
[22,23]. Furthermore,
utilizing magnetic field gradients as in MRI, the spatial
distribution of the TAM tracer can be obtained in a living subject.
By extracting the pO
2
dependent EPR line widths, a three-
dimensional pO
2
map can be generated with a spatial resolution of
1.5–2 mm
3
in only 3–10 min [23]. The technique can be used to
longitudinally monitor changes in pO
2
on the same animal
[19,21,24]. While images from EPRI provide maps of pO
2
, they
lack the anatomic detail as provided by MRI scans. We therefore
designed a combined EPRI+MRI system operating at a common
frequency of 300 MHz in both modalities with the corresponding
magnetic fields at 10 mT (EPRI) and 7 T (MRI). Sequential scans
with the two modalities employing a common resonator enable
obtaining pO
2
maps with anatomic guidance. Additional infor-
mation gathered from MRI such as blood volume, enable to
achieve a more complete understanding of tumor physiology.
In this report, pO
2
and microvessel density in SCC tumors were
longitudinally monitored by using EPRI and MRI to elucidate
rapamycin effect on tumor oxygenation and angiogenesis in vivo.
Methods
Ethics Statement
All animal experiments were carried out in compliance with the
Guide for the care and use of laboratory animal resources (National
Research Council, 1996) and approved by the National Cancer
Institute Animal Care and Use Committee (NCI-CCR-ACUC
(Bethesda), Protocol# RBB-155 and 159).
Cell Culture and Western Blot Analysis
SCCVII cell line was kindly obtained from Dr. T. Philips,
University of California San Francisco (San Francisco, CA), and
was tested in 2011 by IDEXX RADIL (Columbia, MO) using a
panel of microsatellite markers. The SCCVII is a squamous
carcinoma which arose spontaneously in the abdominal wall of a
C3H mouse in the laboratory of Dr. H. Suit, Massachusetts
General Hospital (Boston, MA) [25,26], and was subsequently
adapted for clonogenic growth by Dr. K. Fu, University of
California San Francisco [27].
SCCVII cells were initially grown in RPMI supplemented with
10% FCS to 70% confluency, and following overnight serum
starvation, cells were treated with 100 nM concentration of
rapamycin (LC Laboratories) for the indicated time. Exposure to
Epidermal growth factor (EGF; Sigma Aldrich) was used as a
positive control at 100 ng/mL for 30 min. After treatment, cells
were lysed and total cellular proteins were processed for western
blot analysis for the indicated proteins and appropriate antibodies
(Cell Signaling; GAPDH was from Santa Cruz).
Animals
Female C3H/Hen mice were supplied by the Frederick Cancer
Research Center, Animal Production (Frederick, MD). SCCVII
solid tumors were formed by injecting 5610
5
SCC cells
subcutaneously into the right hind leg of C3H mice. The
experiment was initiated 8 days after tumor cells implantation.
The tumor size during experiments was 550–1500 mm
3
(the
tumor volume (V = length6width
2
6p/6)). Body weight measured
before the experiments was 21–27 g. Mice were anesthetized by
isoflurane (4% for induction and 1.5% for maintaining anesthesia)
in medical air (750 mL/min) and positioned prone with their
tumor-bearing legs placed inside the resonator. During EPRI and
MRI measurements, the breathing rate of the mouse was
monitored with a pressure transducer (SA Instruments Inc.) and
maintained at 60610 breaths per minute. Core body temperature
was also monitored with a non-magnetic rectal temperature probe
(FISO) and maintained at 3761uC with a flow of warm air. For
administration of TAM and ultrasmall superparamagnetic iron
oxide (USPIO, Molday ION from BioPal Inc., Worcester, MA)
solutions, a 30-gauge needle was cannulated into the tail vein and
extended using polyethylene tubing (PE-10).
Rapamycin treatment
Rapamycin (LC laboratories, Woburn, MA) was dissolved in
ethanol, and further diluted in an aqueous solution of 5.2% Tween
80 and 5.2% polyethylene glycol immediately before use.
Rapamycin was injected intraperitoneally to tumor bearing mice
at a dose of 5 or 10 mg/kg body weight/day, and an equal volume
of diluent was injected to control groups. The treatment was
started 8 days after tumor implantation, and the schedule was a
single injection per mouse, per day, consecutively during
experiments.
EPR imaging
Technical details of the EPR scanner operating at 300 MHz,
data acquisition based on the single-point imaging (SPI) modality,
image reconstruction, and the oxygen mapping procedure were
described in earlier reports [22,23,28–30]. After the animal was
placed in the resonator, TAM (Ox063, GE Healthcare) was
injected intravenously as a 1.125 mmol/kg bolus through the
cannula placed in the tail vein. EPR signals were collected
following the RF excitation pulses (60 ns, 80 W, 70u flip angle)
using an analog digital converter (200 megasamples/s). The
repetition time (TR) was 6.0
ms. The FIDs were collected under a
nested looping of the x, y, z gradients and each time point in the
FID underwent phase modulation enabling 3D spatial encoding.
Since FIDs last for a couple of microseconds, it is possible to
generate a sequence of T
2
*
mapping, which allowed pixel-wise
estimation of in vivo pO
2
. The spatial resolution of pO
2
images
measured using EPRI was 1.8 mm, although the pixel resolution
was digitally enhanced in order to co-register with MRI images.
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MRI and co-registration of pO
2
images with anatomic
images
A parallel coil resonator (17 mm i.d. and 25 mm long) with Q
switch was constructed for sequential EPR and MR imaging of the
tumor bearing leg. The basic description of the parallel coil
resonator used for pulsed EPR and 7 T MRI operating at
300 MHz was described in an earlier report [30]. Since required
quality factor (Q value) is different between EPRI and MRI,
switching of Q values of the coil was done by isolating the damping
resistance from the main circuit [28].
MRI scans were conducted using a 7 T scanner controlled with
ParaVision 5.0 (Bruker BioSpin MRI GmbH). After a quick
assessment of the sample position by a fast low-angle shot (FLASH)
tripilot sequence, T
2
*
-weighted anatomical images were obtained
using a fast spin echo sequence (RARE) with an echo time (TE) of
13 ms, TR of 2,500 ms, 14 slices, RARE factor 8, resolution of
0.1160.11 mm, and acquisition time of 80 s. For convenience of
coregistration with EPRI, all MRI images had the same FOV of
2.8 cm and slice thickness of 2 mm. Blood volume calculation was
performed as described previously [31]. Briefly, this technique was
based on the T2* shortening effect and the consequent signal loss
by USPIO injection. Spoiled gradient echo (SPGR) sequence
images were collected as follows: matrix, 2566256; TE, 5.0 ms;
TR, 261.5 ms; slice thickness, 2 mm; scan time, 2 min 14 sec.
These images were obtained before and 5 min after USPIO
injection (1.2
mL/g body weight). Percentage of tumor blood
volume was estimated by the expression 1006(S
pre
2S
post
)/
[S
pre
+S
post
(W
b
/W
t
21)], where S
pre
and S
post
were the signal
intensities of each voxel before and after USPIO injection and W
b
and W
t
were the intra- and extravascular water fractions. Dynamic
contrast enhanced (DCE)-MRI study was carried out using a 1 T
scanner (Bruker ICON). For T1 mapping, coronal RARE images
of three slices passing through the tumor region were obtained
with TR values of 500, 1000, and 3000 ms. Gd-DTPA solution
(50 mM, 5
mL/g body weight) was intravenously injected into tail
vein of mouse 2 min after start of the fast gradient echo scans. The
scan parameters are as follows: TE = 6 ms, TR = 118 ms, tip angle
30u, 2 mm thickness64 slices, 15 sec acquisition time per image,
and 60 repetition. Co-registration of EPR and MRI images was
accomplished using code written in MATLAB (Mathworks) as
described in a previous report [23,32].
Immunohistochemical analysis
Tumor-bearing mice were euthanized, and tumor tissues were
removed from mice. Tumor tissues were fixed with 4%
paraformaldehyde and frozen using ultracold ethanol. Frozen
tumors were sectioned to 10 mm thick using a cryostat, and the
sections were thaw-mounted on glass slides. After blocking non-
specific binding sites with Protein Block Serum-Free reagent (Dako
North America Inc., Carpinteria, CA), the slides were covered by
CD31 antibody (BD Biosciences, San Jose, CA; 1:250) combined
with aSMA antibody (Abcam Inc., Cambridge, MA; 1:250)
overnight at 4uC. The sections were incubated with Alexa Fluor
488 anti-rat and Alexa Fluor 555 anti-rabbit secondary antibody
(Invitrogen, Carlsbad, CA; 1:500). Then they were mounted on
Prolong Gold antifade reagent with DAPI (Invitrogen). Fluores-
cence microscopic observation was performed using an Axiovert
200 inverted fluorescent microscope (Carl Zeiss).
The quantification of CD31 and aSMA was performed
according to the method described by Zhou et al. [18]. Briefly,
tissue sections were viewed at 2006 magnification and more than
three fields per section were captured using Image-Pro Plus Ver.
4.0 imaging software. Then the quantification of vascular density
and pericyte density on each image was performed with histogram
analysis using the ImageJ software package (http://rsb.info.nih.
gov/ij/) and shown as the total number of positive pixels per field.
Paraformaldehyde fixed tissues were paraffin embedded, and
5 micron-thick sections were processed for immunohistochemical
staining for ribosomal S6 protein and its phosphorylated pS6
counterpart following the method as previously described [33].
Statistical analysis
All results were expressed as the mean 6 SEM. The differences
in means of groups were determined by 2-tailed Student’s t test.
The minimum level of significance was set at p,0.05.
Results
To evaluate the effect of rapamycin treatment on SCCVII
tumor growth, tumor sizes of a control group of tumor bearing
mice and two groups of mice treated daily at 5 and 10 mg/kg bw/
day (n = 5–6) were monitored. Rapamycin treatment was initiated
8 days post tumor cell inoculation in the right hind leg. A
significant delay in tumor growth dependent on rapamycin doses
was noticed in agreement with previous reports (Figure 1A) [11].
These results suggest that the SCCVII implants in C3H mice were
sensitive to rapamycin as evidenced by the tumor growth
inhibition.
Monitoring the accumulation of the phosphorylated form of the
ribosomal S6 protein (pS6), which is the most downstream target
of the mTOR pathway, can provide an exquisite surrogate marker
to follow mTOR activity. In cultured SCCVII cells exposed to
rapamycin (100 nM) for different times (0–12 h), an early decrease
in p-S6 was noticed (1 h) while total S6 levels remained unchanged
(Figure 1B). GAPDH was used as loading control. As SCCVII cells
demonstrated sensitivity to rapamycin in vitro, corresponding
xenografts were also assessed by immunohistochemistry for the
status of pS6. As shown in Figure 1C and D, a significant decrease
in immunoreactivity to the phosphorylated form of S6 was noted
in the rapamycin-treated mice compared to untreated controls,
demonstrating that rapamycin achieved its molecular effect in
vivo. These results support the results shown in Figure 1A that the
molecular target of rapamycin in SCCVII cells is being effected
which is responsible for the tumor growth delay.
Based on observations that rapamycin treatment in SCCVII
tumor bearing mice elicits a tumor growth delay correlating with a
decrease in the mTOR dependent signaling markers, we next
conducted non-invasive imaging experiments to longitudinally
monitor tumor oxygen status, tumor anatomy, and tumor blood
volume in control and rapamycin treated mice with SCCVII
implants by using EPRI and MRI. EPRI and MRI have been
recently shown to have the capability to serially and non-invasively
assess changes in tumor pO
2
and microvessel density as a function
of tumor growth or during a treatment course [19,21,23,31].
Figure 2 shows results from such as an experiment with six
adjacent slices of a vehicle-treated control tumor in leg on 12 days
after tumor implantation, each 2 mm thick displayed for T
2
-
weighted anatomy (top row), pO
2
maps using the oxygen sensing
EPR tracer Ox063 (middle row), and blood vessel density using
the blood pool T
2
* contrast media USPIO (bottom row). The data
presented show the capability of the imaging techniques to non-
invasively obtain that pO
2
distribution and microvessel density
which show significant variation across the tumor.
Figure 3 shows results from longitudinal experiments from a
representative control mouse and rapamycin treated mouse.
Figure 3A shows the center slice of anatomy, pO
2
and blood
volume in the SCCVII tumor bearing mouse receiving vehicle as
control on days 0, 2, and 4 (day 0 is 8 days after tumor
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Page 3
implantation, on which the imaging study and the treatment was
initiated). There were a lot of blood vessels observed throughout
the tumor even on day 0, indicating angiogenesis already occurred
on 8 days after tumor implantation. As expected, mice receiving
no treatment exhibited increases in tumor size with the associated
neovascularization supporting the tumor growth but with increas-
ing hypoxia. Results from the mouse treated with rapamycin are
shown in Figure 3B. It can be seen that a significant retardation of
tumor growth (Figure 3B, top row) was accompanied by loss of
tumor blood volume (Figure 3B, bottom row), and the extent of
hypoxia did not increase in contrast to the control. While median
values of pO
2
provide a global assessment, histograms from the
image data reveal additional information. Therefore, the results of
figure 3A and B were analyzed by converting the pO
2
images and
blood volume images as frequency histograms (Figure 3C and D).
The frequency histograms of tumor pO
2
in the control mouse
show a prominent shift leftwards on day 2 and day 4 compared to
day 0 with a significant increase in the number of voxels having
pO
2
values below 5 mmHg (Figure 3C). On the other hand, in the
rapamycin treated mouse, a significant reduction in frequencies
below 5 mm Hg was observed with an appearance of a second
peak around 22 mm Hg, indicating that a significant increase in
the overall tumor oxygen status occurred on day 2 of rapamycin
treatment. The peak around 22 mm Hg decreased on day 4 but
the frequencies below 5 mm Hg noticed on day 0 did not return.
A dramatic decrease in tumor blood volume in rapamycin treated
mice was noticed on day 2 and 4 whereas it slightly increased in
the control mouse (Figure 3D) Such behavior of transient increase
in pO
2
with decrease in microvessel density after treatment was
Figure 1. Effect of rapamycin on SCC tumor growth and mTOR signaling pathway. (A) Tumor sizes of the SCC tumors in the mice leg
treated with vehicle (control,
N
), 5 mg/kg bw/day (m), and 10 mg/kg bw/day (&) rapamycin. (B) Western blot analysis of S6 protein expression and
the abundance of its phosphorylated form in SCC tumor cells treated with rapamycin (100 nM). (C, D) Immunostaining of pS6 in SCC xenograft of
control and rapamycin treated (10 mg/kg bw/day, 2 days).
doi:10.1371/journal.pone.0049456.g001
Figure 2. Anatomy, pO
2
, and blood volume images of SCC
tumor. T
2
-weighted anatomical image (top) of a SCC tumor-bearing
mouse, and the corresponding pO
2
maps (middle) and blood volume
images (bottom) measured by EPRI and MRI. The adjacent center six
slices of the 3D images were displayed, and the every slice has 2 mm
thickness.
doi:10.1371/journal.pone.0049456.g002
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commonly noticed in the case of antiangiogenic agents and this
phenomenon attributed to neovascular normalization [15,16,19].
The longitudinal changes in median tumor pO
2
values in
groups of control and rapamycin treated mice are graphically
displayed in Figure 4A. It can be seen that while the median pO
2
values were similar in the two groups on day 0, rapamycin treated
mice show higher tumor pO
2
values on days 2 and 4. The median
pO
2
in the rapamycin treated group showed a small decrease on
day 6, but it was at the same level as that on day 0. Similarly, the
hypoxic fraction (fraction of tumor with pO
2
,10 mm Hg) showed
that rapamycin treated mice exhibit hypoxia to a lesser extent
compared to control, untreated mice (Figure 4 B). Figure 4C
shows the fractional blood volume in the tumors as a function of
time. A significant decrease in blood volume in rapamycin treated
mice compared to untreated mice was noticed on day 2.
Continuing rapamycin treatment caused a further drop of blood
volume on day 4 and day 6. An empirical analysis of tumor
oxygenation status obtained from EPR imaging and the blood
volume from MRI was done by obtaining the ratio of tumor pO
2
with the fractional blood volume and plotted as a function of time
and the results are shown in Figure 4D. The results show that
oxygen delivery per fractional tumor blood volume in rapamycin
treated mice was significantly more efficient than in control group
of mice.
In order to investigate the underlying mechanism(s) associated
with the observed improved tumor oxygenation, we carried out
DCE-MRI study with Gd-DTPA as a contrast agent. It is well
known that Gd-DTPA uptake is influenced by both tumor
perfusion and vascular permeability. By considering only the
initial rate of the Gd uptake the effects of changes in permeability
Figure 3. Effect of rapamycin treatments on tumor pO
2
and blood volume. Anatomy, pO
2
, and blood volume images of SCC tumor in mice
leg treated with vehicle (A) and 10 mg/kg bw/day rapamycin (B). The center slice of each 3D image is displayed. Treatment was initiated 8 days after
tumor implantation (Day 0). The images of day 0 were obtained before beginning of the treatments. (C, D) Frequency histograms of pO
2
and blood
volume in the SCC tumors of (A) and (B). The values indicate median pO
2
and blood volume in the tumor region.
doi:10.1371/journal.pone.0049456.g003
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on uptake can be minimized [39,40]. Area under the curve (AUC)
of Gd-DTPA concentration in the SCC tumor calculated from the
DCE-MRI results of initial 1 min after injection was 40% larger in
2 days rapamycin treated group (0.18860.017
mM, n = 5)
compared to non-treated control group (0.13460.025
mM,
n = 5), indicating the improvement of blood flow in the SCC
tumor by rapamycin treatments.
Independent microscopic evaluation of tumor vasculature in
control and rapamycin treated mice was carried out from tumor
sections stained with CD31 (green) for microvasculature and
aSMA for pericyte coverage (red) (Figure 5A). A significant
decrease (* p,0.05) in tumor blood vessel density was noticed
(Figure 5B) in rapamycin treated mice compared to untreated
mice in agreement with the blood volume assessment from MRI
experiments. When the histological data was quantitatively
analyzed, it was found that the blood vessel density decreased by
,30% 2 days after treatment with rapamycin. On the other hand,
there was a small but not significant decrease in aSMA staining in
tumors of rapamycin treated mice. The results shown in Figure 5
are consistent with the observations made by Lane et al [34] where
the mTOR inhibitor RAD001 was more effective in reducing
mature vessels with effective aSMA coverage than the anti-
angiogenic agents tested.
In order to examine if the pO
2
increase by rapamycin treatment
enhances outcome of radiotherapy, four different groups of tumor
bearing mice (control; X-ray: rapamycin; rapamycin+X-ray) were
monitored for tumor growth delay (Figure 6). Both mono-therapy
of 5 days rapamycin treatment (closed triangles) and fractionated
5Gy63 days X-irradiation (open squares) suppressed tumor
growth for 2 days. Combination of rapamycin and X-irradiation
resulted in 5 days tumor growth delay (open diamonds). The
‘‘more than additive’’ growth delay may suggest the enhanced
outcome of radiotherapy during vascular normalization window of
rapamycin which transiently increases tumor pO
2
.
Discussion
Increasing evidence supports a strong role for the mTOR
complex as a critical regulator of cellular metabolism, growth, and
proliferation [35,36]. In carcinomas such as SCCVII, this pathway
may be an early and widespread event independent of p53 status
making this an important downstream target for therapy for
mTOR inhibitors such as rapamycin and its analogs [7–10]. The
mTOR pathway, being a part of the PI3K/Akt is considered as a
key determinant in tumor angiogenesis through the expression of
hypoxia related genes VEGF [13,37]. Rapamycin and its analogs
(rapalogs) target the mTOR pathway and induce cell death,
autophagy and also exert antiangiogenic and antivascular effects in
solid tumors [11,14,34,37,38]. Additionally, in preclinical models,
rapamycin was shown to be an effective radiation sensitizer in vivo
[20]. The results from the present imaging study provide non-
invasive evidence for the rapamycin-induced loss in blood vessel
density, but unexpectedly, we observed a concomitant increase in
tumor pO
2
.
The antiangiogenic effects of rapamycin were first observed
using a dorsal skin-fold chamber model using tumor implants in
mice [37]. The antiangiogenic effects were attributed to decreased
production of VEGF and resistance of endothelial cells to VEGF
stimulation. Further studies used the rapalog RAD001 and
compared its effects with known antiangiogenic agents [34]. The
Figure 4. Changes in tumor oxygenation and blood volume. Median pO
2
values (A), percentage of hypoxic fraction (B), and mean blood
volume (C) in the control and rapamycin treated SCC tumors. The values are average of 5 or 6 mice and error bars represent standard deviations.
Oxygen delivery per unit blood volume (D) was calculated by dividing tumor pO
2
by blood volume. * p,0.05 as compared with control, ** p,0.01 as
compared with control, { p,0.05 as compared with rapamycin day 8, {{ p,0.005 as compared with rapamycin day 8.
doi:10.1371/journal.pone.0049456.g004
Rapamycin Improves Tumor Oxygenation
PLOS ONE | www.plosone.org 6 November 2012 | Volume 7 | Issue 11 | e49456
Page 6
results showed that RAD001 was found to be associated with
decreasing the tumor vessel density and the maturity of the tumor
vessels, whereas the antiangiogenic drug vatalanib was found to
impact only the microvascular density but not the vessel maturity
consistent with this class of drugs which impact the VEGF/
VEGFR complex [34]. On the other hand, Zhang et al. reported
that the aSMA level, a marker of mature pericytes, increased in
rapamycin treated tumor compared with non-treated tumor [14].
Other studies have shown that radiation induces activation of
mTOR pathways in the tumor endothelial cells making them
more sensitive to response with rapamycin [20]. However, a more
recent study using a retro-inhibition approach found that HNSCC
cells and not the tumor microenvironment as the target for
rapamycin activity and that the anti-angiogenic effect is a likely
downstream consequence of mTOR inhibition in cancer cells
[13].
Imaging of properties intrinsic to tumor physiology such as
tumor pO
2
and tumor microvessel density made it possible to
sequentially follow rapamycin induced changes during the course
of treatment non-invasively and sequentially during the treatment
course [19,21,23]. The key finding in the present study pertaining
to the rapamycin effect on tumor physiology is that the tumor
microvessel density, when monitored longitudinally showed a
significant decrease whereas a transient increase in tumor pO
2
was
Figure 5. Immunohistochemical analysis of CD31 and aSMA in SCC xenograft. (A) Representative images of control and rapamycin treated
SCC xenograft. Green is CD31, red is aSMA, and blue is DAPI. (B) Percentage of CD31 and aSMA in the SCC tumor of control (n = 3) and rapamycin
treated (n = 4, 10 mg/kg bw/day, 2 days) mice.
doi:10.1371/journal.pone.0049456.g005
Figure 6. Effect of combination of rapamycin and X-irradiation
on tumor growth. Growth kinetics of SCC tumors implanted in mice
leg, treated with vehicle (control,
N
), X-irradiation (5 Gy/day, 3 days, %),
rapamycin (10 mg/kg/day, 5 days, m), and rapamycin and X-irradiation
(e).
doi:10.1371/journal.pone.0049456.g006
Rapamycin Improves Tumor Oxygenation
PLOS ONE | www.plosone.org 7 November 2012 | Volume 7 | Issue 11 | e49456
Page 7
found followed by onset of hypoxia (pO
2
,10 mmHg). It should be
noted that the USPIO-based blood volume assessment may
overestimate the values in tumors because of their leakiness
compared to normal tissues [31]. The observations from
histological experiments were in agreement with the imaging
observations. The rapamycin-induced decrease in CD31 staining
was found to be in agreement with imaging experiments where a
loss in microvessel density was found. However, there was a small
but non-significant decrease in staining of aSMA which reflects the
retention of the integrity of the pericyte coverage of the tumor
vasculature after rapamycin administration. These results indicate
that rapamycin treatment pruned immature blood vessels rather
than mature blood vessels. It is expected that these changes in
tumor microvasculature can cause improvement of blood flow, a
phenomenon known as vascular normalization. The transient
increase in the pO
2
by rapamycin treatment can be attributed to
the increased blood flow in the tumor, which was demonstrated by
a 40% increase in tumor initial uptake of Gd-DTPA 2 days after
rapamycin treatment in the DCE-MRI study.
The identification of transient improvements in tumor oxygen-
ation 2 days after rapamycin treatment provides an opportunity
for chemoradiation modalities where radiation therapy can be
timed to take advantage of increases in tumor pO
2
to elicit
improved response [20]. The results in the present study show
enhancement in tumor radioresponse by rapamycin treatment
(Figure 6). This data suggests that the transiently increased level of
median tumor pO
2
in rapamycin treated mice compared to the
day matched control group may be responsible for the observed
effect of radioresponse with combination treatment. The relatively
smaller effect of radiation with rapamycin (additive), in contrast
with the observed synergistic effect of radiation with sunitinib in
the same tumor xenograft [19], may be explained in terms of the
relatively smaller magnitude difference in tumor pO
2
in rapamy-
cin treated group to the day matched control (,2 mm Hg)
compared to the greater difference in tumor pO
2
in sunitinib
treated group to the control (,5.5 mm Hg). The significant
synergy with mTOR inhibitors including rapamycin and radiation
reported by Shinohara et al [20] may point out the characteristic
influences of the microenvironment of each tumor type as pointed
out in other studies where the synergy was attributed only to
rapamycin targeting the enhanced activity of signaling pathways
controlled by mTOR in the host endothelial cells [41]. Recent
studies with a dual inhibitor of the PI3K and mTOR pathway
found that the period of vascular remodeling is relatively more
sustained than that observed with anti-angiogenic drugs resulting
in substantial therapeutic gain [42]. These studies point to the
importance of longitudinally monitoring such changes to realize
maximal efficacy in combined chemo-radiation treatments.
Imaging studies of the tumor microenvironment can establish a
strategy in preclinical models to identify an optimal treatment
schedule to realize enhanced response to combination treatments.
In summary, results from the current study show that molecular
imaging techniques provide an opportunity to serially monitor
changes in tumor physiology non-invasively and quantitatively and
identify subtle physiological changes in response to rapamycin
treatment. Therefore these techniques have the ability to provide
valuable non-invasive biomarkers which predict treatment out-
come and also identify temporal windows where radiation therapy
can be advantageously combined to elicit improved response.
Author Contributions
Conceived and designed the experiments: KS SM VP JSG JBM MCK.
Performed the experiments: KS SM HY VP. Analyzed the data: KS SM
VP JSG JBM MCK. Contributed reagents/materials/analysis tools: ND
SS JPM. Wrote the paper: KS SM MCK.
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Rapamycin Improves Tumor Oxygenation
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  • Source
    • "At therapeutic concentrations, rapalog drugs primarily inhibit tumor neovascularization through inhibition of mTORC1-dependent VEGF production [2,3] , to blunt tumor angiogene- sis [9,26,27]. However, rapalog treatment is limited by escape of the tumor and vasculature from drug inhibition, and subsequent tumor progression [5,28,29]. "
    [Show abstract] [Hide abstract] ABSTRACT: Tumor neovascularization is targeted by inhibition of vascular endothelial growth factor (VEGF) or the receptor to prevent tumor growth, but drug resistance to angiogenesis inhibition limits clinical efficacy. Inhibition of the phosphoinositide 3 kinase pathway intermediate, mammalian target of rapamycin (mTOR), also inhibits tumor growth and may prevent escape from VEGF receptor inhibitors. mTOR is assembled into two separate multi-molecular complexes, mTORC1 and mTORC2. The direct effect of mTORC2 inhibition on the endothelium and tumor angiogenesis is poorly defined. We used pharmacological inhibitors and RNA interference to determine the function of mTORC2 versus Akt1 and mTORC1 in human endothelial cells (EC). Angiogenic sprouting, EC migration, cytoskeleton re-organization, and signaling events regulating matrix adhesion were studied. Sustained inactivation of mTORC1 activity up-regulated mTORC2-dependent Akt1 activation. In turn, ECs exposed to mTORC1-inhibition were resistant to apoptosis and hyper-responsive to renal cell carcinoma (RCC)-stimulated angiogenesis after relief of the inhibition. Conversely, mTORC1/2 dual inhibition or selective mTORC2 inactivation inhibited angiogenesis in response to RCC cells and VEGF. mTORC2-inactivation decreased EC migration more than Akt1- or mTORC1-inactivation. Mechanistically, mTORC2 inactivation robustly suppressed VEGF-stimulated EC actin polymerization, and inhibited focal adhesion formation and activation of focal adhesion kinase, independent of Akt1. Endothelial mTORC2 regulates angiogenesis, in part by regulation of EC focal adhesion kinase activity, matrix adhesion, and cytoskeletal remodeling, independent of Akt/mTORC1.
    Full-text · Article · Aug 2015 · PLoS ONE
  • Source
    • "Once activated, the Br-IPM moiety can efficiently induce intramolecular 19 39-cross-linkage of DNA, resulting in S139 phosphorylation of histone H2AX and ultimately causing tumor cell death2021. Our lab has developed imaging capabilities to serially map tumor oxygen in vivo and changes in tumor pO 2 distribution in response to treatment using electron paramagnetic resonance imaging (EPRI)222324. Studies from our lab and others using such imaging techniques have found that tumors display both spatial and temporal heterogeneities in pO 2 status. "
    [Show abstract] [Hide abstract] ABSTRACT: BackgroundTH-302 is a hypoxia-activated prodrug (HAP) of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302.Methodology/ResultsThe hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR) oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O2), with minimal effect under aerobic conditions (21% O2). Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500–1500 mm3. Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼550 mm3), significantly delayed tumor growth.Conclusions/SignificanceOur in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the appropriate tumor size and oxygen concentration.
    Full-text · Article · Sep 2014 · PLoS ONE
  • Source
    • "Saito et al32 provided noninvasive evidence of RPM-induced vascular renormalization and the resultant transient increase in tumor oxygenation. The improved oxygenation from RPM treatment provides a temporal window for anticancer therapies to enhance radiotherapy response. "
    [Show abstract] [Hide abstract] ABSTRACT: The mammalian target of rapamycin (mTOR) is a protein kinase that regulates protein translation, cell growth, and apoptosis. Rapamycin (RPM), a specific inhibitor of mTOR, exhibits potent and broad in vitro and in vivo antitumor activity against leukemia, breast cancer, and melanoma. Recent studies showing that RPM sensitizes cancers to chemotherapy and radiation therapy have attracted considerable attention. This study aimed to examine the radiosensitizing effect of RPM in vitro, as well as its mechanism of action. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and colony formation assay showed that 10 nmol/L to 15 nmol/L of RPM had a radiosensitizing effects on pancreatic carcinoma cells in vitro. Furthermore, a low dose of RPM induced autophagy and reduced the number of S-phase cells. When radiation treatment was combined with RPM, the PC-2 cell cycle arrested in the G2/M phase of the cell cycle. Complementary DNA (cDNA) microarray and reverse transcription polymerase chain reaction (RT-PCR) revealed that the expression of DDB1, RAD51, and XRCC5 were downregulated, whereas the expression of PCNA and ABCC4 were upregulated in PC-2 cells. The results demonstrated that RPM effectively enhanced the radiosensitivity of pancreatic carcinoma cells.
    Full-text · Article · Mar 2013 · Drug Design, Development and Therapy
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