European Journal of Radiology 78 (2011) 272–276

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European Journal of Radiology

journal homepage: www.elsevier.com/locate/ejrad

Combination of vascular endothelial growth factor antisense oligonucleotide

therapy and radiotherapy increases the curative effects against maxillofacial VX2
tumors in rabbits
Lin-Feng Zheng 1,2 , Yu-Jie Li 1,2 , Han Wang 3 , Jing-Long Zhao 3 , Xi-Fu Wang 2 ,
Yun-Sheng Hu 2 , Gui-Xiang Zhang ∗
Department of Radiology, Shanghai First People’s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai, China

a r t i c l e i n f o a b s t r a c t

Article history: Purpose: To study the effects of combination of vascular endothelial growth factor (VEGF) antisense
Received 3 September 2010 oligonucleotide therapy and radiotherapy on maxillofacial VX2 tumors in rabbits.
Accepted 29 November 2010 Methods: We used 24 New Zealand white rabbits as a model to induce maxillofacial VX2 tumor. The rabbits
were randomly divided into the following 4 groups: radiotherapy group (group A), treated with 16 Gy
Keywords: of radiotherapy; VEGF antisense oligonucleotide treatment group (group B), treated with an injection of
Vascular endothelial growth factor
150 ␮g of VEGF antisense oligonucleotide into the local tumor; VEGF antisense oligonucleotide combined
Antisense oligonucleotide
with radiotherapy group (group C), treated with an injection of 150 ␮g of VEGF antisense oligonucleotide
Radiotherapy
VX2 tumor
into the local tumor immediately after 16 Gy of radiotherapy; and control group (group D), treated with
Rabbit an injection of 300 ␮l 5% aqueous glucose solution into the local tumor. On days 3 and 14 after treatment,
dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was performed to calculate maximal
enhancement ratio (MER), slope of enhancement (SLE), and tumor volume change. Rabbits were killed
on day 14 to obtain samples for pathological examination and immunohistochemical staining for VEGF.
Results: In group C, tumor volume was significantly reduced on day 14 after treatment, and the difference
was statistically different as compared to that before treatment, on day 3 after treatment and other groups
(P < 0.01). Values of both MER and SLE after treatment were significantly lower than the values before
treatment (P < 0.05). Pathological specimen revealed tumor cell edema, bleeding, necrosis, vascular wall
thickening and occlusion, and decreased VEGF expression. The immunohistochemical score (IHS) of group
C was significantly different from groups A and D respectively (P < 0.05).
Conclusion: Injecting the tumor with VEGF antisense oligonucleotide immediately after radiotherapy
can enhance the curative effect on rabbit maxillofacial VX2 tumor, and DCE-MRI can serve as a reliable
technique for in vivo monitoring.
© 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction angiogenesis, and the vascular endothelial growth factor (VEGF)

Radiotherapy is an important method for the treatment of max-
illofacial tumors [1,2]. In addition to the histological type and
size of tumor, tumor hypoxia also plays an important role in the
effect of radiotherapy on tumors [3–5]. Tumor growth depends on
∗ Corresponding author. Tel.: +86 21 63240090x4166; fax: +86 21 63240825.
E-mail addresses: zhenglinfeng04@yahoo.com.cn (L.-F. Zheng),
yujieli01@yahoo.com.cn (Y.-J. Li), bingowh@hotmail.com (H. Wang),
jinglongz@yahoo.com (J.-L. Zhao), wangxiechen001@163.com (X.-F. Wang),
springmorninghu@163.com (Y.-S. Hu), guixiangzhang@sina.com (G.-X. Zhang).
1
These authors contributed equally to this work.
2
Tel.: +86 21 63240090x4173; fax: +86 21 63240825.
3
Tel.: +86 21 63240090x4169; fax: +86 21 63240825.
is an important cytokine in the process of angiogenesis [6–8].
Hypoxic environment promotes tumor angiogenesis by induc-
ing increased VEGF expression in tumor cells [9]. In addition
to hypoxic environment-induced angiogenesis, radiotherapy also
affects tumor VEGF expression and thus promotes angiogenesis,
leading to tumor resistance to radiotherapy [9,10]. Therefore, radio-
therapy in combination with anti-VEGF therapy shows promise in
improving the curative effect against tumors. In this study, rabbit
maxillofacial VX2 tumor models were used to study the effects of
combination of VEGF antisense oligonucleotides and radiotherapy
on maxillofacial tumors, the effects of this treatment were ana-
lyzed by dynamic contrast-enhanced magnetic resonance imaging
(DCE-MRI) and immunohistochemical and pathohistological exam-
ination.

0720-048X/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.ejrad.2010.11.036
 L.-F. Zheng et al. / European Journal of Radiology 78 (2011) 272–276
2. Materials and methods
2.1. Animal model and groups
All experiments were performed with approval from the animal
ethics committee of our hospital and in accordance with Chi-
nese animal protection and management law guidelines. Healthy
adult New Zealand white rabbits (provided by the Shanghai Baomu
Experimental Animal Farm, Shanghai) of either gender, weighing
1.5–2.0 kg were used for experiments. After the rabbits were anes-
thetized by an intraperitoneal injection of 2% sodium pentobarbital
(40 mg/kg), routine shaving and disinfection of the right maxillo-
facial region of the rabbit were performed; 0.5 ml suspension of
VX2 tumor cells (maintained in our laboratory) at a concentration
of 1 × 108 was extracted using a sterile 1 ml syringe and injected
in the masseter at about 1 cm in front of the right mandibular
angle, following the withdrawal of needles after 1 min retention
[11]. After regaining consciousness, animals received regular feed-
ing, and tumor diameters were measured on alternate days by
using a Vernier caliper. The experimental animals in which the
tumor diameters were 1.0–1.5 cm on day 12 after inoculation were
selected for the present study.
Twenty-four rabbits that met the criteria for inclusion were
randomly divided into 4 groups (n = 6) as follows: radiotherapy
group (group A) received a one-time dose of 16 Gy of radiother-
apy; antisense oligonucleotide treatment group (group B) received
an injection of 150 ␮g VEGF antisense oligonucleotide in the local
tumor; combined treatment group of radiotherapy and antisense
oligonucleotide (group C) received an injection of 150 ␮g of VEGF
antisense oligonucleotide in the local tumor immediately after
16 Gy of radiotherapy; and control group (group D) received an
injection of 300 ␮l 5% aqueous glucose solution in the local tumor.
2.2. Preparation of VEGF antisense oligonucleotide
Liposome (Invitrogen, CA, USA) and 5% glucose (D5W) were
mixed uniformly into 150 ␮l solution in a 2:3 ratio. Subsequently,
D5W was added by uniform mixing into 150 ␮g VEGF antisense
cDNA (pcDNA3.1-ASVEGF) to yield a 150 ␮l volume. The 2 solutions
were rapidly mixed uniformly by inverting it twice to prepare the
final solution.
2.3. Treatment measures
Anesthetized rabbits in group A were fixed on a perspex holder
designed in-house with the tumor surface exposed upward and
were irradiated once using vertical (10.6 Gy) and lateral (5.4 Gy)
field irradiation (16 Gy total). In group B, local tumors were injected
with 150 ␮g of the VEGF antisense oligonucleotide preparation.
Group C rabbits were irradiated (as described for Group A) followed
immediately by injection of 150 ␮g VEGF antisense oligonucleotide
into local tumors. In group D rabbits, 300 ␮l D5W was injected
into tumor. For all groups, DCE-MRI analysis was performed on
days 3 and 14 post-treatment. After the final examination, rabbit
tumor tissues were collected, fixed, embedded, dewaxed, and sliced
for hematoxylin and eosin (HE) and VEGF immunohistochemistry
staining.
2.4. DCE-MRI
MRI scanning and image processing were performed using the
3.0 T excite HD superconducting magnetic resonance imaging sys-
tem and GE ADW 4.3 workstation (GE Medical Systems, Milwaukee,
WI, USA). Knee coil, conventional spin echo (SE), and fast spin
echo (FSE) sequences were used for sagittal and coronal scan,
field of view (FOV) was 12 × 12, matrix 256 × 160, slice thick-
273
ness 3 mm, NEX 2, T1WI (TR/TE 350 ms/15 ms), and T2WI (TR/TE
3000 ms/90 ms). The maximum coronal level of tumor was selected
for DCE-MRI after a plain scan using gradient echo (GRE) sequence,
FOV 12 × 12, matrix 256 × 160, TR 10 ms, TE 2 ms, flip angle 20◦ ,
slice thickness 3 mm, and NEX 2. Magnevist at 1 ml/kg (Bayer Scher-
ing Pharma AG, Berlin, Germany) was injected in rabbit ear vein
at 0.2 ml/s, while a total of 60 levels of images were acquired
over a period of 152 s. Coronal and axial scans after enhance-
ment were obtained after perfusion scan. Post-scan parameters
were equal to pre-enhancement parameters. Tumor location, num-
ber, size, signal, and the adjacent tissues were recorded. On the
T1WI enhancement image, the 3 axial and coronal maximum tumor
diameters (in mm) were measured (labeled A, B, and C, respec-
tively) and tumor volume was calculated according to the formula
V = 1/2 ABC [6]. The series of dynamic contrast-enhanced MR per-
fusion original images were imported to the workstation using
GE FUNCTOOL 4.4.05 software (GE Medical Systems, Milwaukee,
WI, USA), manually setting the tumor region of interest (ROI) at
8–12 mm3 , and placed in a site devoid of blood vessels in which the
tumor was significantly enhanced and necrosis cystic areas could
be identified with the naked eye. Next, a time–signal curve was
drawn during the first pass phase of the contrast agent. The follow-
ing parameters were measured and calculated [12] – Tpre : initial
enhancement time (s), when the curve begins to rise; SIpre : initial
enhancement signal, the relative signal intensity when the curve
begins to rise; Tmax : maximum enhancement time (s), when the
curve reaches the peak; and SImax : maximum enhancement signal,
the relative signal intensity when the curve reaches the peak. The
tumor maximum enhancement ratio (MER) = (SImax − SIpre )/SIpre
and the slope of enhancement (SLE) = MER/(Tmax − Tpre ) were cal-
culated. The ROI before and after treatment was located in the same
position as far as possible.
2.5. Immunohistochemical staining
Mouse anti-rabbit VEGF monoclonal antibodies were used
for immunohistochemical staining (streptavidin peroxidase (SP)
method), the main processes were as follows: the slices were
dewaxed and hydrated by a conventional method; antigen was
retrieved by microwave treatment for 20 min; cooled at room tem-
perature; washed thrice with phosphate-buffered saline (PBS, pH
7.2) for 3 min; 3% hydrogen peroxide (H2 O2 ) was added dropwise,
and the sliced were incubated at room temperature for 10 min to
block endogenous peroxidase; slices were washed 3 times with PBS
for 5 min; 10% calf serum was dropped into, and the slices were
incubated for 20 min in wet box at 37 ◦ C; subsequently, the 10%
calf serum was discarded and the mouse anti-rabbit VEGF mono-
clonal antibodies (1:50, Lab Vision & Neomarkers, Thermo Fisher
Scientific Inc., Fremont, USA) were added and incubated for 2 h in
a wet box at 37 ◦ C; slices were washed 3 times with PBS for 3 min;
secondary antibodies were added (1:100, Lab Vision & Neomark-
ers, Thermo Fisher Scientific Inc., Fremont, USA) and incubated for
10 min at room temperature; slices were washed 3 times with PBS
for 3 min; streptavidin-peroxidase solution was added and incu-
bated for 10 min at room temperature; slices were washed 3 times
with PBS for 5 min; freshly prepared 0.05% DAB containing 0.03%
H2 O2 (Lab Vision & Neomarkers, Thermo Fisher Scientific Inc., Fre-
mont, USA) was stained in the dark at room temperature using
a microscope to control coloration; and finally the reaction was
terminated with PBS. Slices were re-stained with hematoxylin,
conventionally dried, dehydrated, clarified, and mounted. For the
negative control group, PBS replaced the primary antibody and the
remaining steps were identical to those described above.
The VEGF immunohistochemical scores (IHS) [13,14] were cal-
culated as the combined total of positive cell percentage and
staining intensity scores. The absence of positive cells scored 0
274 L.-F. Zheng et al. / European Journal of Radiology 78 (2011) 272–276

Table 1 Table 2
Changes in maximal enhancement ratio (MER) and slope of enhancement (SLE) Changes in tumor volume before and after treatment in each group (mean ± SEM,
before and after treatment in each group. mm3 ).

Group Time point Group Volume of tumor (mm3 )

Pre-treatment Post-treatment day 3 Post-treatment day 14 Pre-treatment Post-treatment day 3 Post-treatment day 14

A group A group 1680 ± 840 1800 ± 800 1530 ± 690*

MER 1.408 ± 0.211 1.799 ± 0.396* 0.950 ± 0.478* B group 1600 ± 640 1700 ± 700 2850 ± 610*
SLE (s−1 ) 0.039 ± 0.018 0.052 ± 0.022* 0.047 ± 0.035* C group 1650 ± 530 1400 ± 500 630 ± 390* , 
B group D group 1670 ± 680 2400 ± 800 4680 ± 750*
MER 1.391 ± 0.208 1.293 ± 0.192 1.299 ± 0.248 *
P < 0.01 compared with pre-treatment and day 3 post-treatment, respectively.
SLE (s−1 ) 0.039 ± 0.009 0.027 ± 0.006* 0.024 ± 0.009* 
P < 0.01 compared with other groups at same time point.
C group
MER 1.563 ± 0.219 1.665 ± 0.140 1.196 ± 0.186*
SLE (s−1 ) 0.043 ± 0.010 0.033 ± 0.013 0.025 ± 0.007*
D group
(P < 0.01). The difference in volume on day 14 between group C and
MER 1.319 ± 0.254 1.272 ± 0.278 1.206 ± 0.171 groups A, B, and D was statistically significant (P < 0.01). See Fig. 1
SLE (s−1 ) 0.041 ± 0.012 0.057 ± 0.032 0.041 ± 0.011 and Table 2.
*
P < 0.05 compared to values before treatment.
3.2. Pathology results
points; 1–10% positive cells scored 1 points; 11–50% scored 2
points; 51–80% scored 3 points; and 81–100% scored 4 points. For In groups A and C, light microscopy revealed large central
positive cell staining: absence of stain scored 0 points; weak stain- areas of necrosis, tumor cell edema, bleeding, partial disappearance
ing scored 1 points; moderate staining scored 2 points; and intense of tumor cell nuclei, large areas of necrotic tumor coagula-
staining scored 3 points. The IHS of the section was calculated as tion, thickening of vascular walls, and partial vessel occlusion
the product of scores of the percentage positive cells and degree of (Fig. 2a and c). In the antisense oligonucleotide alone treat-
staining. ment group (group B), light microscopy revealed clear tumor
boundaries, fewer tumor vessels, varied tumor cell shapes and
2.6. Statistical analysis sizes, and large, intensely stained nuclei (Fig. 2b). In the con-
trol group (group D), light microscopy revealed invasive growth
Experimental data were expressed as mean ± SEM. Using of hard tumors, unclear boundaries between tumor and adjacent
SPSS13.0 software, one-way analysis of variance (ANOVA) was mandibles (2 cases), tumor transition to pale in appearance, irreg-
used to compare the experimental data in each group followed ular bleeding and central tumor necrosis, small well-defined lymph
by the Student’s t-test for paired comparison (Dunett’s tests). P nodes around the tumor, increased tumor vascularit, varied tumor
values < 0.05 were considered significant. shapes and sizes, significant atypia, and pathologic mitotic images
(Fig. 2d).
3. Results
3.3. VEGF Immunohistochemistry
3.1. MRI Results
The VEGF immunoreactive products were found mainly in
Before treatment, the tumors in each group were isointense tumor cell cytoplasm, while positive expression was sometimes
or hypointense on T1WI and hyperintense or slightly hyperin- detected in the nucleus and membranes. VEGF expression was sig-
tense on T2WI and showed significant uniform enhancement on nificantly increased in group A, presenting large coarse dark brown
T1W1 after the contrast agent injection. After various treatments, in granules (Fig. 3a); VEGF expression was decreased in groups B and
groups A and C on the day 3 after treatment, the peripheral tumors C, showing weakly positive changes (light yellow) (Fig. 3b and c);
were more enhanced than before treatment, no enhancement was and tumors in group D mostly presented brown yellow granular
observed in the tumor necrosis central area, and enhancement changes (Fig. 3d). As determined by IHS, VEGF immunoreactive
was slightly reduced on day 14 (Fig. 1a–c, g–i). In group B after expression in group C differed significantly from groups A and D,
treatment, the signal change was not significant compared to pre- respectively (P < 0.01, Fig. 4).
treatment (Fig. 1d–f). No significant changes in tumor size or
necrosis were found in group D (Fig. 1l–n). 4. Discussion
On day 3 after treatment, DCE-MRI dynamic enhancement
revealed increased MER and SLE values in group A (significant The VEGF protein is an important blood vessel-derived growth
compared to pre-treatment values, P < 0.05); reduced SLE values in factor that was first isolated from the tumor ascites of a guinea pig
group B (significant compared to pre-treatment values, P < 0.05); [15]. VEGF plays an important role in tumor angiogenesis [6–8].
and no MER and SLE differences in the other groups. On day Tumor VEGF expression is closely related to radiation therapy
14 after treatment, DCE-MRI revealed decreased MER values in [16,17]. The biological effects of VEGF can be blocked by solu-
groups A and C (significant compared to pre-treatment, P < 0.05); ble anti-VEGF antibodies, VEGF receptor antagonists, VEGF signal
increased SLE values in group A, and decreased SLE values in groups transduction inhibitors, and other measures. Blocking VEGF effects
B and C (both significant compared to pre-treatment, P < 0.05). See may potentially enhance tumor treatments [18–21], and the use
Table 1. of VEGF antisense oligonucleotide may interfere with VEGF tran-
Before treatment, all tumors were similar in volume (differences scription and translation may achieve better treatment results [22].
were statistically insignificant). On day 3 after treatment, tumors Teicher et al. first revealed the antagonistic effects of VEGF could
from groups A, B, and D increased in volume, while group C tumors increase tumor radiosensitivity [18]. Ning et al. [19] used SU5416
decreased, but with no statistical significance. On day 14 after treat- (an inhibitor to the VEGF receptor) and SU6668 (an inhibitor to the
ment, groups A and C tumor volumes were significantly reduced VEGF, FGF, and PDGF receptors) in combination with fractionated
compared to pre-treatment and post-treatment day 3(all P < 0.01), irradiation to treat C3H mice bearing SCC VII carcinomas. SU5416
while control group (group D) volumes were significantly increased and SU6668 were shown to enhance irradiation efficacy. Gorshi
 L.-F. Zheng et al. / European Journal of Radiology 78 (2011) 272–276 275

Fig. 1. Changes in the magnetic resonance images of rabbits in each group before and after treatment (T1WI contrast-enhanced).

et al. [21] found that applying a neutralizing antibody to VEGF-
165 before administering irradiation affected tumor cell radio-
sensitivity.
In this study, the effect of VEGF on tumor growth was examined
by in vivo transfection of eukaryotic expression plasmids con-
taining VEGF antisense oligonucleotide by injection into tumors
immediately following radiotherapy. By injecting after radio-
therapy, the oligonucleotide avoids potential denaturation and
inactivation. Results here show that on the day 14 after treat-
ment, VEGF expression in the combination group was significantly
reduced compared VEGF expression in the control and radiotherapy
only groups, indicating that injection of antisense oligonucleotide
can effectively inhibit tumor VEGF expression. The combina-
tion treatment group effects better tumor volume reduction
than that seen in the radiotherapy only and antisense oligonu-
cleotide only treatment groups, suggesting that VEGF antisense
oligonucleotide therapy produces significant effects on early tumor
growth.

Fig. 2. Result of pathological examination of a tumor specimen after treatment for 14 days in each group (hematoxylin and eosin staining). (a) Group A (100×); (b) group B
(400×); (c) group C (400×); and (d) group D (400×).
276 L.-F. Zheng et al. / European Journal of Radiology 78 (2011) 272–276
Fig. 3. Result of VEGF immunopositive staining after 14 days of treatment in each group (3,3 -diaminobenzidine (DAB) staining, hematoxylin counterstaining, 400×). (a)
Group A; (b) group B; (c) group C; and (d) group D.
Fig. 4. VEGF immunohistochemical scores. *P < 0.01 compared with groups A and D
respectively.
On DCE-MRI, fast scan sequences are used for a rapid and
continuous acquisition after intravenous bolus injection of con-
trast agent and a series of dynamic contrast-enhanced images
can then be obtained, reflecting signal changes when contrast
agents first pass the inspected tissue. This study shows that time-
signal curve parameter MER of DCE-MRI can comprehensively
reflect tumor microvascular perfusion, vascular permeability and
extravascular tissue space size, while SLE indirectly reflects tumor
vascular permeability [12,23]. In this study, after 3 days of radio-
therapy, subjects receiving radiotherapy alone show increased
MER and SLE compared with pre-treatment. This change in MER
and SLE may be associated with local vessel dilatation, improved
blood flow perfusion, and increased vascular permeability follow-
ing tumor radiotherapy. Day 14 tumor MER and SLE values are
lower than day 3 values after radiotherapy. This may be related
to the vascular endothelial cell damage caused by radiotherapy,
as a result, which presenting endangiitis and perivascular inflam-
mation, blood flow perfusion weakeness, and decreased vascular
permeability. Meanwhile, the VEGF expression in surviving tumor
cells is increased under hypoxic conditions, which may in turn
induce the generation of new blood vessels - 2 major causes of
tumor recurrence. While in the combined treatment group 14
days following treatment, both MER and SLE are reduced. On one
hand, this may be related to radiation-induced vascular damage,
poor blood flow perfusion, and reduced vascular permeability;
on the other hand, the antisense oligonucleotide may play a
role by counteracting the radiotherapy-induced increase in VEGF
expression.
In conclusion, the VEGF antisense oligonucleotide can enhance
the early effects of radiotherapy on rabbit maxillofacial VX2 tumors,
and DCE-MRI can be used as an effective method for monitoring in
vivo efficacy.
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