Journal of Cancer Therapy, 2013, 4, 1-6
http://dx.doi.org/10.4236/jct.2013.49A2001 Published Online October 2013 (http://www.scirp.org/journal/jct)
1
Combined Ang-2 and VEGF Targeting Therapies in Renal
Cell Carcinoma*
Nikolett Molnar1, Dietmar W. Siemann1,2
1Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, USA; 2Department of Ra-
diation Oncology, University of Florida College of Medicine, Gainesville, USA.
Received July 23rd, 2013; revised August 22nd, 2013; accepted August 30th, 2013
Copyright © 2013 Nikolett Molnar, Dietmar W. Siemann. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
ABSTRACT
Angiogenesis, the growth of new vessels from pre-existing ones, is an important feature of tumor growth that has been
exploited as a therapeutic target in oncology. Given its key role in facilitating blood vessel sprouting, VEGF has been a
major focus of anti-angiogenic strategies, but the observation of resistance in some clinical trials utilizing such agents
has led to a search for new or complementary targets in angiogenesis process. The Angiopoietin/Tie2 pathway and in
particular the Angiopoietin-2 (Ang-2) ligand which is critically involved in the destabilization of normal vasculature,
has been identified as one such target. The current study investigated the potential benefits of combining an Ang-2 tar-
geted therapy with small molecule VEGF targeted agents (Sunitinib, Cediranib) in a human renal cell carcinoma model.
The results showed that while both Ang-2 and VEGF interference on their own impaired tumor growth and new blood
vessel formation, the combination of agents that targeted both pathways resulted in significantly superior anti-tumor and
anti-angiogenic effects.
Keywords: Angiopoietin-2; Angiogenesis; Anti-Angiogenic Agent; Combination Therapy; Vascular Endothelial
Growth Factor
1. Introduction
Angiogenesis, the formation of new blood vessels from
pre-existing ones, is an important process in normal vas-
cular development and physiological conditions such as
wound healing, reproduction and the menstrual cycle [1].
The importance of angiogenesis not only in physiological
but in pathological conditions has been well established
[2]. A growing tumor cannot sustain its growth without
the initiation and continued maintenance of active an-
giogenesis [3,4]. The resulting vasculature is distinct
from normal vasculature both functionally and structur-
ally and such differences allow for the targeting of tumor
vasculature with limited effects on normal vasculature
[5,6].
Numerous clinical trials that seek to impair the induc-
tion of new blood vessels in tumors are ongoing [7,8].
Many agents targeting Vascular Endothelial Growth Fac-
tor (VEGF) are now commonly used in the clinic; parti-
cularly in diseases such as kidney cancer [9,10]. How-
ever, resistance to such therapies may occur [11-13],
likely due to the redundancy of signaling pathways in-
volved in the activation of sprouting angiogenesis [14].
The search for new or complementary targets in the an-
giogenic process to circumvent resistance and such thera-
pies is therefore being actively pursued [7,11,12].
The Angiopoietin/Tie2 pathway has been shown to be
important both in physiological and pathological angio-
genesis including tumor angiogenesis [15]. Briefly,
Ang-1 and Ang-2 are secreted proteins that interact with
the Tie2 receptor either in a paracrine (Ang-1) or auto-
crine (Ang-2) manner; Ang-1 is expressed and secreted
by peri-endothelial mural cells (smooth muscle cells,
pericytes) while Ang-2 is expressed and secreted by en-
dothelial cells [16]. Both angiopoietins bind the Tie2
receptor with similar affinities at the same site of the
IgG-like and EGF-like domains [17,18]. These ligands,
however, have opposing functions. Ang1-Tie2 signaling
*Financial Support: These investigations were supported in part by a
grant from the National Cancer Institute (Public Health Service Grant
CA089655) and National Institutes of Health (T32 Training Grant
5T32 CA009126-33).
Conflict of Interest: No conflict of interest.

Combined Ang-2 and VEGF Targeting Therapies in Renal Cell Carcinoma 2
controls vessel quiescence, while Ang2-Tie2 association
allows for vessel plasticity [19]. Elevated Ang-2 levels
have been associated with advanced disease, progression
and poor prognosis in several cancers including renal cell
carcinoma [20-22]. Ang-2 serum levels significantly in-
crease in patients compared to healthy individuals, and
patients with more advanced disease show significantly
higher levels of Ang-2 compared to patients with earlier
stage disease [23-27].
Angiogenesis can be considered to be a two-step proc-
ess: 1) the normal vasculature is destabilized by loosening
the endothelial and peri-endothelial cell contacts in the
vasculature (Angiopoietin/Tie2 pathway) at which point
2) pro-angiogenic factors such as VEGF activate the en-
dothelium to proliferate and form new vessels [28]. Cur-
rently the majority of FDA approved anti-angiogenic
agents target the second step of this process. VEGF sig-
naling interference has been investigated in particular but
inhibition of other angiogenesis associated signaling path-
ways is also being pursued [11]. In general, antiangio-
genic agents have been found to be complementary to
conventional cancer therapies, however, there are some
patients who do not respond or stop responding after
prolonged treatment with such agents [11-13]. In lieu of
this observation and given the role of the Angiopoie-
tin/Tie2 axis in angiogenesis, there has been growing
interest in anti-angiogenic treatment approaches that se-
lectively target both Ang-2 and VEGF [15,29-33].
The current study evaluated the combination of an in-
vestigational anti-Ang-2 monoclonal antibody with two
small molecule tyrosine kinase inhibitors against the
VEGF pathway, Sunitinib and Cediranib. The former is a
multikinase inhibitor that is FDA approved for kidney
cancer [34] while the latter is a VEGFR specific small
molecule inhibitor that is currently in clinical develop-
ment [35]. The effects of utilizing these agents alone or
in combination on tumor development and angiogenesis
initiation were evaluated in an aggressive human renal
cell carcinoma model.
2. Materials and Methods
2.1. Reagents
MECA-32 was purchased from BioLegend (San Diego,
CA), AlexaFluor 594 was purchased from Invitrogen
(Grand Island, NY). VectaShield mounting medium with
DAPI was purchased from Vector Labs Inc. (Burlingame,
CA). Tissue-Tek OCT Compound was purchased from
Sakura Finetek (Torrance, CA). 2-methylbutane was ob-
tained from Thermo Fisher Scientific (Waltham, MA).
2.2. Cell Culture
The human clear cell renal cell carcinoma cell line
Caki-2 was received as a gift from Dr. Susan Knox
(Stanford University). Caki-2 cells were grown in Dul-
becco’s modified minimum essential medium (DMEM,
Invitrogen) supplemented with 10% fetal bovine serum
(FBS, Invitrogen), 1% penicillin-streptomycin (Invitro-
gen), and 1% 200-mmol/L L-glutamine (Invitrogen).
Cells were maintained at 37˚C in a 5% CO2-incubator.
The cells were used between passages 2 and 10.
2.3. Drug Preparation
Anti-Angiopoietin-2 monoclonal antibody was kindly
provided by MedImmune, LLC. Stock solutions (5 mg/
ml) of the antibody were diluted to working concentra-
tions in sodium citrate buffer solution. Stock solutions
were kept at −80˚C and working concentrations at 4˚C.
Cediranib (AstraZeneca, Wilmington, DE) was stored at
4˚C and prepared fresh daily in 10% volume Tween 80
and 1M HEPES. Sunitinib (LC Laboratories, Woburn,
MA) was stored at −20˚C and prepared fresh daily in
stock and diluent buffers of citric acid monohydrate and
sodium citrate dihydrate at pH 6.8 and 3.2 respectively at
1:7 stock to diluent solution (~pH 3.3) and acidified to
pH 1.0. Sunitinib was dissolved, and the solution ad-
justed to pH 3.5.
2.4. Intradermal Assay
All in vivo procedures were conducted in agreement with
a protocol approved by the University of Florida Insti-
tutional Animal Care and Use Committee. Female athy-
mic nu/nu mice were injected intradermally with 105
Caki-2 cells (10 l volume) at four sites on the ventral
surface. Beginning the day prior to tumor cell injection,
mice were treated with 1) daily oral gavage of either
Sunitinib (10 mg/kg) or Cediranib (2 mg/kg), 2) IP
injection of anti-Ang-2 antibody (2 mg/kg) every 3 days
or 3) the combination of either Sunitinib or Cediranib
plus the anti-Ang-2 antibody. At the end of the treatment
(six days post tumor cell inoculation) the mice were then
euthanized. Tumors were measured using calipers and
tumor volumes (mm3) were calculated using the formula:
12
tumor volume6diameterdiameterheight


Skin flaps were then removed and vessels growing
into tumor nodules were counted using a Leica MZ16F
dissecting microscope with Leica KL 1500 LCD fiber
optic illuminator (Leica Microsystems Inc., Buffalo
Grove, IL) at 2.5 × original magnification (1-3). Images
were captured with a Retiga EXi Fast1394 digital CCD
camera (QImaging, British Columbia, Canada) and
OpenLab5 software (PerkinElmer Inc., Waltham, MA).
Statistical significance between control and treated
groups was determined using the Mann-Whitney U-Test
at p < 0.05.

Combined Ang-2 and VEGF Targeting Therapies in Renal Cell Carcinoma 3
2.5. Immunohistochemistry
Intradermal tumors were freshly frozen in OCT and me-
thylbutane and sectioned at 5 m thickness using a Leica
CM 3050S cryostat (Leica Microsystems Inc., Buffalo
Grove, IL). Sections were placed on superfrost plus gold
slides (Thermo Fisher Scientific Inc., Waltham, MA) and
kept at −80˚C until immunohistochemical staining. Tis-
sue sections were fixed with acetone for 10 min, blocked
in 2% normal horse serum in 1 × TBS, and incubated
overnight at 4˚C with MECA-32. Secondary antibody
AlexaFluor 594 was added onto slides for 1 hr. Tissue
sections were imaged with a Zeiss Axioplan 2 imaging
microscope (Carl Zeiss, Inc., Thornwood, NY) with
EXFO X-Cite 120 light source (Lumen Dynamics Group
Inc., Ontario, Canada). Images were taken with a Retiga
EXi Fast digital CCD camera (Qimaging, British Co-
lumbia, Canada) and processed using OpenLab5 soft-
ware (PerkinElmer Inc., Waltham, MA); Rhodamine for
MECA-32/AlexaFluor594 and DAPI filters were used.
Up to ten random fields were examined on each tumor
section and the number of vessels in each field was
counted using a 20 × objective. Statistical significance
between control and treated groups was determined using
the Mann-Whitney U-Test at p < 0.05.
3. Results
3.1. Treatments Targeting Both Ang-2 and
VEGF Result in Significantly Greater
Impairment of Tumor Growth than Either
Therapy Alone
Figure 1(a) shows that treatment with either the anti-
Ang-2 antibody or the VEGFR1-3 inhibitor Cediranib
resulted in significant reductions in tumor volumes com-
pared to those of untreated mice, 3.5- and 3.8-fold (p <
0.05) respectively. However, the combination of these
two therapies resulted in an even greater impairment of
tumor growth; at the end of the treatment period tumors
treated with the anti-Ang-2 antibody plus Cediranib were
~54.6-fold (p < 0.0001) smaller than untreated control
tumors. Furthermore, the tumors of mice with the com-
bination of agents were also significantly smaller than
those of mice treated solely with the anti-Ang-2 antibody
(15.6-fold, p < 0.0001) or Cediranib (14-fold, p < 0.01).
Similar results were seen when the anti-Ang-2 antibody
and the multikinase inhibitor Sunitinib were combined
(Figure 1(b)). Both agents administered individually im-
paired tumor growth; tumor volumes assessed at the end
of the treatment period were found to be 1.5-fold (p =
0.06) 4.6-fold (p < 0.0001) smaller that untreated control
tumors for anti-Ang-2 antibody and Sunitinib treatments
respectively. However, the combination of the two agents
once again led to a greater reduction in tumor volume
(15.8 fold, p < 0.0001) compared to control tumors as
(a) (b)
Figure 1. Effect of Ang-2 and VEGF treatment on tumor
growth. Mice were injected intradermally with Caki-2 renal
cell carcinoma cells and beginning the day prior to tumor
cell injection, were treated with (a) Cediranib (2 mg/kg), (b)
Sunitinib (10 mg/kg), ((a) and (b)) anti-Ang-2 antibody (2
mg/kg), or the combination of either (a) Cediranib plus the
anti-Ang-2 antibody or (b) Sunitinib plus the anti-Ang-2
antibody. Tumor volumes were assessed 6 days after tumor
cell inoculation. Line, median; bar 10 - 90 percentile; Con-
trol (0) (n = 20), anti-Ang-2 antibody (A) (n = 16),
Cediranib (C) (n = 16), Sunitinib (n = 16), anti-Ang-2 anti-
body + Cediranib (A + C) (n = 16), anti-Ang-2 antibody +
Sunitinib (A+S) (n = 20). *p < 0.05; **p < 0.01; ***p < 0.0001;
Mann-Whitney U-Test.
well as tumors in mice treated with only the anti-Ang-2
antibody (10.7-fold, p < 0.0001) or Sunitinib (3.4-fold, p <
0.0001).
3.2. Combined Ang-2 and VEGF Targeting
Significantly Impairs Tumor Cell
Induced Angiogenesis
To evaluate the effect of Ang-2 and VEGF directed the-
rapies administered alone or in combination on Caki-2
renal cell carcinoma cell induced angiogenesis an in-
tradermal angiogenesis assay was used (Figure 2). The
results showed that while the anti-Ang-2 antibody and
Cediranib treatments individually reduced the number of
vessels growing into the tumor nodules by 1.6- (p < 0.01)
and 1.5-fold (p < 0.01) respectively, the combination
treatment led to an even greater reduction in blood vessel
number (2.7-fold (p < 0.0001) compared to control)
(Figure 2(a)). Similar results were seen when the anti-
Ang-2 antibody was combined with Sunitinib (Figure
2(b)). While the anti-Ang-2 antibody and Sunitinib each
decreased the number of vessels induced by Caki-2 tu-
mor cells (1.7- and 1.8-fold (p < 0.0001) respectively),
combining the therapies led to a significantly greater
reduction in the number of blood vessels induced than
was achieved with either agent alone (3.5-fold (p <

Combined Ang-2 and VEGF Targeting Therapies in Renal Cell Carcinoma 4
0.0001), compared to control tumors). Furthermore, in
both studies (Figures 2(a) and (b)), the combination
treat- ment was significantly more effective at reducing
the formation of tumor cell induced blood vessels than
either the anti-Ang-2 antibody or the VEGF directed
therapy when used alone.
The impact of VEGF and Ang-2 directed therapy on
the vasculature within Caki-2 tumors was evaluated us-
ing immunohistochemistry (Figure 3). The results showed
that treatment with the anti-Ang-2 antibody or Cediranib
reduced the number of blood vessels within the tumor
nodules 2.5- and 1.4-fold (p < 0.01) respectively and the
combination treatment led to a 3.5-fold (p < 0.01) reduc-
tion in intratumor blood vessels (Figure 3(a)). A similar
analysis performed on the tumors of mice treated with
the anti-Ang-2 antibody alone or in combination with
Sunitinib (Figure 3(b)) showed that the combination
reduced the number of blood vessels detected in the tu-
mor nodules 3.7-fold (p < 0.05) compared to the 2- (p <
0.05) and 1.7-fold (p = 0.057) decrease in tumor blood
(a)
(b)
Figure 2. Effect of Ang-2 and VEGF treatments on blood
vessels induction by Caki-2 tumor cells. Mice were treated
as described in Figure 1 and the number of peripheral ves-
sels growing into the tumor nodules was counted 6 days
after tumor cell inoculation. Line, median; bar 10-90 per-
centile; Control (0) (n = 16), anti-Ang-2 antibody (A) (n =
16), Cediranib (C) (n= 16), Sunitinib (n = 12), anti-Ang-2
antibody + Cediranib (A + C) (n=16), anti-Ang-2 antibody +
Sunitinib (A + S) (n = 16); **p < 0.01; ***p < 0.0001; Mann-
Whitney U-Test. Representative images were taken at 2.5×
magnification.
(a)
(b)
Figure 3. Effect of Ang-2 and VEGF treatments on tumor
core blood vessels assessed by immunohistochemistry. Mice
were treated as described in Figure 1. Six days post tumor
cell inoculation frozen tumor sections were stained with
MECA-32 and up to 10 random fields/tumor were evalu-
ated. Line, median number of vessels; bar 10 - 90 percentile;
Control (0) (n = 4 - 6), anti-Ang-2 antibody (A) (n = 4 - 6),
Cediranib (C) (n = 6), Sunitinib (n = 4), anti-Ang-2 antibody
+ Cediranib (A + C) (n = 6), anti-Ang-2 antibody + Sunit-
inib (A + S) (n = 4) *p < 0.05; **p < 0.01; Mann-Whitney
U-Test. Representative images of the median of each group
are shown. Images taken with a Zeiss Axioplan Imaging2
microscope with a 20× objective; scale bar = 140 μm.
vessels noted in the tumors of mice treated with the
anti-Ang-2 antibody or Sunitinib alone.
4. Discussion
Angiogenesis is an important feature of tumor growth
that has been considered to be a potential target for can-
cer therapy for decades. Angiogenesis can be separated
into two main events: 1) the destabilization of normal
vasculature, or the loosening of endothelial and peri-
endothelial cell contacts, and 2) the activation of the en-
dothelium to proliferate and form new vessels. The An-
giopoietin/Tie2 axis is responsible for the first step in
angiogenesis or the vessel destabilization while pro-an-
giogenic factors such as VEGF activate the endothelial
cells. Currently FDA approved anti-angiogenic agents to
target the VEGF pathway and has been shown to com-
plement conventional therapies such as chemotherapy
[36], however the issues of lack of patient response and
tumor rebound due to acquired resistance have raised

Combined Ang-2 and VEGF Targeting Therapies in Renal Cell Carcinoma 5
concerns in the clinic [11-13].
Another strategy to interfere with tumor angiogenesis
is to target the Angiopoietin/Tie2 pathway [15]. Ang-2 is
abundantly present in many tumors and its expression
appears to correlate with poor disease prognosis [22]. It
is also conceivable that Ang-2 and VEGF targeting may
be complementary and that Ang-2/Tie2 targeting may
circumvent patient resistance to VEGF targeting therapy.
While endothelial cell activation to form sprouts occurs
in response to a variety of pro-angiogenic factors in addi-
tion to VEGF, the initial destabilization of vasculature
through the Ang-2/Tie2 axis is currently known as a non-
redundant pathway. In the current study the effects of
inhibiting these two pathways through the combination
of VEGF targeted agents and an anti-Ang-2 monoclonal
antibody were evaluated in an aggressive and highly
vascularized, VHL mutated, human renal cell carcinoma
model. Two different VEGF targeted agents were evalu-
ated, the small molecule multi tyrosine kinase inhibitor
Sunitinib that is FDA approved for treatment of metas-
tatic kidney cancer and the VEGFR specific small mol
cule inhibitor Cediranib that is in clinical development
for a variety of solid tumors.
The results of the present study support the notion that
the efficacies of Ang-2 and VEGF targeted therapies may
be complementary. When either Cediranib or Sunitinib
was combined with the anti-Ang-2 antibody, the combi-
nation treatment showed superior anti-tumor and anti-
angiogenic effects compared to any of the agents used on
their own (Figures 1 and 2). The enhanced treatment
efficacy was likely not only a consequence of a reduction
in the ability of the renal cells to induce the initiation of
vessel growth toward the tumor mass (Figure 2), but also
a consequence of an impairment of vascular development
within the tumors themselves (Figure 3). Taking these
findings together lends additional support to therapeutic
intervention strategies seeking to maximize antitumor ef-
ficacy through combination treatments directed at the
multiple components comprising tumor angiogenesis.
5. Acknowledgements
The authors thank MedImmune, LLC for kindly provi-
ding the anti-Ang-2 monoclonal antibody and Marda
Jorgensen (University of Florida Tissue Core) for assis-
tance with the immunohistochemistry studies.
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