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roAntiangiogenic therapy, Caki-1 renal cell carcinoma, Cediranib (Recentin, AZD2171).
INTRODUCTION
Neovascularization, a rare event in adults confined almost ex-
clusively to the female reproduction system, is an essential
feature of solid tumors (1). It is widely accepted that contin-
ued tumor growth depends on nutrient supply from a network
of microvessels that may originate from angiogenesis, vascu-
logenesis, vessel intussusception, vascular mimicry, or any
combination thereof (2, 3). Angiogenesis involves the migra-
tion and proliferation of endothelial cells from existing ves-
sels (4) triggered by the release of such stimulators as the
endothelial cell–specific mitogen vascular endothelial
growth factor (VEGF), which is secreted as a 45-kDa homo
dimer protein (5). The soluble isoform VEGF165 is com-
monly expressed in a wide variety of human and animal tu-
mors (6) and currently is believed to be the key mediator of
tumor angiogenesis (5). In patients, VEGF overexpression
has been documented in most types of cancers, and this over-
expression often has been associated with poor prognosis (7,
8). This has made the VEGF pathway an attractive target for
therapeutic interventions in the treatment of patients with
cancer.
Renal cell carcinoma (RCC) is the most common malig-
nancy of the kidney and accounts for about 2% of all adult
malignancies (9, 10). Unless discovered at an early stage,
RCC has a very unfavorable treatment outcome after conven-
tional therapeutic interventions and is fatal in nearly 80% of
its patients (10). The RCC is a highly vascularized neoplasm
(11), and increased serum/urine VEGF levels have been asso-
ciated with malignant progression and poor treatment out-
come (12, 13). Consequently, RCC may be an excellent
site to investigate VEGF-targeted antiangiogenic therapies.
A number of approaches have been devised to interfere
with the VEGF signaling cascade (14, 15); targeting the
VEGF protein- or receptor(s) (VEGFR[s])-associated tyro-
sine kinases has emerged as a key therapeutic strategy.
This approach has recently led to clinical validation of the
value of antiangiogenic therapy (16, 17) and the first approval
of the antiangiogenic therapeutics bevacizumab (Avastin,
Reprint requests to: Dietmar W. Siemann, Ph.D., Department of
Radiation Oncology, University of Florida, 2000 S.W. Archer
Road, Gainesville, FL 32610. Tel: (352) 265-0287; Fax: (352)
265-0759; E-mail: siemadw@ufl.edu
Supported in part by a grant from AstraZeneca and US Public
Health Service Grant CA089655 from the U.S. National Cancer
Institute.
Conflict of interest: These studies were supported in part by a re-
search grant to Dr. Siemann from AstraZeneca. Dr. Juliane Ju¨rgen-
smeier is an employee of AstraZeneca.
Acknowledgments—The authors thank S. Lepler and C. Pampo for
excellent technical support.
Received June 30, 2008, and in revised form Sept 3, 2008.
Accepted for publication Oct 20, 2008.
897BIOLOGY CONTRIBUTION
THE VASCULAR ENDOTHELIAL GRO
KINASE INHIBITOR CEDIRANIB (RECE
CELL FUNCTION AND GROWTH OF H
DIETMAR W. SIEMANN, PH.D.,* W. D. BRAZELL
*Department of Radiation Oncology, University of Florida, Gai
Macclesfield, Ches
Purpose: The goal of this study was to examine the ther
(VEGF) signaling inhibitor cediranib in a human mod
Methods and Materials: The effects of cediranib trea
migration, and tube formation), as well as in vivo angio
Results: In vitro, cediranib significantly impaired the pro
ity to form tubes, but had no effect on the proliferati
reduced Caki-1 tumor cell–induced angiogenesis, redu
tumor xenografts.
Conclusions: The present results are consistent with t
indirect (i.e., antiangiogenic) antitumor effect, rather
suggest that inhibition of VEGF signaling with cedi
 2009 Elsevier Inc.
doi:10.1016/j.ijTH FACTOR RECEPTOR-2 TYROSINE
IN; AZD2171) INHIBITS ENDOTHELIAL
MAN RENALTUMOR XENOGRAFTS
PH.D.,* AND JULIANE M. JU¨RGENSMEIER, PH.D.y
sville, FL; and yCancer Bioscience, AstraZeneca, Alderley Park,
e, United Kingdom
eutic potential of the vascular endothelial growth factor
f renal cell carcinoma (Caki-1).
ent on in vitro endothelial cell function (proliferation,
nesis and tumor growth, were determined.
eration andmigration of endothelial cells and their abil-
of Caki-1 tumor cells. In vivo, cediranib significantly
d tumor perfusion, and inhibited the growth of Caki-1
notion that inhibition of VEGF signaling leads to an
n a direct effect on tumor cells. These results further
nib may impair the growth of renal cell carcinoma.
Int. J. Radiation Oncology Biol. Phys., Vol. 73, No. 3, pp. 897–903, 2009
Copyright  2009 Elsevier Inc.
Printed in the USA. All rights reserved
0360-3016/09/$–see front matter
bp.2008.10.031

Genentech, San Francisco, CA) and sunitinib (Sutent, Pfizer
Inc., New York, NY; SU11248). Sunitinib and sorafenib
(Nexavar, Bayer Pharmaceuticals, West Haven, CT), multi-
targeted receptor tyrosine kinase inhibitors, have now been
approved by the Food and Drug Administration (FDA), as
well as in the European Union, for the treatment of patients
with RCC. The goal of the present study was to evaluate
the efficacy of cediranib (Recentin; AstraZeneca Pharmaceu-
ticals, Macclesfield, UK), an oral, highly potent, and selective
VEGF signaling inhibitor of VEGFR-1, -2, and -3 tyrosine
kinases (18) in a human RCC (Caki-1) model.
METHODS AND MATERIALS
Cell culture
The clear-cell RCC cell line Caki-1 was originally received as
a gift from Dr. Susan Knox (Stanford University, Palo Alto, CA).
Caki-1 cells were grown in Dulbecco’s modified minimum essential
medium (D-MEM, Invitrogen, Grand Island, NY) supplemented
with 10% fetal bovine serum (Invitrogen, Grand Island, NY), 1%
penicillin-streptomycin (Invitrogen), and 1% 200-mmol/L L-gluta-
mine (Invitrogen). Human microvascular endothelial cells from
the lung (HMVEC-L) were obtained from Clonetics (San Diego,
CA). The HMVEC-L cells were grown in EBM-2-MV (Clonetics)
supplemented with 5% fetal bovine serum.
Drug preparation
Cediranib was provided by AstraZeneca Pharmaceuticals. For in
vitro investigations, 10 mM of stock solutions prepared in dimethyl
sulfoxide were serially diluted in sterile saline such that dimethyl
sulfoxide exposure in cell cultures was less than 0.1%. For in vivo
studies, stock solutions of drug (10 mM) were prepared by suspend-
ing the agent in 10% (vol/vol) Tween-80 (Invitrogen, Grand Island,
NY) and N-2-hydroxyethylpiperazine propanesulfonic acid. Work-
ing dilutions were made by serial dilution of the stock solution in
sterile saline. All drug preparations were kept refrigerated and in
the dark and used within 1 week of preparation.
In vitro cell growth
The HMVEC-L or Caki-1 cells (1  104) were seeded in triplicate
into 60-mm tissue culture dishes and allowed to attach overnight.
The next day, drug was added to the medium at the appropriate
dose as a solution of 10 ml/ml of medium. At various times later,
plates were trypsinized and cells were counted.
Endothelial cell migration
The HMVEC-L cells (1  104) were seeded in sextuplet into six-
well plates and allowed to reach confluence. A scrape 2-mm wide
was then made across the entire length of each well. Cediranib
was added to each well at the appropriate dose in a volume of 10
ml/ml. Forty-eight hours later, cells were stained with crystal violet
and viewed at original magnification 5, and the number of endo-
thelial cells that had migrated into the scraped area was determined.
Endothelial cell tube formation
The HMVEC-L cells (6–8  104) were plated into 24-well dishes
precoated with 200 ml of matrigel. Photos of endothelial tubes at
original magnification 5 were obtained 24 hours after plating the
898 I. J. Radiation Oncology d Biology d Physicsendothelial cells.Caki-1 xenografts
Tumors were initiated by implanting (1  106) Caki-1 tumor cells
in the left hind calf muscle of 6–8-week-old female athymic nude
mice (NCR nu/nu). All mice were provided sterilized food and water
ad libitum and housed in a barrier facility with 12-hour light and
dark cycles. All procedures were conducted at the University of
Florida, Gainesville, FL, according to guidelines laid out by the In-
stitutional Animal Care and Use Committee. When tumors reached
approximately 200 mm3, the animals were randomly assigned to the
various treatment groups.
In vivo drug administration
Cediranib (6 mg/kg/d) was administered by means of oral gavage
for a 2-week period (Monday to Friday) after tumors reached a vol-
ume of 200 mm3.
Intradermal angiogenesis assay
Caki-1 cells (1  105) were inoculated intradermally in a volume
of 10 ml at four sites on the ventral surface of nude mice. One drop of
0.4% trypan blue was added to the cell suspension, making it lightly
colored, simplifying subsequent location of the sites of injection.
Three days later, the mice were euthanized, the skin was carefully
separated from the underlying muscle, and the number of blood ves-
sels intersecting each inoculate was counted by using a dissecting
microscope. The resultant data for each treatment group were pooled
for statistical analysis (Wilcoxon’s rank-sum test). Statistical signif-
icance at p < 0.05 was used.
Vessel density: CD31 immunohistochemistry
Frozen sections of tumors were cut on a cryostat, air dried, and
fixed in acetone/methanol at 4C for 10 minutes. Tumor microves-
sels were stained using a mouse monoclonal antibody to the CD31
(PECAM-1) antigen found on endothelial cells (Beckman Coulter,
Brea, CA), applied overnight at 4C at a dilution of 1:50. A second-
ary antibody conjugated with Cy3 (Jackson ImmunoResearch Lab-
oratories, Inc., West Grove, PA) was applied for 1 hour at room
temperature. Staining was followed by standard washing, then slides
were allowed to air dry before storage at 4C.
Tumor perfusion, patent blood vessels, Hoechst-33342
The fluorescent dye Hoechst-33342 (bisBenzimide; Sigma, Saint
Louis, MO) was prepared in 0.9% sterile saline immediately before
intravenous administration (40 mg/kg) (18). One minute after
Hoechst-33342 injection, the mice were euthanized and the tumors
were resected and immediately immersed in liquid nitrogen for sub-
sequent frozen sectioning. The sections were studied under UV illu-
mination by using a fluorescent microscope. Blood vessel outlines
were identified by the surrounding halo of fluorescent Hoechst-
33342–labeled cells (19).
Vessel counts
For each tumor sample, 10-mm cryostat sections were cut at three
different levels between one pole and the equatorial plane. Vessel
counts were performed by using a Chalkley point array for ran-
dom-sample analysis (20). Briefly, three sections were cut per tumor
and 10 areas were viewed (at objective magnification 10) per sec-
tion. A 25-point Chalkley grid was positioned over the field of view,
and any points within the fluorescent areas were scored as positive.
Data from three controls and six tumors from cediranib-treated mice
Volume 73, Number 3, 2009were pooled and presented.