Antiangiogenic gene therapy: Disruption of neovascular networks mediated by inducible caspase-9 delivered with a transcriptionally targeted adenoviral vector

Article (PDF Available)inGene Therapy 12(4):320-9 · March 2005with23 Reads
DOI: 10.1038/sj.gt.3302306 · Source: PubMed
Abstract
The activation of an inducible caspase (iCaspase-9) mediates apoptosis of neovascular endothelial cells, and overcomes the prosurvival effect of vascular endothelial growth factor or basic fibroblast growth factor. The potential utilization of direct activation of caspases as an antiangiogenic strategy for treatment of angiogenesis-dependent diseases (eg cancer) requires expression of the inducible caspase primarily in the tumor endothelium. The objective of this work was to develop and characterize a transcriptionally targeted adenoviral vector that mediates expression of iCaspase-9 specifically in neovascular endothelial cells. We observed that adenoviral vectors containing the human VEGFR2 promoter induced reporter gene expression primarily in proliferating human dermal microvascular endothelial cells (HDMEC). HDMEC transduced with recombinant adenoviral vectors containing iCaspase-9 under regulation of the VEGFR2 promoter (Ad-hVEGFR2-iCaspase-9) and exposed to a cell-permeable dimerizer drug (AP20187), presented higher caspase-3 activity and apoptosis than controls (P < or = 0.05). Using the SCID Mouse Model of Human Angiogenesis, we observed that local delivery of Ad-hVEGFR2-iCaspase-9 followed by intraperitoneal injection of AP20187 resulted in endothelial cell apoptosis and local ablation of microvessels. We believe that this constitutes the first report of a transcriptionally targeted antiangiogenic adenoviral vector that mediates neovascular disruption upon activation of a caspase-based artificial death switch.
RESEARCH ARTICLE
Antiangiogenic gene therapy: disruption
of neovascular networks mediated by inducible
caspase-9 delivered with a transcriptionally
targeted adenoviral vector
W Song
1
, Q Sun
1
, Z Dong
1
, DM Spencer
2
,GNu
´
n
˜
ez
3
and JE No
¨
r
1
1
Angiogenesis Research Laboratory, Department of Restorative Sciences, University of Michigan School of Dentistry, Ann Arbor, MI,
USA;
2
Department of Immunology, Baylor College of Medicine, Houston, TX, USA; and
3
Department of Pathology, University of
Michigan School of Medicine, Ann Arbor, MI, USA
The activation of an inducible caspase (iCaspase-9) med-
iates apoptosis of neovascular endothelial cells, and over-
comes the prosurvival effect of vascular endothelial growth
factor or basic fibroblast growth factor. The potential
utilization of direct activation of caspases as an antiangio-
genic strategy for treatment of angiogenesis-dependent
diseases (eg cancer) requires expression of the inducible
caspase primarily in the tumor endothelium. The objective of
this work was to develop and characterize a transcriptionally
targeted adenoviral vector that mediates expression of
iCaspase-9 specifically in neovascular endothelial cells. We
observed that adenoviral vectors containing the human
VEGFR2 promoter induced reporter gene expression pri-
marily in proliferating human dermal microvascular endothe-
lial cells (HDMEC). HDMEC transduced with recombinant
adenoviral vectors containing iCaspase-9 under regulation of
the VEGFR2 promoter (Ad-hVEGFR2-iCaspase-9) and
exposed to a cell-permeable dimerizer drug (AP20187),
presented higher caspase-3 activity and apoptosis than
controls (Pp0.05). Using the SCID Mouse Model of Human
Angiogenesis, we observed that local delivery of Ad-
hVEGFR2-iCaspase-9 followed by intraperitoneal injection
of AP20187 resulted in endothelial cell apoptosis and local
ablation of microvessels. We believe that this constitutes the
first report of a transcriptionally targeted antiangiogenic
adenoviral vector that mediates neovascular disruption upon
activation of a caspase-based artificial death switch.
Gene Therapy (2005) 12, 320–329. doi:10.1038/sj.gt.3302306
Published online 23 December 2004
Keywords: angiogenesis; neovascularization; apoptosis; suicide gene; cell death
Introduction
Angiogenesis is a requirement for the growth of solid
tumors.
1
However, it is becoming increasingly evident
that each tumor cell uses a distinct combination of
proangiogenic factors to acquire and maintain their own
microvascular network.
2–4
Vascular endothelial growth
factor (VEGF) and basic fibroblast growth factor (bFGF)
are two of the most predominant proangiogenic factors
in tumors, and have been shown to function as strong
inducers of endothelial cell survival.
5–8
We have recently
demonstrated that the direct activation of caspase-9
induces apoptosis of proliferating endothelial cells, that
the endothelial cell survival factors VEGF and bFGF do
not protect these cells against apoptosis, and that
activated iCaspase-9 is sufficient to disrupt blood vessels
in vivo.
9
These experiments established proof-of-princi-
ple data to support the concept that direct activation of
a proapoptotic caspase in endothelial cells might be
beneficial for the treatment of solid tumors.
9
However,
the utilization of a caspase-based artificial death switch
as an antiangiogenic therapeutic strategy would require
that the therapeutic gene be expressed only in neovas-
cular endothelial cells, and not expressed in mature
blood vessels.
Recombinant adenoviruses have been used for gene
therapy because of their stability and infection effi-
ciency in vivo, and relative low risk for secondary
mutagenesis.
10,11
Several strategies have been devel-
oped to enhance the safety of adenovirus-mediated
gene therapy. First, one can design vectors that
incorporate cell- or tissue-specific promoters and
enhancers to limit expression of the therapeutic
gene.
12,13
Second, a targeted adenoviral vector can be
developed by removing its interaction with native
receptors and introducing a cell- or tissue-specific
ligand to the virus.
14
The use of transcriptional
and/or receptor-targeting strategies may allow for
the use of lower viral titers, and perhaps reduce the
chance for an acute immune response. And third, the
useofasuicidegenesuchasaninduciblecaspase
(iCaspase) may be considered a ‘safety switch’ for gene
therapy since the infected cells will be induced to
undergo apoptosis.
15–17
Tissue specificity of an adenoviral vector to neovas-
cular endothelial cells might be increased by the
Received 12 January 2004; accepted 12 April 2004; published online
23 December 2004
Correspondence: Dr JE No
¨
r, Angiogenesis Research Laboratory, Uni-
versity of Michigan School of Dentistry, 1011 N University, Rm. 2309,
Ann Arbor, MI 48109-1078, USA
Gene Therapy (2005) 12, 320–329
&
2005 Nature Publishing Group All rights reserved 0969-7128/05
$
30.00
www.nature.com/gt
utilization of the promoter and/or enhancer of vascular
endothelial growth factor receptor 2 (VEGFR2), also
called KDR or Flk-1. VEGFR2 is poorly expressed in
mature blood vessels, however, its expression is upre-
gulated in the proliferating endothelium of several
tumors.
18–20
It has been reported that a 939-bp mouse
VEGFR2 promoter fragment and a 430-bp mouse
enhancer fragment mediated strong endothelium-speci-
fic reporter gene expression in mammary adenocarcino-
mas grown in transgenic mice, while reporter gene
expression was absent from most blood vessels in normal
tissues.
20
Furthermore, a 494 bp fragment (226 to +268)
of the human VEGFR2 promoter was shown to be
capable of driving expression primarily in endothelial
cells following retroviral delivery.
21,22
Here, we tested
both the human and the mouse VEGFR2 transcriptional
elements to identify which one mediates stronger and
more specific expression of reporter genes transduced
with an adenoviral vector into neovascular endothelial
cells.
‘Artificial death switches’ based on chemically in-
duced dimerization (CID) of caspases with lipid-perme-
able and nontoxic dimerizer drugs have been
described.
13,17,23,24
In this approach, a protein can be
fused to a CID-binding domain that will mediate
noncovalent crosslinking by high-affinity interactions
with a dimerizer compound.
15
When the catalytic
domain of caspase-9 is fused to one or more CID-binding
domains, the resulting fusion protein (inducible caspase-
9, named iCaspase-9) can be activated with a dimerizer
compound (eg AP20187) and initiate an irreversible
signaling pathway that results in cell death.
9,13,17,24
In a previous report, we have characterized a stable
endothelial cell line expressing iCaspase-9, and demon-
strated that activation of this caspase-based artificial
death switch was sufficient to induce apoptosis.
9
Here,
we used a transcriptional targeting strategy to generate
an adenoviral vector containing the human VEGFR2
promoter and showed that it drives expression of
reporter genes primarily in neovascular endothelial cells.
We showed that activation of adenovirally delivered
iCaspase-9 with the dimerizer compound AP20187
activated endogenous caspase-3 and induced endothelial
cell apoptosis in vitro. Furthermore, we showed that
activation of iCaspase-9 with AP20187 was sufficient to
induce apoptosis of neovascular endothelial cells, and to
cause a marked decrease in tissue microvessel density
in vivo. Here, we combined two of the three strategies
described above (ie transcriptional targeting of the vector
and use of an artificial death switch) to develop a novel
adenovirus-mediated antiangiogenic strategy, and de-
monstrated its efficacy in vivo.
Results
Adenoviruses containing the human VEGFR2
promoter mediated expression of reporter genes
primarily in proliferating endothelial cells
We decided to target transcriptionally our adenoviral
vectors with the regulatory elements of VEGFR2, because
this receptor is expressed primarily in proliferating
endothelial cells.
20,25
We first compared the intensity of
reporter gene expression under the regulation of either
human or mouse VEGFR2 transcriptional elements
(Figure 1a) after transient transfection of the constructs
into proliferating human dermal microvascular endothe-
lial cells (HDMEC). We observed that the human
VEGFR2 promoter construct allowed for higher reporter
gene expression levels in proliferating HDMEC, as
Figure 1 Human VEGFR2 promoter mediates preferential reporter gene
expression in human endothelial cells. (a) Schematic diagram depicting the
luciferase reporter constructs used in this study. (b) Luciferase gene
expression mediated by human VEGFR2 promoter, mouse VEGFR2
promoter, or mouse VEGFR2 promoter and mouse VEGFR2 enhancer in
HDMECs. Constructs were transiently transfected into HDMECs with
Lipofectin, and luciferase activity was evaluated after 24–72 h. (c) The
specificity of expression mediated by human VEGFR2 promoter in primary
human endothelial cells was evaluated by transfecting the pGL2-
hVEGFR2 promoter-Luc into HDMEC, human dermal fibroblasts
(HDF), mouse embryonic fibroblasts (MEF), NIH3T3, two oral squamous
cell carcinoma cell lines (UM-SCC-17B and OSCC-3), and a breast
carcinoma cell line (SK-BR-3) with Lipofectin. Data is expressed as fold
induction of expression mediated by pGL2-hVEGFR2 promoter-Luc
overexpression mediated by pGL2-Basic (promoter-less control), from
triplicate samples per condition. HDMEC transfected with pGL2-Control
(SV40 promoter and enhancer driving luciferase expression) were the
positive controls.
Caspase-mediated antiangiogenic gene therapy
W Song et al
321
Gene Therapy
compared with either mouse VEGFR2 promoter alone or
mouse VEGFR2 promoter and enhancer together (Figure
1b). To further evaluate the tissue specificity of this
transcriptional element, we tested the ability of the
human VEGFR2 promoter to drive specific expression in
proliferating HDMEC by transfecting the reporter con-
structs into a panel of cell lines. We observed that the
activity of the human VEGFR2 promoter is 98-fold higher
than a promoter-less control plasmid (ie pGL2-Basic) in
HDMEC, and comparable with the activity observed
with the nonspecific SV40 promoter from the pGL2-
Control plasmid (Figure 1c). In contrast, the activity of
the human VEGFR2 promoter was minimal in all other
cell lines evaluated, including 1.6-fold induction (human
dermal fibroblasts), 1.6-fold induction (mouse embryonic
fibroblasts), 7.0-fold induction (breast cancer cell line,
SK-BR-3), and 2.3- and 3.8-fold induction (squamous cell
carcinoma cell lines, UM-SCC17B and OSCC-3, respec-
tively) (Figure 1c). Taken together, these data demon-
strated that the human VEGFR2 promoter was able to
drive strong and specific expression in proliferating
HDMEC in vitro, and suggested that this promoter might
be the basis of a viable strategy to build transcriptionally
targeted antiangiogenic adenoviral vectors.
Therefore, we designed and characterized three
recombinant adenoviruses to evaluate the expression
patterns of a reporter gene (ie LacZ) in proliferating
endothelial cells (Figure 2a). We observed that the
human VEGFR2 promoter mediated relatively strong
LacZ expression in HDMEC, which was comparable to
the expression observed when the nonspecific CMV
promoter was used (Figure 2b). Importantly, VEGFR2-
driven expression retained specificity to neovascular
endothelial cells, while CMV was clearly nonspecific
(Figure 3). Further, we observed that the human VEGFR2
promoter-driven expression of LacZ was relatively long
lasting and significantly higher than baseline even 14
days after infection in vitro (Figure 2c).
To confirm that the recombinant adenoviruses con-
structed with human VEGFR2 promoter retained a
similar cell-type specificity of reporter gene expression
that we obtained with our transient transfection experi-
ments, we transduced HDMEC, human dermal fibro-
blasts, and two human squamous cell carcinoma cell
lines (ie UM-SCC-17B and OSCC-3) and evaluated LacZ
expression in vitro and in vivo. With recombinant Ad-
hVEGFR2-LacZ adenoviruses, we observed staining of
proliferating HDMEC but not staining of the remaining
cell types evaluated here (Figure 3). In contrast, all cell
lines expressed LacZ when the CMV promoter was used
(Figure 3).
To evaluate if adenovirally transduced endothelial
cells retained their ability to differentiate into mature and
functional human blood vessels in mice, we transduced
HDMEC in vitro with Ad-hVEGFR2-LacZ (or promoter-
less, Ad-LacZ) 48 h prior to transplantation, seeded the
cells in biodegradable scaffolds, and implanted them
subcutaneously in immunodeficient mice using the SCID
Mouse Model of Human Angiogenesis.
9,26
At 14 days
after implantation, we observed that the blood vessels
inside the scaffolds were positive for b-galactosidase
(b-gal), which demonstrates that they were lined with the
human endothelial cells that we implanted, and that
adenoviral transduction of the cells did not block their
ability to differentiate into blood vessels in vivo (Figure
4a–c). Interestingly, the endothelial cells lining functional
blood vessels inside the scaffold stained positive for
b-gal, but the blood vessels in the fascia surrounding the
implants were not stained (Figure 4b). These findings
demonstrated that the staining is specific to transduced
endothelial cells. Transduction of the endothelial
cells with promoter-less (Ad-LacZ) adenoviral vector
did not induce detectable staining of tissue sections
(Figure 4d).
To further evaluate the practical applicability of this
system under more physiological conditions, we per-
formed experiments in which we first implanted
untransduced HDMEC into the scaffolds, waited for 14
days, and then injected the adenoviruses locally to
Figure 2 Adenoviruses containing the VEGFR2 promoter mediated
expression of reporter genes at a level comparable to the nonspecific
CMV promoter. (a) Schematic diagram depicting the LacZ reporter
constructs used in this study. (b) b-Gal activity mediated by human
VEGFR2 or controls in endothelial cells. HDMEC were transduced with
adenoviral vectors, and b-gal activity was evaluated after 2 days. (c) b-Gal
activity mediated by human VEGFR2, 0–14 days after infection. Data
represent mean values (7s.d.) of triplicate samples per condition.
Caspase-mediated antiangiogenic gene therapy
W Song et al
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Gene Therapy
evaluate the specificity of human VEGFR2 promoter-
driven expression in vivo. We observed that Ad-
hVEGFR2-LacZ allowed for endothelial cell-specific
expression under the experimental conditions described
here, since only the cells lining blood vessels were
stained positively for b-gal (Figure 4e). Delivery of
promoter-less control adenoviruses did not allow for
observable staining of the tissue sections (Figure 4f).
These data demonstrated that the human VEGFR2
promoter was effective in inducing endothelial cell-
specific expression in vivo, and served as the basis for the
development of the transcriptionally targeted adenoviral
vectors for endothelial cell-specific delivery of iCaspase-9
described below.
Expression and dimerization-induced processing of
adenovirally transduced iCaspase-9 in proliferating
endothelial cells
To begin the characterization of the recombinant adeno-
viruses that expressed iCaspase-9 under the human
VEGFR2 promoter, we performed a dose–response
experiment and observed that increasing the multiplicity
of infection (M.O.I.) mediated increasing expression
levels of iCaspase-9 up to a M.O.I. of 200 (Figure 5a).
We observed that 250 M.O.I. or more was inherently
cytotoxic to primary HDMEC with our adenoviruses
(unpublished observations); therefore, we limited the
maximum M.O.I. to 200 in all experiments included here.
Figure 3 Adenoviruses containing the human VEGFR2 promoter mediated expression of reporter genes primarily in proliferating endothelial cells in vitro.
Representative photomicrographs depicting LacZ staining mediated by human VEGFR2 promoter, CMV, or promoter-less controls. HDMEC, HDF,
UMSCC-17B, or OSCC-3 cells were transduced with the adenoviral vectors described above, and b-gal activity was evaluated after 2 days.
Figure 4 Adenoviruses containing the human VEGFR2 promoter mediated expression of reporter genes primarily in microvascular endothelial cells in
vivo. Representative photomicrographs depicting LacZ staining mediated by Ad-hVEGFR2-LacZ or Ad-LacZ. (a) Lower magnification view ( 100) of
implant populated with HDMEC transduced with Ad-hVEGFR2-LacZ before implantation. (b) Higher magnification view ( 400) of implant (and
surrounding tissues) populated with HDMEC transduced with Ad-hVEGFR2-LacZ before implantation. Arrows point to microvessels lined with HDMEC
transduced with Ad-hVEGFR2-LacZ before implantation (in vitro), and arrowheads point to host microvessels. (c) Representative photomicrograph of
implant containing HDMEC transduced with Ad-hVEGFR2-LacZ, or (d) transduced with Ad-LacZ. (e) Representative photomicrograph of implant
injected with Ad-hVEGFR2-LacZ, or (f) with Ad-LacZ 14 days after implantation in the SCID mouse. (e, f) Each implant was injected in vivo with
5 10
10
viral particles.
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In a time course experiment, we observed that 48 h after
infection, the expression of iCaspase-9 reached its
maximum level and remained at a similar expression
level for at least 96 h in vitro (Figure 5b). Notably, in the
absence of dimerizer drug, there was minimal detectable
processing of iCaspase-9 (not shown).
To determine if adenovirally transduced iCaspase-9 is
processed upon chemically induced dimerization, we
treated HDMEC infected with 20 or 200 M.O.I. Ad-
iCaspase-9 (promoter-less) or Ad-hVEGFR2-iCaspase-9
with the dimerizer drug AP20187.
27
We observed that the
chimeric iCaspase-9 is processed in an AP20187-depen-
dent manner (Figure 5c), while no significant changes
were observed in the expression levels of endogenous
caspase-9 (Figure 5c). To confirm the specificity of
iCaspase-9 expression mediated by Ad-hVEGFR2-iCas-
pase-9, we infected HDMEC, NIH3T3, and UM-SCC-17B.
We observed that only HDMEC expressed iCaspase-9,
and that it was consistently processed upon exposure of
the cells to AP20187 (Figure 5d).
Activation of adenovirally transduced iCaspase-9
induces apoptosis and caspase-3 activity
in proliferating endothelial cells
To study the expression of adenovirally transduced
iCaspase-9 in proliferating endothelial cells in vitro,we
performed immunostaining of HDMEC transduced
with Ad-hVEGFR2-iCaspase-9 (Figure 6a), Ad-iCas-
pase-9 (Figure 6b), or no virus control (Figure 6c),
observing that approximately 50% of the cells were
positive for iCaspase-9 (Figure 6d).
To evaluate if AP20187-induced processing of iCas-
pase-9 resulted in the induction of endothelial cell
apoptosis, we exposed HDMEC to Ad-hVEGFR2-iCas-
pase-9 or control viruses, and treated the cells with the
dimerizer drug AP20187. Thereafter, we observed a
significant increase in the percentage of apoptotic
endothelial cells exposed to Ad-hVEGFR2-iCaspase-9
and treated with AP20187, but not in the absence of the
dimerizer drug (Figure 6e). To study if the activation of
iCaspase-9 transduced into HDMEC is capable of
activating the endogenous proapoptotic signaling path-
way, we exposed HDMEC to Ad-hVEGFR2-iCaspase-9
or control adenoviruses and measured caspase-3 activity.
We observed that HDMEC exposed to the virus and
treated with AP20187 presented an increase in caspase-3
activity, which was not observed when no promoter
adenoviral controls were transduced or when the cells
were not exposed to AP20187 (Figure 6f). These data
demonstrated that exposure of endothelial cells trans-
duced with Ad-hVEGFR2-iCaspase-9 to AP20187 is
sufficient to initiate a caspase-3-dependent signaling
pathway that results in endothelial cell apoptosis in vitro.
Ad-hVEGFR2-iCaspase-9 induces endothelial
cell apoptosis and is antiangiogenic through
an AP20187-dependent process in vivo
To evaluate if adenovirally transduced iCaspase-9 is
effective in decreasing tissue microvascular density in
vivo, we implanted untransduced HDMEC into SCID
mice as described,
9,26
allowed them to differentiate into
functional blood vessels for 14 days,
26
and then delivered
Ad-hVEGFR2-iCaspase-9 by a single intraimplant injec-
tion of the virus. Starting 2 days after delivery of
adenoviruses, mice were injected with AP20187 or PBS
intraperitonealy for 3 consecutive days. We observed that
microvessel density was decreased significantly (Po
0.05?) in AP20187-treated mice that were infected with
Ad-hKDR-iCaspase-9 (Figure 7e, f, and i), but not in
control mice (Figure 7a–d, and i). To confirm that the
Figure 5 Expression of adenovirally transduced iCaspase-9 in endothelial
cells. Expression of iCaspase-9 or endogenous caspase-9 analyzed by
immunoblotting of lysates obtained from HDMEC, UM-SCC-17B, or
NIH3T3 transduced with Ad-hVEGFR2-iCaspase-9 or the promoter-less
vector Ad-iCaspase-9. Controls were HDMEC transfected with pSH1-
iCaspase-9. (a) Dose-dependency assay in which HDMEC were transduced
with 0–200 M.O.I. Ad-hVEGFR2-iCaspase-9. (b) Time course assay in
which HDMEC were transduced with 200 M.O.I. Ad-hVEGFR2-
iCaspase-9 or Ad-iCaspase-9, and iCaspase-9 expression was evaluated
24–96 h after infection. (c) AP20187-induced processing of iCaspase-9,
and expression of endogenous caspase-9, in HDMECs as determined by
immunoblotting of cells exposed to 0–100 n
M AP20187 for 96 h. (d)
Expression and AP20187-induced processing of iCaspase-9 in HDMEC,
UM-SCC-17B, and NIH3T3 as determined by immunoblotting of cells
transduced with 0–200 M.O.I. Ad-hVEGFR2-iCaspase-9 or Ad-iCaspase-
9, and exposed to 0–100 n
M AP20187 for 96 h.
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Figure 6 Activation of adenovirally transduced iCaspase-9 induces apoptosis and caspase-3 activity in proliferating endothelial cells. Representative
photomicrographs ( 400) of cytospins depicting HDMEC transduced with (a) Ad-hVEGFR2-iCaspase-9, (b) Ad- iCaspase-9, or (c) no virus control and
immunostained with anti-HA antibody (to detect iCaspase-9 expression). (d) Graph depicting number of HA-positive HDMEC per high power field
( 400) after infection with Ad-hVEGFR2-iCaspase-9, Ad-iCaspase-9, or no virus control. (e) Graph depicting the percentage of apoptotic HDMEC after
transduction with 20 or 200 M.O.I. Ad-hVEGFR2-iCaspase-9 or Ad-iCaspase-9, and exposure to 0–100 n
M AP20187 for 96 h. Apoptotic cells were
identified by Annexin V staining followed by flow cytometry. (f) Caspase-3 activity was determined in lysates from cells exposed to AP20187 for 96 h.
Symbols used: HDMEC transduced with Ad-iCaspase-9 and exposed to 0 (n) or 100 nM AP20187 (m); HDMEC transduced with Ad-hVEGFR2-
iCaspase-9 and exposed to 0 (&) or 100 n
M AP20187 (); untransduced HDMEC (J); or 1 ng of purified recombinant human caspase-3 (K). Data
represent mean values (7s.d.) of triplicate samples and results were reproducible in at least three independent experiments.
Figure 7 Local delivery of Ad-hVEGFR2-iCaspase-9 induces endothelial cell apoptosis and is antiangiogenic through an AP20187-dependent process in
vivo. (a–h) Representative photomicrographs of implants in which 5 10
10
Ad-hVEGFR2-iCaspase-9 or Ad-iCaspase-9 viral particles were delivered in
vivo by local injection. (a–f) Factor VIII immunostaining to study the effects mediated by Ad-hVEGFR2-iCaspase-9 or Ad-iCaspase-9 on the local
microvasculature. (a–c) Representative fields of mice injected daily for 3 days with control PBS, or (d–h) with 2 mg/kg AP20187. These implants were
retrieved from mice that received three daily intraperitoneal injections of AP20187. (g, h) TUNEL staining of implants that were injected with Ad-
iCaspase-9 or Ad-hVEGFR2-iCaspase-9. Black arrows point to TUNEL-positive endothelial cells lining the walls of microvessels. Photomicrographs are at
400 magnification, except for panels (c) and (f) that are at 100 magnification. (i) Graph depicting the average number of microvessels (7s.d.) per high
power field ( 200) in implants in which 5 10
10
Ad-hVEGFR2-iCaspase-9 or Ad-iCaspase-9 viral particles were delivered in vivo by local injection,
retrieved from mice that received three daily intraperitoneal injections of PBS or 2 mg/kg AP20187. Microvessel density data were calculated from 12
implants per group, from five randomly selected microscopic fields per implant.
Caspase-mediated antiangiogenic gene therapy
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decrease in microvessel density was caused by endothe-
lial cell apoptosis, we performed in situ TUNEL assays.
We observed the appearance of TUNEL-positive en-
dothelial cells in mice exposed to Ad-hVEGFR2-iCas-
pase-9 and treated with AP21087 (Figure 7h), but not in
when Ad-iCaspase-9 was delivered to the implants
(Figure 7g). Thus, Ad-hVEGFR2-iCaspase-9 induces
apoptosis of endothelial cells and disruption of micro-
vessels in vivo upon conditional induction of caspase-9
activity with a dimerizer drug.
Discussion
Deregulated angiogenesis is an integral component of
the pathobiology of cancer.
1,28
The microvascular net-
work might be a promising target for cancer gene
therapy because: (A) neovascular endothelial cells are
strategically accessible to the gene therapy vectors
delivered through the blood stream; and (B) it is
estimated that each microvascular endothelial cell sup-
ports up to 100 tumor cells.
29
The primary objective of
antiangiogenic strategies proposed for the treatment of
cancer is to eliminate pathologic microvessel networks
while sparing the remaining vasculature. Recent ad-
vances in the design of vectors for gene therapy
combined with the development of tightly regulated
‘artificial death switches’ led to the novel antiangiogenic
gene therapy strategy presented here. We developed and
characterized a recombinant adenoviral vector capable of
mediating expression of iCaspase-9 primarily in neovas-
cular endothelial cells, and demonstrated that activation
of this chimeric protein is sufficient to disrupt angiogenic
blood vessels in vivo.
Transcriptional targeting of adenoviral vectors is a
strategy that has been used extensively to direct
expression of therapeutic genes to their target cell
population, and to improve safety.
12,13
Several neovas-
cular endothelial cell-specific markers have been char-
acterized, such as VEGFR2, Tie, and VE-Cadherin.
30–33
For this study, we selected VEGFR2 for transcriptionally
targeting our viral vectors because this receptor is not
expressed in most vascular endothelial cells from adult
organisms but it is strongly and specifically upregulated
in tumor microvessel networks.
20,30,34
Here, we com-
pared the ability of the human VEGFR2 promoter,
21,22
and the murine VEGFR2 promoter/enhancer
20
to drive
expression of our recombinant adenoviruses primarily in
proliferating neovascular human endothelial cells. We
observed that both the human and the murine VEGFR2
transcription regulatory sequences drove expression
specifically in proliferating endothelial cells, which is in
agreement with previous reports. However, the human
VEGFR2 promoter mediated highest expression levels in
proliferating HDMEC, when compared to the murine
transcriptional elements tested here. The recombinant
adenoviral vectors constructed with the human VEGFR2
promoter retained the specificity of expression in
HDMEC in vitro and in angiogenic microvessels in vivo,
which is consistent with our initial observations in
experiments involving the transfection of luciferase-
based plasmids. These findings suggest that the use of
the VEGFR2 promoter might allow for enhancing the
safety of a gene therapy-based antiangiogenic strategy by
limiting expression of the therapeutic gene to the target
cells, that is, the endothelial cells of proliferating
microvessels.
Others have shown that inducible proapoptotic
caspases might be incorporated into gene therapy
approaches for cancer.
13,24
We decided to use an
inducible system for our proposed antiangiogenic gene
therapy strategy for the following reasons: (A) It allows
for control of the effects of the therapeutic gene. The
therapeutic gene is only activated when the dimerizer
drug is delivered to the recipient of the adenoviruses.
Therefore, if a potential deleterious effect is identified
after delivery of the adenoviruses, the therapy can still be
halted simply by not delivering the activating (dimer-
izer) drug. And, (B) Cells that are transduced with the
adenovirus will undergo apoptosis upon exposure to the
dimerizer drug. Therefore, the risk of viral-induced cell
transformation is minimized by the fact that the
transduced cells will be eliminated by apoptosis.
We observed a dose-dependent increase in iCaspase-9
expression with adenoviral titers of up to 200 M.O.I., and
a tightly regulated processing of the iCaspase-9 protein
upon its activation with the cell-permeable dimerizer
drug AP20187. This processing resulted in an increase in
the percentage of apoptotic cells that was observed
specifically in endothelial cells treated with AP20187, but
not in other cell types (data not shown) exposed to the
same drug. Neither did we observe apoptosis when
promoter-less adenoviral vectors were used in the same
cells. Interestingly, cells that were transduced with Ad-
hVEGFR2-iCaspase-9 and exposed to AP20187 presented
a significant increase in the activity of endogenous
caspase-3. These data confirmed the ability of transduced
iCaspase-9 to activate the endogenous proapoptotic
signaling pathway upon dimerization with AP20187 in
neovascular endothelial cells.
Interestingly, approximately 44% of the endothelial
cells infected with Ad-hVEGFR2-iCaspase-9 and ex-
posed to AP20187 undergo apoptosis. This was some-
what lower than we expected initially, because we had
observed before that approximately 90% of the endothe-
lial cells stably expressing iCaspase-9 undergo apoptosis
upon exposure to AP20187,
9
and that about 90% of the
endothelial cells were positive for b-gal when Ad-
hVEGFR2-LacZ was used here. These puzzling findings
were perhaps clarified when we performed immunos-
taining in endothelial cells infected with Ad-hVEGFR2-
iCaspase-9 and observed that only about 48% of the cells
actually expressed iCaspase-9. Our interpretation of
these results is that a subset of cells transduced at the
highest level underwent apoptosis in a CID-independent
fashion and that most of the remaining cells that express
iCaspase-9 become apoptotic when exposed to AP20187,
implying that the expression of the reporter gene (in this
case LacZ) may not be directly correlated with the
expression of the therapeutic gene (ie iCaspase-9).
Nevertheless, we observed a dramatic decrease in
microvessel density in the implants treated with Ad-
hVEGFR2-iCaspase-9 and injected with AP20187, which
was in contrast with the microvessel density observed
upon the delivery of promoter-less adenoviruses or
control injections. This was correlated with the presence
of apoptotic (ie TUNEL-positive) endothelial cells in
microvessels from implants treated with Ad-hVEGFR2-
iCaspase-9 and AP20187. In contrast, no TUNEL staining
was observed when implants were injected with
Caspase-mediated antiangiogenic gene therapy
W Song et al
326
Gene Therapy
promoter-less adenoviruses (ie Ad-iCaspase-9) in mice
treated with AP20187, or when implants injected with
Ad-hVEGFR2-iCaspase-9 in mice treated with PBS.
These findings correlate well with emerging evidence
that suggest that microvascular disruption does not
require that every single endothelial cell undergo
apoptosis. It is speculated that hemorrhage and blood
flow perturbations caused by the apoptosis of some
endothelial cells lining the walls of the microvessels may
be enough to induce signaling cascades that result in the
disruption of the vascular network.
In summary, we describe here the development and
characterization of a novel recombinant adenoviral
vector designed for the treatment of pathologies that
are characterized by aberrant angiogenesis. We demon-
strated its specificity of infection in proliferating en-
dothelial cells and also its efficacy in inducing
endothelial cell apoptosis and eliminating blood vessels
in vivo. We believe that the recombinant adenoviral
vector presented here can be used to induce selective and
regulated disruption of angiogenic neovascular net-
works, and therefore we are currently testing its safety
and efficacy for treatment of solid tumors. This vector
could become a powerful new tool for antiangiogenic
gene therapy of cancer.
Materials and methods
VEGFR2 promoter/enhancer cloning and sequencing
A 494 bp human VEGFR2 promoter fragment containing
sequence from 226 to +268 was amplified by PCR from
human genomic DNA.
21,22
The sequences of PCR
primers were 5
0
-TACAGGTTAACAAAGTTGTTGCT
CTGGGATGTTCTC-3
0
and 5
0
-AATGAAGCTTGGGAG
CCGGTTCTTTCTCCCAGCGC-3
0
. A 957 bp mouse
VEGFR2 (Flk-1) promoter fragment containing sequence
from +1 to +939 was amplified by PCR from mouse
genomic DNA.
20
The sequences of PCR primers
were 5
0
-TACCGACCCAGCCAGGAAGTTC-3
0
and 5
0
-GT
GCCCAGCGCGAGGTGCAGG-3
0
. A 447 bp mouse
VEGFR2 enhancer fragment containing the minimal
sequence from +1 to +430 was amplified by PCR from
mouse genomic DNA.
20
The sequences of PCR primers
were 5
0
-TACAGGTTAACAAAGTTGTTGCTCTGGGA
TGTTCTC-3
0
and 5
0
-AATGAAGCTTGGGAGCCGGTT
CTTTCTCCCAGCGC-3
0
. The PCR conditions were set
to denature at 951C for 1 min, then 941C for 30 s, 681C for
3 min, total 30 cycles, followed by extension at 681C
for 3 min. The PCR products of human VEGFR2
promoter, mouse VEGFR2 promoter and enhancer
were subcloned into pGEM-T Easy vector (Promega,
Madison, WI, USA), and sequenced to confirm absence
of mutations.
Luciferase assays
The hVEGFR2 promoter fragment was released from
pGEM-T vector by digestion with HpaI and HindIII, and
was then inserted into pGL2-Basic (Promega) to generate
pGL2-hVEGFR2-luc. The mouse VEGFR2 promoter
fragment was released from pGEM-T vector by digestion
with KpnI and HindIII, and was then inserted into pGL2-
Basic to generate pGL2-mVEGFR2-luc. The mouse
VEGFR2 enhancer fragment was released from pGEM-
T vector by digestion with BamH1 and SalI, and was then
inserted into pGL2-mVEGFR2-luc to generate pGL2-
mVEGFR2-promoter/enhancer-luc. Transient transfec-
tions were carried out with 1.0 mg of the appropriate
reporter construct along with 20 ng of pRL-TK (Rluc,
encoding Renilla luciferase, Promega) with Lipofectin
(Invitrogen Corp., Carlsbad, CA, USA). Cell extracts
were prepared 24 h after transfection with lysis buffer
(Promega). Luciferase activity was measured with the
Dual Luciferase Reporter Assay System (Promega) in a
luminometer (Turner Designs Instrument, Sunnyvale,
CA, USA). The ratio of luciferase activity to Renilla
luciferase activity in each sample served as a measure of
normalized luciferase activity. The normalized luciferase
activity was divided by that of pGL2-Basic and ex-
pressed as relative luciferase activity. Data for each
construct were presented as the mean of triplicate wells,
and was reproducible in three independent experiments.
Generation of adenoviruses
Adenoviral shuttle plasmids were constructed with the
backbone of pACpL+loxP-SSP or pACCMVpLpA(-)loxP-
SSP (University of Michigan Vector Core). The LacZ
fragment was released from pCMV-SPORT-Bgal (Invitro-
gen) by digestion with EcoRI and BamHI. It was then
inserted into the shuttle plasmids to generate pAC-lacZ
(no promoter) and pAC-CMV-lacZ. The iCaspase-9
fragment was released from pSH1-iCaspase-9
13
by
digestion with NotI and EcoRI. It was then inserted into
pACpL+loxP-SSP to generate pAC-iCaspase-9 (no pro-
moter). The hVEGFR2 promoter fragment was released
from pGEM-T vector by digestion with HpaI and HindIII.
It was then inserted into pAC-lacZ or pAC-iCaspase-9 to
generate pAC-hVEGFR2-lacZ and pAC-hVEGFR2-
iCasp9. The University of Michigan Vector Core gener-
ated Ad-hVEGFR2-lacZ, Ad-iCaspase-9, Ad-hVEGFR2-
iCaspase-9, and we generated Ad-LacZ and Ad-CMV-
LacZ adenoviruses using the Cre-loxP system.
b-gal staining and b-gal assay
Ad-LacZ, Ad-hVEGFR2-lacZ, and Ad-CMV-LacZ were
used to infect HDMEC (Clonetics, Walkersville, MD,
USA), human dermal fibroblasts (HDF), or two human
squamous cell carcinoma cell lines (UM-SCC-17B, gift of
T Carey; and OSCC-3, gift of M Lingen) at a M.O.I. of
0–200. After 48 h, cells were stained with 1 mg/ml b-gal
in the dishes, or cell extracts were prepared with the lysis
buffer (Promega). b-Gal activity was measured with the
b-gal Enzyme Assay System (Promega) in a Spectro-
fluoroluminometer (GENious, Tecan, Gro
¨
dig, Austria).
Data were presented as the mean (7s.d.) of triplicate
wells, in three independent experiments.
Western blotting
HDMEC were infected with Ad-iCaspase-9 or Ad-
hVEGFR2-iCaspase-9 at 0–200 M.O.I. After 48 h, cells
were treated with 0–100 n
M AP20187 (Ariad Pharmaceu-
ticals, Cambridge, MA, USA). After a 96-h exposure to
AP20187, protein lysates were prepared with lysis buffer
(10 m
M Tris-Cl (pH 8.0), 140 mM NaCl, 0.1% SDS, 1% NP-
40). Cell debris was removed by centrifugation, and
supernatants were boiled in 1:1 sample buffer (50 m
M
Tris-Cl (pH 6.8), 2% SDS, 10% glycerol, 0.01% bromo-
phenol blue, 100 m
M DTT). Proteins were separated on
10% SDS-PAGE gels, membranes were probed with
monoclonal mouse anti-caspase-9 antibody (Oncogene,
Caspase-mediated antiangiogenic gene therapy
W Song et al
327
Gene Therapy
San Diego, CA, USA), or monoclonal mouse anti-HA
antibody (Babco, Berkeley, CA, USA) followed by
exposure to appropriate peroxidase-coupled secondary
antibodies and exposure to ECL (Amersham, Piscataway,
NJ, USA). Blots were reprobed with monoclonal mouse
anti-GAPDH antibody (Chemicon, Temecula, CA, USA)
to control for equal loading.
CytoSpins and HA immunostaining
HDMEC were infected with Ad-iCaspase-9 or Ad-
hVEGFR2-iCaspase-9 at 0–200 M.O.I. After 48 h, cells
were harvested and 10
5
cells were centrifuged against a
histological slide and immediately fixed in methanol for
15 min at room temperature. Cells were incubated with
3% H
2
O
2
for 5 min at room temperature, blocked and
incubated with anti-HA antibody (Babco) at 1:100
dilution for 1 h at 371C. Then slides were incubated with
biotinylated secondary antibody for 20 min, streptavidin-
HRP for 15 min, and with AEC substrate for 7 min. Slides
were counterstained with hematoxylin for 3 s.
Flow cytometry
HDMEC and UM-SCC-17B were infected with Ad-
iCaspase-9 or Ad-hVEGFR2-iCaspase-9 at 0–200 M.O.I.
After 24 h, cells were exposed to 0–100 n
M AP20187 for
0–96 h. Cells were harvested, washed, stained with
40 mg/ml Annexin V (BD Biosciences Clontech, Palo
Alto, CA, USA), and evaluated by flow cytometry as
described.
9
Fluorometric assay of caspase-3 activity
HDMEC were seeded in six-well plates 18 h before
infection. Cells were infected with Ad-iCaspase-9 or Ad-
hKDR-iCaspase-9 at 20 or 200 M.O.I. for 24 h. Cells were
treated with 0 or 100 n
M AP20187 for additional 96 h.
Both attached and floating cells were retrieved and lysed
in the lysis buffer (50 n
M HEPES, 1 mM DTT, 0.1 mM
EDTA and 0.1% CHAPS (pH 7.4)). Cell extracts were
resuspended in the assay buffer (100 m
M NaCl, 50 mM
HEPES, 10 mM DTT, 1 mM EDTA, 10% glycerol, 0.1%
CHAPS). The reactions were carried out at 371Cin
presence of 10 m
M Ac-DEDV-AMC (Alexis Biochemicals,
San Diego, CA, USA). The fluorometric activity was
monitored in a Spectrofluoroluminometer (TECAN) at
excitation wavelength of 360 nm and emission 465 nm
for up to 180 min. Recombinant human caspase-3
(Alexis) was used as positive control. Data were obtained
from triplicate wells per cell and condition from three
independent experiments.
SCID mouse model of human angiogenesis
Functional human microvessels were induced in severe
combined immunodeficient (SCID) mice (CB-17 SCID;
Taconic, Germantown, NY, USA), as described.
9,26
Briefly,
1 10
6
HDMEC were seeded in poly-(L-lactic acid)
(PLLA, Medisorb, Cincinnati, OH, USA) biodegradable
scaffolds, and two scaffolds were implanted subcuta-
neously into the dorsum of each SCID mouse. HDMEC
were infected with Ad-LacZ, Ad-hVEGFR2-lacZ, Ad-
iCaspase-9, or Ad-hVEGFR2-iCaspase-9 at 200 M.O.I. for
48 h before they were harvested and seeded in the
scaffolds. Alternatively, scaffolds containing untrans-
duced HDMEC were implanted in the mice, and
5 10
10
particles/scaffold Ad-LacZ, Ad-hVEGFR2-lacZ,
Ad-iCaspase-9, or Ad-hVEGFR2-iCaspase-9 were in-
jected locally 10 days after implantation. All mice were
euthanized 14 days after implantation. Prior to euthana-
sia, the animals received a daily intraperitoneal injection
of 2 mg/kg AP20187 (Ariad) in a solution of 10% PEG
400 and 1.7% Tween 20 for 3 consecutive days. The
implants were retrieved and fixed in 10% neutral-
buffered formalin for 30 min at 41C, and processed for
b-gal staining or for histology. The care and treatment of
mice were in accordance with University of Michigan’s
institutional guidelines.
b-Gal staining for paraffin-embedded tissue sections
Immediately after retrieval, implants removed from the
SCID mice were immersed in washing buffer (0.1
M
NaPO
3
(pH 8.3), 2 mM MgCl
2
, 0.1% sodium deoxycho-
late, 0.02% Triton X-100) and stained overnight in the
dark with the staining solution (1 mg/ml X-gal, 5 m
M
K3Fe(CN)6, 5 mM K4Fe(CN)6-3H
2
O) at 41C. Next day,
implants were rinsed thoroughly in H
2
O, then fixed in
10% neutral-buffered formalin for additional 1 h, and
processed for histology. After deparafinization, the slides
were stained with 0.1% safranin O for 3 min, rinsed
briefly in H
2
O, dehydrated in 95% followed by 100%
ethanol for 30 s each, and cleared in xylene for 30 s before
mounting.
Immunolocalization of microvessels and in situ TUNEL
assay
Microvessels were identified by incubating tissue sec-
tions with polyclonal rabbit anti-human Factor VIII
antibody (Lab Vision Corp., Freemont, CA, USA).
26,35
The number of stained microvessels was counted in five
randomly selected fields per implant at 200, from 12
implants per condition. TUNEL staining (ApopTag
peroxidase in situ apoptosis detection kit; Chemicon)
was used according to the manufacturer’s instruc-
tions.
7,9,35
Statistical analysis
Statistical significance was determined at the Pp0.05
level, using one-way ANOVA and the Student–New-
man–Keuls test.
Acknowledgements
We thank Ruben Hernandez-Alcoceba for help in
designing the adenovirus vectors; Michael Clarke for
helpful discussions and for critical review of this manu-
script; Victor Rivera for critical review of this manuscript;
and John Westman and Chris Strayhorn for help with
the histology. We also thank ARIAD Pharmaceuticals
(www.ariad.com/regulationkits) for the dimerizing
agent AP20187. This research was supported in part by
Grant DE14601 from NIH, and grant from the American
Dental Association Health Foundation (JEN); Grants
CA70057 (GN) and CA77266 (DMS) from the NIH; and
by Grant 2 P30 CA46592-14 from NIH to the University
of Michigan Comprehensive Cancer Center.
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    • "Initial evaluation of the Robo4-driven Luc expression in endothelial cells in vitro demonstrated relatively low levels of promoter activity in comparison with AdH5CMVLuc. However, compared with other promoters tested to date (Cefai et al., 2005; Greenberger et al., 2004; Reynolds et al., 2001; Savontaus et al., 2002; Song et al., 2005; Takayama et al., 2007; Wung et al., 2005; Yang et al., 2006), the Robo4 promoter displayed significantly less liver tropism and higher endothelial-specific expression in vivo. Remarkably, the lung endothelial expression was specific and high, while the liver expression was barely detectable by immunohistochemistry analysis . "
    [Show abstract] [Hide abstract] ABSTRACT: Adenovirus serotype 5 (Ad5) vectors are well suited for gene therapy. However, tissue-selective transduction by systemically administered Ad5-based vectors is confounded by viral particle sequestration in the liver. Hexon-modified Ad5 expressing reporter gene under transcriptional control by the immediate/early cytomegalovirus (CMV) or the Roundabout 4 receptor (Robo4) enhancer/promoter was characterized by growth in cell culture, stability in vitro, gene transfer in the presence of human coagulation factor X, and biodistribution in mice. The obtained data demonstrate the utility of the Robo4 promoter in an Ad5 vector context. Substitution of the hypervariable region 7 (HVR7) of the Ad5 hexon with HVR7 from Ad serotype 3 resulted in decreased liver tropism and dramatically altered biodistribution of gene expression. The results of these studies suggest that the combination of liver detargeting using a genetic modification of hexon with an endothelium-specific transcriptional control element produces an additive effect in the improvement of Ad5 biodistribution.
    Full-text · Article · Dec 2013
    • "Activation of adenovirally delivered iCaspase-9 with the dimerizer compound AP20187 activated endogenous caspase-3 and induced endothelial cell apoptosis in vitro [74,153]. Furthermore, we showed that activation of iCaspase-9 with AP20187 was sufficient to induce apoptosis of neovascular endothelial cells, and to cause a marked decrease in tissue microvessel density in vivo [74,153]. When we infected proliferating human endothelial cells and human tumor cells of different linages with the same recombinant adenovirus, we observed that the vector includes the VRGFR-2 promoter induced apoptosis of proliferating human endothelial cells, but not human tumor cells in vitro [153]. "
    [Show abstract] [Hide abstract] ABSTRACT: It is well known that angiogenesis plays a critical role in the pathobiology of tumors. Recent clinical trials have shown that inhibition of angiogenesis can be an effective therapeutic strategy for patients with cancer. However, one of the outstanding issues in anti-angiogenic treatment for cancer is the development of toxicities related to off-target effects of drugs. Transcriptional targeting of tumor endothelial cells involves the use of specific promoters for selective expression of therapeutic genes in the endothelial cells lining the blood vessels of tumors. Recently, several genes that are expressed specifically in tumor-associated endothelial cells have been identified and characterized. These discoveries have enhanced the prospectus of transcriptionally targeting tumor endothelial cells for cancer gene therapy. In this manuscript, we review the promoters, vectors, and therapeutic genes that have been used for transcriptional targeting of tumor endothelial cells, and discuss the prospects of such approaches for cancer gene therapy.
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    • "In this model, we seed primary human endothelial cells together with head and neck cancer cells in highly porous biodegradable scaffolds, implant them in the subcutaneous of SCID mice, and observe tumor progression and tumor angiogenesis. Here, we used luciferase-tagged OSCC3 cells to allow us to evaluate tumor progression noninvasively over time, as described242526. To evaluate the effect of Bcl-2 expression levels in endothelial cells on tumor cell invasion, we removed the primary tumors 35 days after implantation of the cells and evaluated local recurrence of the tumors by in vivo bioluminescence for a period of 3 weeks (Figure 2A). "
    [Show abstract] [Hide abstract] ABSTRACT: Field cancerization involves the lateral spread of premalignant or malignant disease and contributes to the recurrence of head and neck tumors. The overall hypothesis underlying this work is that endothelial cells actively participate in tumor cell invasion by secreting chemokines and creating a chemotactic gradient for tumor cells. Here we demonstrate that conditioned medium from head and neck tumor cells enhance Bcl-2 expression in neovascular endothelial cells. Oral squamous cell carcinoma-3 (OSCC3) and Kaposi's sarcoma (SLK) show enhanced invasiveness when cocultured with pools of human dermal microvascular endothelial cells stably expressing Bcl-2 (HDMEC-Bcl-2), compared to cocultures with empty vector controls (HDMEC-LXSN). Xenografted OSCC3 tumors vascularized with HDMEC-Bcl-2 presented higher local invasion than OSCC3 tumors vascularized with control HDMEC-LXSN. CXCL1 and CXCL8 were upregulated in primary endothelial cells exposed to vascular endothelial growth factor (VEGF), as well as in HDMEC-Bcl-2. Notably, blockade of CXCR2 signaling, but not CXCR1, inhibited OSCC3 and SLK invasion toward endothelial cells. These data demonstrate that CXC chemokines secreted by endothelial cells induce tumor cell invasion and suggest that the process of lateral spread of tumor cells observed in field cancerization is guided by chemotactic signals that originated from endothelial cells.
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