Group Title: Molecular Cancer 2006, 5:32
Title: Intra-arterial adenoviral mediated tumor transfection in a novel model of cancer gene therapy
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Title: Intra-arterial adenoviral mediated tumor transfection in a novel model of cancer gene therapy
Series Title: Molecular Cancer 2006, 5:32
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Creator: Cabrera G
Porvasnik SL
DiCorleto PE
Siemionow M
Goldman CK
Publication Date: 38938
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Volume ID: VID00001
Source Institution: University of Florida
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Molecular Cancer


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BioMed Central


Research


Intra-arterial adenoviral mediated tumor transfection in a novel
model of cancer gene therapy
Gustavo Cabrera*', Stacy L Porvasnik2, Paul E DiCorleto3, Maria Siemionow4
and Corey K Goldman5


Address: 'Gene Therapy Laboratory, National Cancer Institute, Mexico City, Mexico, 2Powel Gene Therapy Center, The University of Florida,
Gainesville, USA, 3Department of Cell Biology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, USA, 4Department of
Plastic and Reconstructive Surgery, The Cleveland Clinic Foundation, Cleveland, USA and 5Department of Vascular Medicine, Ochsner Clinic
Foundation, New Orleans, USA
Email: Gustavo Cabrera* g.cabrera@yahoo.com; Stacy L Porvasnik slporvas@yahoo.com; Paul E DiCorleto dicorlp@ccf.org;
Maria Siemionow siemiom@ccf.org; Corey K Goldman coreykeith@aol.com
* Corresponding author


Published: 09 August 2006
Molecular Cancer 2006, 5:32 doi:10.1 186/1476-4598-5-32


Received: 16 January 2006
Accepted: 09 August 2006


This article is available from: http://www.molecular-cancer.com/content/5/1/32
2006 Cabrera et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: The aim of the present study was to develop and characterize a novel in vivo cancer
gene therapy model in which intra-arterial adenoviral gene delivery can be characterized. In this
model, the rat cremaster muscle serves as the site for tumor growth and provides convenient and
isolated access to the tumor parenchyma with discrete control of arterial and venous access for
delivery of agents.
Results: Utilizing adenovirus encoding the green fluorescent protein we demonstrated broad
tumor transfection. We also observed a dose dependant increment in luciferase activity at the
tumor site using an adenovirus encoding the luciferase reporter gene. Finally, we tested the intra-
arterial adenovirus dwelling time required to achieve optimal tumor transfection and observed a
minimum time of 30 minutes.
Conclusion: We conclude that adenovirus mediated tumor transfection grown in the cremaster
muscle of athymic nude rats via an intra-arterial route could be achieved. This model allows
definition of the variables that affect intra-arterial tumor transfection. This particular study suggests
that allowing a defined intra-tumor dwelling time by controlling the blood flow of the affected organ
during vector infusion can optimize intra-arterial adenoviral delivery.


Background
The therapeutic efficacy of novel cancer therapeutic agents
including genetic vectors that directly target tumor cells
greatly depends on their adequate distribution to and
within the tumor mass [1-6]. The capacity of adenoviral
vectors to reach and transfect the highest possible number
of cells that constitute the tumor mass is thus critical for
the success of adenoviral cancer gene therapy [5,6]. Vari-


ous routes and methods to deliver genetic vectors to the
tumor mass have been used [7-9]. The route and method
chosen may have a profound effect on tumor transfection
efficiency and thus on therapeutic efficacy. Direct intra-
arterial delivery of vector has also been utilized and repre-
sents a viable clinical and experimental route [10-16].
Other delivery methods employed include intraperito-
neal, intra-tumor, intravenous and intravesical adminis-


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tration routes [7-16]. While some clinical trials have been
positive, further improvement should include a system-
atic assessment of the variables that affect intra-arterial
tumor transfection. To date, substantial work has been
done to understand the variables that affect tumor drug
bioavailability [1-4]. Three properties of tumors result in
poor distribution of macromolecules in tumors: 1) a het-
erogeneous disposition of blood vessels within the tumor;
2) elevated tumor interstitial pressure; and 3) large trans-
port distances in the tumor interstitium [1-4]. Various in
vivo models have been used to characterize tumor vascu-
lature and macromolecular tumor dynamics [17,18].
Among these, the rat cremaster muscle has been used as a
model to study microcirculatory hemodynamics in vari-
ous pathologic and physiologic conditions [19-22]. The
rat cremaster muscle has three important properties that
make it an attractive site for the growth of solid tumors
and to study the dynamics involved in the intra-arterial
delivery of gene vectors for cancer gene therapy. Firstly,
the rat's cremaster muscle is fed by one principal artery
and drained by one main vein [23]. Secondly, the micro-
surgical dissection and manipulation of these vessels ena-
ble simultaneous access to the tumor site and vascular
inflow and outflow. Thirdly, the cremaster muscle is con-
stituted by well-vascularized skeletal muscle that provides
a useful substrate for the growth of tumor masses. We thus
tested the hypothesis that tumor masses grown on the cre-
master muscle of male athymic nude rats could be trans-
fected via the intra-arterial route. In these studies, we
describe the tumor cremaster model and demonstrate the
time and viral particle number dependence for adenoviral
gene transfer.

Results
Tumor growth on the cremaster of homozygous athymic
male nude rats
We initially tested if a tumor mass could be grown in the
cremaster muscle of athymic male nude rats by inoculat-
ing 5 x 104 cells of the human bladder carcinoma cell line
T24 into the muscle and documented tumor growth at
days 10, 20 and 30 post cell injection. As seen in Figure 1,
the tumor masses grew beyond 5 mm in diameter. Con-
current with tumor growth we observed recruitment of
new blood vessels. As shown in Figure 2, we observed con-
centric growth of blood vessels that infiltrate the tumor as
well as peritumoral edema.

Spatial distribution of intra-arterial adenoviral mediated
tumor transfection
We then studied if tumors grown in the cremaster muscle
of athymic nude rats could be transfected using recom-
binant adenoviral vectors via the intra-arterial route. Cre-
master muscle bearing tumor was transfected with Ad5-
CMV-GFP for 1 hour and examined for the presence and
distribution of the expressed reporter green fluorescent


Ye- -WV

Figure I
Tumor growth on the cremaster muscle of athymic
nude male rats. The cremaster muscle of athymic male
nude rats were inoculated with 5 x 104 cells of the human
bladder carcinoma cell line T24. Tumor growth was followed
at days 10, 20 and 30 post cell injection.


protein (GFP) within the tumor mass seventy-two hours
post Ad5-CMV-GFP transduction. The tumor vasculature
was counterstained using intra-arterial infusion of red flu-
orescent 1 micron diameter beads and mounted for fluo-
rescent confocal microscopy as described in material and
methods. As seen in Figure 3, panels B and D, confocal
microscopy revealed diffuse transfection of the tumor
mass. A concentric neo-angiogenic response elicited
within the tumor mass was also observed. Muscle trans-
fection was also observed throughout the muscle pedicle
(data not shown).

Dose dependence of in vivo adenovirus gene transfection
Once we visualized the spatial distribution of green fluo-
rescent reporter gene expression in tumors we used a sim-
ilar recombinant adenovirus carrying the Luc reporter
gene (Ad-CMV-Luc) to quantify the profile of tumor trans-


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Figure 2
Angiogenic response elicited by tumor masses growing in the cremaster muscle of athymic nude rats. The cre-
master muscle of athymic male nude rats were inoculated with 5 x 104 human T24 bladder carcinoma cells. Ten days after
tumor cell inoculation rats were sacrificed and mounted for photographic documentation. Concurrent with tumor growth we
observed recruitment of new blood vessels that grew in a concentric pattern. Peri-tumoral edema characteristic of tumor
growth can be observed. A. I x magnification of the cremaster muscle bearing tumors on both sides of the principal artery, B.
2x magnification of left tumor, C. 2x magnification of the right tumor.


fection. The data presented in Figure 4 demonstrate
increasing luciferase activity in the tumor mass after in
vivo adenoviral transfection using doses of 1 x 108, 1 x
109 1 x 1010 plaque forming units (PFU's) in a fixed vol-
ume of 200 microliters. Each viral transfection was per-
formed with a vascular isolation-adenovirus dwelling
time of 60 minutes.

Temporal dependence on incubation time for adenoviral
mediated intra-arterial tumor transfection
In the initial experiments the cremaster vascular supply
isolation time in the presence of adenovirus was 60 min-
utes. This time frame was chosen based on standard ex
vivo adenoviral transfection protocols in which adenovi-
rus is allowed to be in contact with the target cells for no
less than 60 minutes [22,23]. In an applied in vivo clinical
scenario, arterial blood flow interruption would be kept
to a minimum yet maximum tumor transfection would be
desired. We sought to determine a minimum intra-cre-
masteric virus dwelling time required to achieve accepta-
ble reporter gene transfection with 60 minutes being the
maximal blood interruption time. Two shorter durations


of vascular isolation were assessed; 5 minutes and 30 min-
utes. To this end, tumor-bearing animals were infused
with Ad-CMV-Luc using the surgical method described
previously. After the specified dwelling time had elapsed,
arterial and venous circulation was re-established and the
samples were processed as described in materials and
methods. The data presented in Figure 6 suggest that an
intra-cremaster dwelling time of 5 minutes yields 21%
transfection efficacy when compared to the 60 minute
group while an intracremaster adenovirus dwelling time
of 30 minutes yielded a 91% transfection efficacy when
compared to the 60 minute group. No statistical signifi-
cance was observed between the 30 minute group and the
60 minute group (p > 0.05).

Discussion
The efficacy of novel cancer treatment modalities includ-
ing the use of gene therapy strategies depends on their
ability to deliver genetic information to the tumor mass in
adequate quantities [1-6]. In general, cancer gene therapy
strategy success greatly depends on achieving high tumor
transfection profiles in addition to an effective anticancer


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5000 -

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3000 -

2000 -

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Figure 3
Intra-arterial adenoviral mediated transfection of
tumors grown in the cremaster muscle of athymic
nude rats. Tumors from two different animals are shown.
The cremaster muscle of athymic male nude rats were inocu-
lated with 5 x 104 cells of the human bladder carcinoma cell
line T24. Ten days after tumor cell implantation, the cremas-
ter muscle flap was intra-arterially infused with I x 109 pfus
of a recombinant adenovirus encoding the green fluorescent
protein gene (Ad5-CMV-GFP) and were left to incubate for I
hour. Seventy two hours after viral infusion cremasters were
set up for standard micro-photography and fluorescent con-
focal microscopy and photography as described in materials
and methods. Texas red fluorescent micro beads were used
to counter stain the muscle and tumor vasculature. Panel A:
2X Photographic image of a tumor using a standard stereo-
scopic microscope. Panel B Confocal fluorescent photogra-
phy of the same tumor. Panel C: 2X Photographic image of a
tumor using a standard stereoscopic microscope on a second
animal. Panel D: Confocal fluorescent photography of the
same tumor. Green fluorescent protein can be seen
expressed throughout the tumor mass. A concentric neoang-
iogenic response can be seen as well.


10. 109 100


Figure 4
Dose curve effect on adenoviral mediated intra-arte-
rial tumor transfection. Tumor bearing animals were
infused with I x 108(n = 8), I x 109(n= 8) and I x 10'0(n=
8) of Ad-CMV-Luc using the surgical approach described in
materials and methods. After intra-arterial adenoviral infu-
sion was completed, the cremasters bearing tumors were left
to incubate for I hour. After the time had elapsed, clamps
were released and rats were left to live for 72 hrs. The rats
were then sacrificed and the samples were assayed for luci-
ferase activity as described in materials and methods. The
results show dose curve dependant luciferase activity at the
tumor site.



strategy [5,6]. In human and animal studies, an intra-arte-
rial route of gene therapy vector administration represents
a viable method in order to transfect the tumor paren-
chyma and normal tissue [9-16]. The lack of success of
some intra-arterial gene therapy trials may be related to
delivery issues rather than payload bioactivity. A system-
atic assessment of the variables that affect tumor transfec-
tion via the intra-arterial route in a cancer gene therapy
context would be useful to refine the delivery technique to
accomplish efficient tumor vector expression. In this
regard, substantial research has been done to define the
variables that optimize molecular bioavailability [1-4,17].
For a blood-borne therapeutic agent to reach the neoplas-
tic cells, it must enter the blood circulation, cross the ves-
sel wall, move across the extracellular matrix, and finally
reach the tumor cell [1-4]. In addition to these anatomic
impediments, three physiological barriers are responsible
for the poor bioavailability of macromolecules in tumors:
1) a heterogeneous tumor vascular supply limits the deliv-
ery of therapeutic molecules to well vascularized or well-
perfused regions of the tumor; 2) elevated tumor intersti-
tial pressure reduces the penetration of the therapeutic
macromolecules whereby a high pressure outward radial
gradient leaves tumor regions without drug; and 3) large
transport distances within the tumor interstitium slow
down the transit of the macromolecules and thus make
distal regions of a tumor hard to reach [1-4]. The rat cre-
master muscle has been extensively used to study tumor
molecular dynamics, structural properties of the tumor


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vasculature and microcirculatory dynamics of various
pathophysiologic conditions [18-23]. In addition, the rat
cremaster muscle has several properties that make it an
attractive site for the growth of tumors and for its use as an
endovascular cancer gene therapy model. As seen in Fig-
ure 5, the cremasters vascular anatomy enables the surgi-
cal dissection, manipulation and isolation of the iliac and
common femoral arteries and veins which permits the
direct infusion of agents into the pudo-epigastric artery
that feeds the cremaster muscle. Another desirable prop-
erty of the cremaster muscle relates to well vascularized
skeletal muscle. This is essential for growth ofvascularized
tumor masses and thus to access the tumors parenchyma
via the intra-arterial route. Finally, the inside aspect of the
cremaster tube muscle provides a protected encased envi-
ronment in which the tumors can grow. As seen in Figures
1 and 2, the careful surgical manipulation of the cremaster


after several weeks of tumor growth did not damage the
integrity of the tumor mass, the tumor vasculature or the
surrounding tissue. This issue is of relevance if further
measurements such as microcirculatory hemodynamics,
tumor dimensions and tumor vasculature measurements
are to be done. In order to characterize a novel in vivo
intravascular cancer gene therapy model we initially tested
if a tumor mass could be grown in the cremaster muscle
of athymic male nude rats and if the masses grew beyond
a few millimeters. The growth of tumors beyond a few
millimeters is relevant for two main reasons: 1) Growth
beyond 2 mm in diameter requires neo-angiogenesis and
thus enables the intravascular access to the tumors paren-
chyma [24] and 2) growth beyond several millimeters in
diameter during a period of 30 days would allow the
model to be used in anti-cancer studies. Additionally, the
need to test the therapeutic efficacy of emerging anti-can-


Figure 5
Anatomy of the cremaster muscle in the athymic male nude rat. A. Diagram depicting the blood vessel anatomy and
the intra-arterial access to the cremaster muscle and the tumor site. B. Actual anatomy of the vessels that feed and drain the
cremaster muscle of male rat.




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- Control Mok
-Conirol Mock


Figure 6
Effect of incubation time on
intra-arterial tumor transfer
bearing animals were infused wii
using the surgical approach desc
ods. After intra-arterial adenovil
the cremasters bearing tumors
either 5 minutes (n = 8), 30 mini
= 8). After the times had elapsed
rats were left to live for 72 hrs.
and the samples were processes
and methods. The data present
adenoviral mediated tumor tran
optimally with a dwelling time o



cer agents on tumor masses la
in diameter for tumor regress
[25]. Vascularized tumor mass
ter of homozygous athymic
beyond a few millimeters as se
Once established that vascular
generated, we wanted to dete
plish intra-arterial tumor tra
vectors. We therefore exami
expressed green fluorescent p
Confocal microscopy revealed
tumor mass. As seen in Figure
neo-angiogenic response was
microscopy and when the vas
with red fluorescent microbea
also observed throughout the
shown). We then tested the
tumor transfection using an
Luciferase reporter gene (Ad
sented in Figure 4 indicate tha
tumor mass via the intra-arteri
adenoviral vectors was achieve
ence of luciferase activity ir
observed increment in luci
tumors correlates with the inc
rus infused. As demonstrated I


animal models, intra-arterial delivery of genetic vectors to
the cancer affected organ remains a viable route [9-16]. In
the present model, we initially tested Ad5-CMV-GFP and
Ad-CMV-Luc tumor transfection by closing the vascular
circuitry of the cremaster muscle for 60 minutes, thus
allowing the virus to bind to its receptor and internalize.
This time frame was utilized based on standard adenoviral
protocols in which the studied cells are allowed to be in
contact with adenovirus for no less than 60 minutes
[26,27]. In a clinical scenario the interruption of arterial
blood flow to a given organ for 60 minutes would not
I seem optimal due to prolonged anoxia. Arterial blood
0min 30 min 5smin flow interruption of the target organ would thus have to
be kept to a minimum yet enough time could be allotted
adenoviral mediated in order to allow maximum tumor transfection. We thus
action. Cremaster tumor tested how varying the intra cremaster virus dwelling time
th I x 10 Ad-CMV-Luc affected tumor transfection. We selected three incubation
ribed in materials and meth- times; 5 minutes, 30 minutes and 60 minutes and an ade-
ral infusion was completed, noviral dose of 1 x 1010. As seen in Figure 6, allowing the
were left to incubate for virus to dwell within the cremaster for 30 minutes yielded
utes (n = 8) or 60 minutes (n transfection profiles similar to the 60 minute incubation
Sclamps were released and time group. Arterial blood flow interruption for a period
The rats were then sacrificed r
I as described in materials of 30 minutes may be viable for various tumor types since
d suggest that intra-arterial the treated normal tissues would be exposed to decreased
section could be achieved anoxia time and still allow adenoviral binding and inter-
f 30 minutes, nalization. An intra- cremaster dwelling time of 5 minutes
revealed relatively low adenoviral transfection of the
tumor mass when compared to the 30- and 60-minute
incubation times. These findings have implications for
rger than a few millimeters anoxia sensitive organs including the kidney and brain
ion studies has been stated whereby a 30-minute in vivo occlusion time is unlikely to
es developed in the cremas- be acceptable, and thus the optimal virus dwelling time to
male nude rats and grew achieve optimal viral transfection should be defined on a
en in our studies (Figure 1). per organ basis. In addition to the three physiologic barri-
ized tumor masses could be ers that affect macromolecular tumor transport discussed
ermine if we could accom- above, optimal virus-tumor interaction time should be
nsfection using adenoviral defined if therapeutic tumor transfection efficiency is to
ined the presence of the be achieved. Additional research needs to be done in
rotein in the tumor mass. order to study the variables, conditions, and mechanics
diffuse transfection of the such as temperature and injection pressure in the intra-
3 panels A-D a concentric arterial route in order to accomplish the optimal delivery.
observed using stereoscopic Our results suggest that the intra-organ virus dwelling
:ulature was counterstained time is a relevant variable that has an influence on tumor
ds. Muscle transfection was transfection efficiency in vivo.


Muscle pedicle (data not
dose dependence effect on
adenovirus encoding the
-CMV-Luc). The data pre-
t in vivo transfection of the
ial infusion of recombinant
ed as reflected by the pres-
n the tumor masses. The
erase activity within the
remental doses of adenovi-
by clinical trials and various


Conclusion
Our study demonstrates the in vivo dependence of aden-
oviral contact (dwell) time and dosage on transfection. A
novel model is also described that could be of utility to
further study variables that affect vector delivery to the
tumor mass via the intra-arterial route and thus refine the
optimal conditions to enhance tumor transfection.
Finally, the present model could have utility to test valida-
tion of emerging anti-tumor vasculature or anticancer
agents employing in vivo tissue targeting and differential


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vascular or tissue toxicities relative to the hosts normal
vasculature and tissue.

Methods
The Internal Review Board and Animal Research Commit-
tee of the Cleveland Clinic Foundation approved all ani-
mal experiments. In addition, all animals used in this
study received the humane care in compliance with the
Guide for the Care and Use of Laboratory Animals pub-
lished by the National Institutes of Health.

Animals
Athymic homozygous male nude rats with a weight of 100
- 110 gm were obtained from Harlan Sprague Dawley Inc.
(Indianapolis, IN). Animals were kept under standard
rodent laboratory housing conditions with 12 hours day/
night cycles and given standard rodent chow diets (Nutri-
tion International Inc., Brentwood, MO) and water ad
libitum.

Cell lines
The human bladder carcinoma cell line T24 was obtained
from American Type Culture Collection (Rockville, MD).
The cell line was grown in McCoys 5a medium (Sigma, St.
Louis, MO) supplemented with 10% heat-inactivated
fetal bovine serum (Hyclone, Logan, UT) and 2 mmol/L-
glutamine, 100 U/mL penicillin G sodium, streptomycin
sulfate, 0.25 g/mL.

Adenoviral vectors
Ad5-CMV-GFP encodes the green fluorescent protein
(GFP) gene driven by the cytomegalovirus promoter
obtained from Q-Biogene Inc. (Montreal, Canada). The
adenovirus Ad-CMV-Luc encodes the luciferase gene
driven by the cytomegalovirus (CMV) promoter and was
a kind gift from Dr. David Curiel at the University of Ala-
bama at Birmingham. Adenoviral preparations and titer-
ing were performed as previously described (7).

Tumor cell inoculation into the cremaster muscle
Tumor cell inoculation into the cremaster muscle was
done under general anesthesia with an intraperitoneal
injection of sodium pentobarbital (50 mg/kg) (Abbott
Laboratories, Chicago, IL). The skin was diagonally
incised from the middle of the scrotum to the inguinal lig-
ament. The cremaster muscle was dissected free from the
scrotum as previously described [23]. Briefly, the testis
and spermatic cord were freed from the interior of the
muscle tube flap through a horizontal incision in the
anterior surface of the cremaster muscle at the level of the
inguinal ring and were subsequently guided back into the
abdominal cavity. The muscle tube was left inverted for
tumor cell inoculation. Under microsurgical observation
(Zeiss S3 OPMI operating microscope, Carl Zeiss, Gottin-
gen, Germany), 5 x 104 human T24 bladder carcinoma


cells resuspended in 50 ptl of phosphate buffer saline
(PBS) solution (Long Island City, GIBCO) were injected
into the cremaster muscle wall using a 1 mL insulin
syringe (Becton & Dickinson Corp. Franklin, NJ) with a 30
gauge 1/2 inch needle (Becton & Dickinson Corp.). Two
intramuscular inoculations, one on either side of the
main artery and vein of the muscle tube flap were per-
formed on the right cremaster muscles. Subsequently, the
cremaster muscle tube was returned to its normal anatom-
ical position and placed back in the scrotal bag. The skin
was sutured with 5-0 Vicryl (Ethicon Inc. Somerville, NJ)
and the animals were given antibiotics (5,000 IU/kg sub-
cutaneous) Penicillin G Benzathine and Penicillin G Pro-
caine (G.C. Hartford Mfg. Corp., Syracuse, NY), 5 ml/S.C.
of Ringer's solution (Baxter Corp. Dearfield, IL) and anal-
gesics, Acetaminophen 110 mg/kg/PO (McNeil-PCC Inc.,
Fort Washington, PA). For the tumor growth curve, ani-
mals were sacrificed at days 10, 20 and 30 following
tumor cell implantation by an intraperitoneal overdose of
sodium pentobarbital. Collected tumor samples were
measured, representative photographs were taken and the
tumors were then sectioned and counter stained with
hematoxylin and eosin for histopathologic evaluation.

Intra-arterial delivery of recombinant adenoviral vectors
Intra-arterial infusion of the viral vectors was performed
10 days following tumor inoculation. Under sodium
pentobarbital anesthesia, the previously placed skin
sutures were carefully cut and the cremaster muscle was
dissected free from the scrotum. The cremaster muscle flap
and its supplying pudo-epigastric vascular pedicle were
dissected to its origin at the iliac vessels. The external iliac
artery, the proximal femoral artery and vein were clamped
with microsurgical aneurysm clamps (Accurate Surgical &
Scientific Instruments Corp., Westbury, NY) to create a
cremaster muscle end-organ tube flap. Using a 1 mL tuber-
culin syringe with a 30 gauge 1/2 inch needle, the cremas-
ter muscle tube flap was primed with 200 ptl of PBS
solution via the external iliac artery.

AdS-CMV-GFP infusion and confocal microscopy
For the intra-arterial delivery of recombinant adenovirus
encoding the green fluorescent protein (Ad5-CMV-GFP),
1 x 109 pfu's in a total volume of 200 ptl using a 1 mL
tuberculin syringe with a 30 gauge 1/2 inch needle were
used in all animals (n = 4). Briefly, the femoral artery was
clamped with microsurgical aneurism clamps in order to
direct the infused solution into the pudo-epigastric artery
as shown in Figure 5, panel A. The iliac artery was also
clamped to stop arterial blood flow and allow injection
into the artery to proceed without bleeding. Immediately
after adenoviral infusion was completed, the external iliac
vein was clamped to avoid retrograde venous blood flow
into the muscle flap. The puncture site at the iliac artery
was sutured with 10-0 nylon microsurgical suture (Surgi-


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cal Specialities Corp., Reading, PA). The muscle flaps were
then left to incubate for 1 hour wrapped with a moist
gauze. After 1 hour had elapsed, clamps were removed,
circulation into the cremaster muscle flap was re-estab-
lished and the muscle tube was inserted into a subcutane-
ous tunnel in the anteromedial aspect of the ipsilateral
limb and the animals were returned to their cages for
observation.

Confocal microscopy
Seventy-two hours following Ad5-CMV-GFP infusion,
animals were sacrificed with an intraperitoneal overdose
of sodium pentobarbital and the cremaster muscle tube
flap was carefully withdrawn from the subcutaneous tun-
nel of the limb. A round flat muscle flap with an axial pat-
tern of vessels was created by vertically transecting the
frontal wall of the cremaster muscle tube from the
inguinal ring to the tip of the tube using a thermal cautery.
The animal was secured on a specially designed Plexiglass
tissue bath as previously described [10] and the cremaster
muscle was spread out with 5-0 Ethibond (ETHICON
Inc.) sutures over a cover glass. The external iliac artery
and the proximal femoral artery and vein were dissected
and clamped with microsurgical aneurism clamps isolat-
ing the vascular access into the muscle flap. Using a 1 mL
insulin syringe with a 30 gauge 1/2 inch needle, the cre-
master muscle tube flap was primed with 200 tl of PBS
solution via the external iliac artery. Subsequently, 200 ptl
of a solution containing 1- micron diameter Texas Red flu-
orescent microbeads (Molecular Probes Inc., Eugene, OR)
were infused using a 1 mL insulin syringe with a 24 gauge
0.75 inch Angiocath. This permitted red counter staining
of the cremaster and tumor vasculature. After fluorescent
microbead infusion was completed, the cremaster muscle
pedicle was ligated at its iliac vessel origin with 5-0 Ethi-
bond (ETHICON) and transected with a thermal cautery.
The free flat cremaster muscle flap was then fixed with a
10% buffered formalin solution, for 1 hour and was
placed on a tissue slide for confocal microscopy. Confocal
microscopy images were collected using a Leica TCS-NT
laser scanning spectrophotometric confocal microscope
(Leica Microsystems AG, Mannheim, Germany).

Adenoviral dose curve effect
For the dose curve intra-arterial delivery of recombinant
adenovirus encoding the luciferase reporter gene (Ad-
CMV-Luc) experiments, three groups of cremaster tumor
bearing rats were administered the following doses ofAd-
CMV-Luc; 1 x 108 (n = 8), 1 x 109 (n = 8) and 1 x 1010 (n
= 8) following the surgical procedure described above.
Seventy two hours following intra-arterial Ad-CMV-Luc
delivery, animals were sacrificed with an intraperitoneal
overdose of sodium pentobarbital. Tumors were collected
in 1.5 mL polypropylene tubes and luciferase assay was
performed as described below.


Intra-cremaster adenoviral dwelling time
For the incubation time course experiments, cremaster
tumor bearing rats were administered 1 x 1010 pfu's of Ad-
CMV-Luc as previously described. The animals were then
divided into three groups. In group I (n = 8), the muscle
flaps were allowed to incubate for 5 minutes, in group II
(n = 8) the muscle flaps were allowed to incubate for 30
minutes and in group III (n = 8) the muscle flaps were
allowed to incubate for 1 hour. After the various times had
elapsed, the vascular clamps were released, circulation
was re-established, the muscle flaps were processed as pre-
viously described and the animals were placed in warm
cages for observation. Seventy two hours following intra-
arterial Ad-CMV-Luc delivery, animals were sacrificed
with an intraperitoneal overdose of sodium pentobarbi-
tal. Tumors were collected in 1.5 mL polypropylene tubes
and luciferase assay was performed as described below.
For luciferase activity determination, tumors were col-
lected in 1.5 mL polypropylene tubes and resuspended in
200 ptl of luciferase lysis buffer (Promega Inc., Madison,
WI). Tumors were then lysed using a manual tissue
homogenizer, protein concentration was determined
using the Bradford method and luciferase assay was done
as indicated by the manufacturer using a Turner Designs
TD-20/20 luminometer (Turner Designs, Sunnyvale, CA).
Controls consisted of negative untreated tumors (n = 8)
and mock (n = 8) groups. The negative control group con-
sisted of untreated tumors that were collected at day 13
post cell implantation and assayed for luciferase activity.
The mock control groups consisted of tumors that at day
10 after cell implantation were infused with viral preser-
vation media, the cremasters were sutured as previously
described and collected 72 hours post mock transfection
for luciferase assay.

Competing interests
The authors) declare that they have no competing inter-
ests.

Authors' contributions
GC: Conceptualized the model, carried out the adenoviral
methodologies, performed the animal surgeries, proc-
essed the tissue, carried out the luciferase assays and wrote
the manuscript. SLP: contributed with animal surgeries
and aided in tissue processing, MS: participated with revis-
ing the manuscript, PD, participated with the experimen-
tal design and revising the manuscript, CG: participated
with the experimental design and revising the manuscript,

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