Intra-arterial adenoviral mediated tumor transfection in a novel model of cancer gene therapy
© Cabrera et al; licensee BioMed Central Ltd. 2006
Received: 16 January 2006
Accepted: 09 August 2006
Published: 09 August 2006
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.
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.
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.
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]. Various 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 represents a viable clinical and experimental route [10–16]. Other delivery methods employed include intraperitoneal, intra-tumor, intravenous and intravesical administration routes [7–16]. While some clinical trials have been positive, further improvement should include a systematic 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 heterogeneous disposition of blood vessels within the tumor; 2) elevated tumor interstitial pressure; and 3) large transport distances in the tumor interstitium [1–4]. Various in vivo models have been used to characterize tumor vasculature and macromolecular tumor dynamics [17, 18]. Among these, the rat cremaster muscle has been used as a model to study microcirculatory hemodynamics in various 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 . Secondly, the microsurgical dissection and manipulation of these vessels enable simultaneous access to the tumor site and vascular inflow and outflow. Thirdly, the cremaster muscle is constituted 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 cremaster muscle of male athymic nude rats could be transfected 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.
Tumor growth on the cremaster of homozygous athymic male nude rats
Spatial distribution of intra-arterial adenoviral mediated tumor transfection
Dose dependence of in vivo adenovirus gene transfection
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 minutes. This time frame was chosen based on standard ex vivo adenoviral transfection protocols in which adenovirus 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-cremasteric virus dwelling time required to achieve acceptable reporter gene transfection with 60 minutes being the maximal blood interruption time. Two shorter durations of vascular isolation were assessed; 5 minutes and 30 minutes. 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 significance was observed between the 30 minute group and the 60 minute group (p > 0.05).
Our study demonstrates the in vivo dependence of adenoviral 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 validation of emerging anti-tumor vasculature or anticancer agents employing in vivo tissue targeting and differential vascular or tissue toxicities relative to the hosts normal vasculature and tissue.
The Internal Review Board and Animal Research Committee of the Cleveland Clinic Foundation approved all animal 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 published by the National Institutes of Health.
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 (Nutrition International Inc., Brentwood, MO) and water ad libitum.
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.
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 Alabama at Birmingham. Adenoviral preparations and titering 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 ligament. The cremaster muscle was dissected free from the scrotum as previously described . 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, Gottingen, Germany), 5 × 104 human T24 bladder carcinoma cells resuspended in 50 μl 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 performed on the right cremaster muscles. Subsequently, the cremaster muscle tube was returned to its normal anatomical 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 subcutaneous) Penicillin G Benzathine and Penicillin G Procaine (G.C. Hartford Mfg. Corp., Syracuse, NY), 5 ml/S.C. of Ringer's solution (Baxter Corp. Dearfield, IL) and analgesics, Acetaminophen 110 mg/kg/PO (McNeil-PCC Inc., Fort Washington, PA). For the tumor growth curve, animals 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 tuberculin syringe with a 30 gauge 1/2 inch needle, the cremaster muscle tube flap was primed with 200 μl of PBS solution via the external iliac artery.
Ad5-CMV-GFP infusion and confocal microscopy
For the intra-arterial delivery of recombinant adenovirus encoding the green fluorescent protein (Ad5-CMV-GFP), 1 × 109 pfu's in a total volume of 200 μl 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 (Surgical 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-established and the muscle tube was inserted into a subcutaneous tunnel in the anteromedial aspect of the ipsilateral limb and the animals were returned to their cages for observation.
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 tunnel of the limb. A round flat muscle flap with an axial pattern 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  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 isolating the vascular access into the muscle flap. Using a 1 mL insulin syringe with a 30 gauge 1/2 inch needle, the cremaster muscle tube flap was primed with 200 μl of PBS solution via the external iliac artery. Subsequently, 200 μl of a solution containing 1- micron diameter Texas Red fluorescent 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 Ethibond (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 of Ad-CMV-Luc; 1 × 108 (n = 8), 1 × 109 (n = 8) and 1 × 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 × 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 previously 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 pentobarbital. Tumors were collected in 1.5 mL polypropylene tubes and luciferase assay was performed as described below. For luciferase activity determination, tumors were collected in 1.5 mL polypropylene tubes and resuspended in 200 μl 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 consisted 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 preservation media, the cremasters were sutured as previously described and collected 72 hours post mock transfection for luciferase assay.
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