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J Thorac Cardiovasc Surg 2003;126:28-38
© 2003 The American Association for Thoracic Surgery
General thoracic surgery |
a Thoracic Service, Department of Surgery, Memorial Sloan-Kettering Cancer Center,, New York, NY, USA
b Department of Cardiothoracic Surgery,, New York, NY, USA
c Division of Pulmonary and Critical Care Medicine, New York, NY, USA
d Department of Genetic Medicine, Weill Medical College of Cornell University, New York, NY, USA
Received for publication August 6, 2002; revisions received September 12, 2002; accepted for publication September 23, 2002.
* Address for reprints: Robert J. Korst, MD, Department of Cardiothoracic Surgery, M 404, Weill Medical College of Cornell University, 525 East 68th Street, New York, NY 10021, USA
rjk2002{at}med.cornell.edu
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Abstract |
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METHODS: An adenovirus vector encoding the human pigment epithelium-derived factor cDNA (AdPEDF) was used to transduce human lung cancer cells in vitro. Transgene expression was assessed using Western analysis. Three different murine flank tumors (2 lung, 1 colon) were then established in syngeneic mice and treated intratumorally with phosphate-buffered saline, AdPEDF, or an empty vector (AdNull). Endpoints measured included transgene expression, tumor size, and animal survival, as well as microvessel density within the tumor. Additionally, a murine pulmonary metastasis model was established by intravenous injection of a syngeneic colon adenocarcinoma cell line expressing a marker gene (ß-galactosidase). One day later, treatment (phosphate-buffered saline, AdNull, or AdPEDF) was administered intrapleurally. Tumor burden (gross and histologic inspection, lung weight, and ß-galactosidase expression) was then evaluated 13 days after vector dosing, and survival was recorded.
RESULTS: AdPEDF-derived expression of pigment epithelium-derived factor was demonstrated in vitro and in vivo. In syngeneic murine lung cancer flank tumors, intratumoral administration of AdPEDF significantly inhibited tumor growth (P < .01), prolonged mouse survival (P < .01), and decreased microvessel density (P < .01) compared with control groups. In the pulmonary metastasis model, AdPEDF-treated mice exhibited significantly reduced lung lesions, lung weight (P < .0005), ß-galactosidase expression (P < .05), and animal survival was prolonged (P < .05).
CONCLUSION: Gene transfer of pigment epithelium-derived factor suppresses tumor vascularization and growth, while prolonging survival in syngeneic murine models of thoracic malignancies.
Angiogenesis is known to be a series of linked and sequential steps that ultimately leads to the development of a neovascular blood supply to a tumor mass and is crucial for the continuous growth of tumors and the development of metastases.1-3 Tumor masses less than 0.5 mm in diameter receive necessary nutrients and oxygen through diffusion; however, any growth beyond 0.5 mm requires neovascularization.2 Therefore, expansion of tumors beyond a microscopic size is dependent on two cellular components growing in tandem: tumor cells and microvascular endothelial cells.3 To accommodate their continued proliferation, tumor cells secrete proangiogenic factors (eg, vascular endothelial growth factor), which induce endothelial cell proliferation, migration, and eventually capillary tube formation.1-3 Because angiogenesis is essential for tumor growth and metastasis formation, efforts to discover effective antiangiogenic compounds are warranted.
Pigment epithelium-derived factor (PEDF), a 50-kDa protein first purified from the conditioned media of human retinal pigment epithelial cells,4 has neuroprotective activities.5-7 In addition, PEDF has recently been shown to be a potent inhibitor of both choroidal and retinal angiogenesis in animal models.8-12 A phase I clinical trial evaluating intravitreal adenovirus (Ad)-mediated gene transfer of the human PEDF cDNA in patients with age-related macular degeneration has been proposed based on these data.13
Given this background, we hypothesized that PEDF may exhibit antitumor properties, based on its ability to inhibit angiogenesis. The objectives of this study are to determine if (1) intratumoral administration of an Ad vector encoding the human PEDF cDNA (AdPEDF) will result in PEDF expression and inhibit the growth of syngeneic murine lung cancers in an established flank tumor model; (2) the antitumor effect of AdPEDF will be preserved in 3 different strains of immunocompetent mice bearing 3 different established syngeneic tumors; (3) intrapleural delivery of AdPEDF will result in significantly reduced tumor burden and prolonged survival in an orthotopic murine pulmonary metastasis model; (4) tumors injected with AdPEDF will exhibit evidence of angiogenesis inhibition.
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Methods |
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Cell culture
CT26 is an undifferentiated colon adenocarcinoma (H-2d, syngeneic to BALB/c mice) originally derived by intrarectal injections of N-nitro-N-methylurethane in a female BALB/c mouse. CT26.CL25 was derived from CT26 cells modified to express the Escherichia coli ß-galactosidase (ß-gal) gene (both cell lines kindly provided by N.P. Restifo, National Cancer Institute, Bethesda, Md). Lewis lung carcinoma (LLC; H-2b, syngeneic to C57BL/6), KLN205 (H-2d, a murine lung squamous cell carcinoma syngeneic to DBA/2 mice), and A549 cells, a human lung carcinoma, were obtained from the American Type Culture Collection (ATCC; Manassas, Va). CT26 was maintained in complete (10% fetal bovine serum, 100 µg/mL streptomycin sulfate and 100 U/mL penicillin G) RPMI 1640. CT26.CL25 was maintained in similar medium plus 400 µg/mL G418 (Invitrogen Corporation, Carlsbad, Calif). LLC and A549 cells were maintained in complete Dulbecco’s modified Eagle’s medium (DMEM). KLN205 cells were maintained in complete minimum essential medium with the addition of 0.1 mM nonessential amino acids (Invitrogen).
Adenovirus vectors
All Ad vectors used in this study are replication-deficient, E1-, E3-, serotype 5 vectors, and were propagated in human embryonic kidney (293) cells (ATCC), purified by 2 rounds of cesium chloride density gradient ultracentrifugations, then dialyzed as previously described.14,15 Viral particle concentration was determined by ultraviolet absorbance at 260 nm.16 AdPEDF contains an expression cassette with the cytomegalovirus early/immediate gene promotor/enhancer driving the human PEDF cDNA (GenVec Inc, Gaithersburg, Md).9 AdNull is a similar vector but contains no transgene.17
Western analysis
To confirm production of PEDF, A549 cells were transduced with AdNull or AdPEDF at an MOI (multiplicity of infection) of 104 particles per cell, then plated at a concentration of 5 x 105 cells/100 µL of serum-free DMEM into each well of a 96-well plate (Becton Dickinson, Franklin Lakes, NJ). Cells "transduced" with phosphate-buffered saline (PBS) served as further controls (sham transduced). After 48 hours of incubation at 37°C, the overlying supernatant was collected and subjected to Western analysis. Briefly, samples were resolved using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, then transferred to a nitrocellulose membrane (Bio-Rad, Hercules, Calif) for Western analysis using a monoclonal rabbit anti-human PEDF antibody (Chemicon International, Temula, Calif) as the primary antibody and a donkey anti-rabbit immunoglobulin G (IgG; Jackson ImmunoResearch Laboratories, West Grove, Pa) as the secondary antibody. Immunoreactivity was detected by chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). Recombinant human PEDF peptide (residues 196 to 347, 46 kDa) was used as a positive control (100 ng; Chemicon International).
Subcutaneous flank tumor model and vector administration
Tumor cells (2 x 105 CT26, 5 x 105 LLC, or 2.5 x 105 KLN205) were injected subcutaneously into the flanks of syngeneic mice. When the tumors reached approximately 30 mm2 (day 7 to 12), PBS, AdNull (5 x 1010 particles/100 µL PBS), or AdPEDF (5 x 1010 particles/100 µL PBS) was injected intratumorally. Because some tumors grew into oblong nodules, as opposed to spheres, the size of the flank tumor was assessed in situ every 2 to 3 days by measuring the largest perpendicular diameters using microcalipers, and recorded as average tumor area (mm2). When the animals appeared moribund or the tumor growth exceeded 15 mm in largest diameter, the mice were sacrificed, and this time point was defined as death for survival analysis.
To confirm PEDF production in vivo, established CT26 flank tumors were injected with PBS, AdNull, or AdPEDF as described above. Three days after intratumoral vector administration, flank tumors were harvested, homogenized in lysis buffer, centrifuged, and concentrated using Microcon centrifugal filter units (Millipore, Bedford, Mass) to eliminate proteins larger than 100 kDa and smaller than 10 kDa. After concentration, the filtrate was subjected to Western analysis as described above.
Pulmonary metastasis model and intrapleural vector administration
A murine pulmonary metastasis model was established by injecting 3 x 105 CT26.CL25 cells suspended in 100 µL of PBS into the right internal jugular vein of BALB/c mice. Vectors were then administered into the right pleural cavity 1 day after the intravenous tumor bolus.18 Intrapleural administration of dilute (1:5 in PBS) methylene blue dye (1 mL) was initially performed to evaluate whether interpleural communication exists in BALB/c mice. To perform intrapleural gene delivery, mice were anesthetized via intraperitoneal injection with a cocktail of ketamine (100 mg/kg) and xylazine (10 mg/kg). The trachea was cannulated with a 22-gauge angiocatheter (Becton Dickinson) and mechanical ventilation was achieved with a small animal ventilator (Harvard Apparatus, Holliston, Mass). Next, a 5-mm right anterolateral thoracotomy was performed at approximately the level of the fifth intercostal space. Either PBS, AdNull (1010 particles/100 µL PBS), or AdPEDF (1010 particles/100 µL PBS) was injected into the hemithorax, followed by a single simple absorbable suture to close the interspace. The skin was closed with absorbable 4-0 suture. Once spontaneous respirations were noted, the intratracheal angiocatheter was removed, mechanical ventilation discontinued, and the pretracheal incision sutured. Tumor burden was then quantified 13 days after vector dosing and animal survival was recorded.
Quantification of tumor burden in the lungs
To quantify lung tumor burden 3 different endpoints were examined: lung weight, gross and microscopic inspection, as well as lung ß-gal expression. Thirteen days after vector dosing, animals were sacrificed at designated time points, and the lungs were harvested en bloc and dissected free from the thymus, heart, and other mediastinal structures. For each mouse, the right and left lungs were isolated and each immediately weighed (wet weight). Next, lungs were homogenized in lysis buffer, and ß-gal gene expression was measured in a luminometer (Monolight 3010C, PharMingen, San Diego, Calif) using the Galacto-Light Plus kit (Tropix, Bedford, Mass). In addition, the total protein was quantified for each lung using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, Calif). ß-gal expression in the lung is reported as total activity in relative light units (RLU) normalized to total protein.
Lungs from additional animals were stained with India ink and subjected to gross inspection.19 Briefly, the animals were sacrificed, the trachea was cannulated in situ with a 22-gauge angiocatheter, and 1 mL of 15% India ink (Sanford, Bellwood, Ill) solution was slowly infused into the lungs. Next, the intrathoracic organs were harvested en bloc and placed in Fekete’s solution (70% ethanol, 10% formaldehyde, 5% acetic acid) at 4°C overnight. Gross photographs were taken the next day. Finally, lungs were fixed and stained for histologic evaluation. To accomplish this, the animals were sacrificed, the trachea was cannulated in situ with a 22-gauge angiocatheter, and 1 mL of 4% paraformaldehyde (PFA) was slowly infused into the lungs. Next, the intrathoracic organs were harvested en bloc and placed in a 4% PFA at 4°C overnight. The lungs were then embedded in paraffin, and butterfly-shaped sections (5 µm) were cut and stained with hematoxylin-eosin, followed by evaluation using photomicroscopy.
Evaluation of microvessel density
Six days after injection of LLC flank tumors with PBS, AdNull, or AdPEDF, tumors were harvested, embedded in optimum cutting temperature compound (Sakura Finetek, Torrance, Calif), snap-frozen in a 2-methylbutane/dry ice bath, and 8 µm frozen sections were cut. Immunohistochemical staining for endothelial cells was then performed to determine microvessel density. Briefly, frozen sections were dried for 90 minutes, blocked with normal rabbit serum (Vector Laboratories, Burlingame, Calif) for 30 minutes, washed with PBS, and incubated with rat anti-mouse CD34 monoclonal antibody (Pharmingen, San Diego, Calif) for 3 hours. Slides were then washed with PBS, incubated with biotinylated rabbit anti-rat IgG antibody (Vector Laboratories) for 60 minutes, washed again, and incubated with an avidin-horseradish peroxidase complex (Ventana Medical Systems, Tucson, Ariz) for 8 minutes. Following a final wash, slides were exposed to 3'3' diaminobenzidine chromogen substrate for 8 minutes, washed, stained with hematoxylin for background staining, rehydrated, and mounted for microscopy. To perform a quantitative assessment of microvessel density, 2 different, blinded observers counted the number of vessels per high power field (HPF; 40x). This was performed for 6 HPFs per treatment group. Data are reported as the mean (±standard error) number of vessels counted per HPF for the 3 treatment groups.
Statistical analysis
All data are reported as mean ± standard error. Statistical significance between means was determined using the unpaired, two-tailed Student t test. Survival evaluation was carried out using Kaplan-Meier analysis, with the P value determined by log-rank analysis (SPSS).
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Results |
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Intratumoral administration of AdPEDF inhibits the growth of established flank tumors and prolongs mouse survival
The ability of PEDF gene transfer to inhibit tumor growth in vivo was first evaluated by injecting established subcutaneous flank tumors with AdPEDF and following their growth over time. Experiments were performed in three syngeneic tumors (2 lung and 1 colon) in three different but corresponding syngeneic strains of mice. The administration of AdPEDF significantly inhibited the growth of CT26 tumors when compared with either AdNull or PBS (Figure 2, A,
AdPEDF versus AdNull, P < .01). In addition, intratumoral administration of AdPEDF resulted in prolonged survival of animals (Figure 2, B, AdPEDF versus AdNull, P < .005).
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Intrapleural delivery of AdPEDF decreases tumor burden and prolongs survival in a murine pulmonary metastasis model
To enhance the ability to quantify tumor burden in the lungs, pulmonary metastases were induced using a cell line that expresses a marker gene. BALB/c mice were injected intravenously with CT26.CL25 cells and lungs were harvested on subsequent days in the absence of any treatment. At each time point, both wet lung weight and ß-gal gene expression were used to quantify lung tumor burden. Following injection of tumor, but no therapy, both left and right lung weights increased incrementally as a function of time (Figures 3, A and B).
Similarly, ß-gal gene expression increased in a corresponding fashion (Figures 3, C and D).
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Discussion |
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Given the antiangiogenic properties of PEDF, this study is based on the hypothesis that in vivo PEDF gene transfer using an Ad vector may inhibit tumor growth. Consistent with this hypothesis, the data demonstrate that Ad-mediated transfer of the human PEDF cDNA to established murine tumors inhibits tumor growth and prolongs animal survival; this inhibition correlates with a decrease in tumor vascularization. Furthermore, intrapleural administration of AdPEDF results in suppression of growth of pulmonary metastases and enhances host survival.
AdPEDF as an antineoplastic agent
Angiogenesis is a complex, multistep process that is essential for the growth of tumors and their metastasis.1-3 Because PEDF has been shown to be a potent inhibitor of angiogenesis, its role as an antitumor agent is intuitive. In this regard, the present study clearly demonstrates the antineoplastic effect of PEDF in two separate, syngeneic models of thoracic malignancies. Lung cancer and metastatic colon carcinoma to the lungs were chosen as models to evaluate AdPEDF, given that these are common thoracic malignancies where novel therapies are desirable.
Intrapleural gene delivery as a strategy to express proteins in the pulmonary parenchyma is a novel approach that has only recently been described.18 As gene expression is seen primarily in the periphery of the lung using this technique, penetration of the vector through the visceral pleura represents the most likely mechanism. The observation that instillation of AdPEDF into 1 pleural space results in tumor suppression bilaterally is best explained by the communication between hemithoraces in the BALB/c mouse,18 which was readily demonstrable with methylene blue dye. In this regard, intrapleural administration of AdPEDF for pulmonary metastases in humans will likely require bilateral dosing. In addition, the effect of the relatively large size of the human pleural space (compared with the mouse) may impact on the excellent results seen in this murine model. Despite these potential limitations, intrapleural therapy for metastatic lung disease is a novel strategy with potential clinical application.18
An Ad vector was chosen as the delivery vehicle in this study for the following reasons. First, Ad vectors are well known for their ability to induce high levels of gene expression after multiple routes of administration22; second, Ad vectors induce transient transgene expression, a quality that is desirable in an antineoplastic therapy; and third, Ad vectors, when administered intratumorally, induce localized transgene expression, potentially limiting any systemic toxicity.22 Whether systemically administered PEDF will also inhibit tumor growth is unknown but needs to be evaluated, as does its potential toxicity. Despite this, the concept of local antiangiogenesis remains an important one for the treatment of locally advanced tumors of the chest, including lung cancer and pulmonary metastases, as well as pleural tumors, where local disease progression is often problematic.
The antitumor effect of AdPEDF may be due to inhibition of angiogenesis
The following evidence suggests that the antitumor effect of PEDF is due to its antiangiogenesis properties. First, AdPEDF has previously been shown to inhibit angiogenesis in a rat corneal pocket assay8 and in murine models of induced retinal and choroidal neovascularization.9-12 Second, immunostaining of tumor microvasculature in the present study demonstrated decreased microvessel density in AdPEDF-treated tumors. Third, consistent with the antineoplastic effect of antiangiogenesis agents,1-3 tumors treated with AdPEDF did not regress and the animals were not cured, although growth was inhibited and survival was significantly prolonged. Whether or not other mechanisms of tumor growth inhibition (eg, induction of tumor cell apoptosis or tumor immunity) by AdPEDF plays a role in its antineoplastic effect was not directly assessed in this study but warrants further investigation. Finally, although AdPEDF provided a survival benefit, this effect was modest, implying that the tumors eventually are able to circumvent the antiangiogenesis induced by PEDF. As a result, future investigation will involve strategies to "attack" the angiogenesis process at multiple levels to make the antitumor effect last longer. Given the transient nature of the antineoplastic effect of AdPEDF, such studies are needed prior to investigating this approach in a clinical setting.
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Acknowledgments |
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Footnotes |
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60 kDa in lanes 1 to 3 is nonspecific.
