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Carcinogenesis Advance Access originally published online on April 13, 2007
Carcinogenesis 2007 28(9):2053-2058; doi:10.1093/carcin/bgm091
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

The use of a cyclooxygenase-2 inhibitor (Nepafenac) in an ocular and metastatic animal model of uveal melanoma

Jean-Claude A. Marshall*, Bruno F. Fernandes, Sebastian Di Cesare, Shawn C. Maloney, Patrick T. Logan, Emilia Antecka and Miguel N. Burnier, Jr

The Henry C. Witelson Ophthalmic Pathology Laboratory and Registry, McGill University Health Center, 3775 University Street, Lyman Duff Building, Room 216, Montreal, Quebec H3A 2B4, Canada

* To whom correspondence should be addressed. Tel: +1 514 398 3456 ext. 4; Fax: +1 514 398 5728; Email: jeanclaude.marshall{at}gmail.com


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The expression of cyclooxygenase-2 (COX-2) has been reported as an indicator of poor prognosis in a wide variety of human tumors, including colon, breast and uveal melanoma (UM). COX-2 inhibitors have shown promise in controlling the malignancy of several types of tumors. Previous studies have demonstrated the efficacy of a COX-2 inhibitor on the proliferation rates of human UM cells. The goal of this experiment was to investigate the efficiency of Nepafenac, a topically administered COX-2 inhibitor, in a rabbit model of UM. The animals were divided into two groups of 14 animals for the duration of the 12-week experiment. One animal per group was killed each week to evaluate disease progression and for histopathological studies. The experimental group received drops containing 0.3% Nepafenac solution. Intraocular tumor growth was evaluated weekly by fundoscopic examination and each animal was weighed prior to examination. Blood samples were taken weekly from all rabbits to detect circulating malignant cells (CMCs) throughout the experiment. After the second week of inoculation, the experimental group weighed significantly more than the control group. The control group developed more intraocular tumors and presented with metastases and higher detectable levels of CMCs before the treated group. These results indicate that the topical administration of a COX-2 inhibitor delayed the progression of this malignancy in our animal model. A clinical trail using an anti-COX-2 inhibitor for patients with UM should be considered.

Abbreviations: CMC, circulating malignant cell; COX-2, cyclooxygenase-2; CsA, cyclosporin A; PCR, polymerase chain reaction; UM, uveal melanoma


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Uveal melanoma (UM) is the most common primary intraocular tumor in adults. The ability of ophthalmologists to currently diagnose the primary tumor has increased from an average accuracy of 87.5% in 1980 (1) to ~99.5% in 1990 (2). Local treatment has improved alongside diagnosis, with the development of conservative radiotherapy versus the previous standard of treatment, enucleation. Despite advances in diagnosis and treatment of the primary tumor, the mortality rates of UM have not significantly changed over the past decades (3). The survival rates at 5, 10 and 15 years after diagnosis are 65, 52 and 46%, respectively (4,5). The principal target organ for metastasis is the liver, involved in 71.4–87% of patients with metastatic disease (6–8). However, a recent study showed that almost 40% of patients present with non-liver sites of first metastasis, with lung being the most common (24.4%) (9). Unfortunately, if liver metastases are diagnosed, treatment options are limited and life expectancy is short. After the first evidence of liver disease, the median survival is <6 months (10). It is therefore apparent that novel targets for local or systemic therapy that will affect patient prognosis are needed and must be explored.

Cyclooxygenase-2 (COX-2) has previously been correlated with features of worse prognosis in UM patients (11) and it's expression has been reported in a wide variety of malignant tumors (12–14). COX-2 is an inducible enzyme expressed in response to a variety of inflammatory and mitogenic stimuli (15). The expression of COX-2 has been linked to various processes including tumor proliferation (16), immunosuppression (17) and metastasis (18,19). Specific COX-2 inhibitors are currently in use for patients diagnosed with a genetic disorder that predisposes patients to colonic adenocarcinomas called familial adenomatous polyposis (20). The effectiveness of these selective inhibitors has been investigated and shows promise for use as an adjuvant therapy in many tumor types (21). These inhibitors have not been previously tested for use in UM patients.

Nepafenac is a COX-2 inhibitor that is currently approved by the FDA in the USA for the treatment of ocular inflammatory processes such as uveitis (22–24). The drug has been shown to have a good penetrance in the eye and is formulated for topical use (22). Topically administered Nepafenac may avoid some of the safety issues that have been reported for systemic COX-2 inhibitors such as increased risk for acute myocardial infarction (25). We therefore sought to investigate the potential use of this COX-2 inhibitor in a previously established xenograft animal model of UM (26).

Several UM animal models are currently in existence (27–29); however, to the best of our knowledge, our model is the only model that implants human UM cells into the suprachoroidal space of the rabbit eye (26). Although this requires immunosuppression of the animals, it also gives rise to large intraocular tumors and subsequent formation of lung and liver metastases. Therefore, this model is ideal for characterizing the effects of a topically administered COX-2 inhibitor on the formation of the intraocular tumor and metastases in these animals.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
The animal model was carried out in compliance with the Association of Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. The approval of both the Animal Care Committee and the Ethics Subcommittee at the McGill University was obtained prior to all experiments.

Animals
Twenty-eight male New Zealand albino rabbits (Charles River Canada, St Constant, Québec, Canada) were randomly divided into two groups, control and experimental, with mean initial weights of 3.2 ± 0.18 kg and 3.2 ± 0.17 kg, respectively. Male animals were used to avoid any effects on the experimental study due to possible hormonal changes. The animals were immunosuppressed using daily intramuscular injections of cyclosporin A (CsA) (Sandimmune 50 mg/ml, Novartis Pharmaceuticals Canada, Dorval, Québec, Canada) in order to avoid rejection of the human cells. CsA administration was maintained throughout the 12-week experiment to prevent tumor regression. The dosage schedule recommended in previous studies was employed: 15 mg/kg/day, 3 days before cell inoculation and during 4 weeks thereafter, followed by 10 mg/kg/day during the last 8 weeks of the experiment (30). CsA doses were adjusted weekly according to the animal weight to compensate for decreased animal weight and possible toxicity.

Cell line and cell injection procedure
The injection procedure and subsequent animal handling were carried out as described previously (26). The 92.1 primary human UM cell line (31), kindly provided by Dr Antonia Saornil from the Instituto Universitario de Oftalmobiología Aplicada, University of Valladolid, was used. This selection was based on previous studies performed in our laboratory where these cells showed to have a high proliferative and invasive potential in vitro (32). The cells were maintained at 37°C in a humidified 5% CO2-enriched atmosphere (Thermo Forma Series II Water Jacketed CO2 Incubator, Fisher Scientific Limited, Ontario, Canada). The cells were cultured in RPMI-1640 medium (Invitrogen, Burlington, Ontario, Canada), supplemented with 5% heat inactivated fetal bovine serum (Invitrogen), 1% fungizone (Invitrogen) and 1% penicillin–streptomycin (Invitrogen). One million cells (cellular viability >98%) suspended in 0.1 ml of RPMI-1640 media were injected into the suprachoroidal space of the right rabbit eye according to a previously described technique (30). Ketamine (35 mg/kg; Vetalar, Vetrepharm Canada, Belleville, Ontario, Canada) and xylazine (5 mg/kg; Anased, Novopharm Limited, Toronto, Ontario, Canada) were used as anesthetics during the surgical procedure.

Drug administration
Animals from both groups were dosed twice a day. The control group received two drops twice a day of the vector in the inoculated eye. The experimental group received two drops twice a day of 0.3% formulation of Nepafenac. The vector and drug formulations were both provided by Alcon Canada (Mississauga, Canada).

Follow-up
Animal weight.
During the 12-week experiment, the animals were monitored daily for signs of CsA toxicity including regurgitation, diarrhea, dehydration and weight loss. A statistical analysis of the rabbit weights from both groups was performed using the statistical software package SAS. Data were analyzed as a repeated measures design using mixed models, with time (weeks) as the within-subject factor and treatment group as the between-subject factor. For all statistical evaluations, P values of ≤0.05 were considered to indicate significance.

Fundoscopy.
Indirect ophthalmoscopy of dilated pupils using Tropicamide (Mydriacyl, Alcon Canada) was performed before cell inoculation, to rule out any existing ocular pathologies, and weekly after cell inoculation to clinically document intraocular tumor development.

Euthanasia.
In order to document the time-course of the disease, particularly the development of metastasis, one animal per group was euthanized per week starting on the second week after the inoculation of cells into the eye. The selection criterion was based on the appearance of the animal, signs of CsA toxicity and veterinary recommendations. The remaining rabbits of each group (N = 4) were killed at the end of the experiment. The method of euthanasia was exsanguination following anesthesia using intramuscular ketamine and xylazine (35 and 5 mg/kg). An autopsy was performed on every animal that was killed. The enucleated eyes and other organs with possible metastatic disease such as lungs, livers and kidneys were collected, macroscopically examined and preserved in 10% phosphate-buffered formalin.

Immunohistopathological studies
Formalin-fixed, paraffin-embedded sections of the collected specimens were stained with hematoxylin and eosin for histopathologic assessment. Immunohistochemistry using the HMB-45 monoclonal antibody (1:50 dilution; DakoCytomation, Mississauga, Ontario, Canada) and the COX-2 monoclonal antibody (1:500 dilution; Zymed Laboratories, San Francisco, CA; clone COX 229) were performed using the Ventana BenchMark (Ventana Medical Systems, Tucson, AZ) fully automated machine. Cytospins of the 92.1 cell line that was used for injections into the eyes were also stained with both antibodies. The fully automated processing of the barcode-labeled slides included baking of the slides, solvent-free deparaffinization and antigen retrieval. The COX-2 staining was ranked for intensity as weak, moderate or high and for the percentage of cells in each tumor by two independent pathologists. Conflicting results were resolved by mutual agreement.

Two different statistical tests were used to determine significance of metastatic formation between the two groups. The statistical software package SAS and R were used. The first method was a cumulative incidence analysis and the second was Kaplan–Meier curve and groups were compared with log-rank test.

Blood sample processing and real-time quantitative polymerase chain reaction analysis
Peripheral blood samples were obtained weekly from each rabbit and at the moment of euthanasia by cardiac puncture. RNA from the blood was extracted using the RiboPure blood kit (Ambion, Austin, TX) as per the manufacturer's recommendations. A minimum of 4 ml of blood per rabbit was processed weekly and the acquired RNA was subsequently frozen at –80°C until used for quantitative real-time polymerase chain reaction (PCR) analysis. The presence of circulating malignant cells (CMCs) was determined by real-time PCR performed using Quantitect one step SYBR Green PCR (Qiagen, Mississauga, Ontario, Canada) as per the manufacturer's instructions. A Chromo4 thermocycler (MJ Research, Waltham, MA, USA) was used for all experiments and all results were analyzed using the GeneEx software. QuantiTect primer assay pairs (Qiagen) for both Melan A and the housekeeper gene Beta actin were used for each gene product of interest as per the manufacturer's instructions.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Survival rate, weight and general animal condition
The animals remained in good condition throughout the experiment, presenting only minor signs of CsA toxicity including occasional diarrhea or, in some cases, hypersalivation. One of the major indicators of animal health was weight loss or gain. As previously observed, an initial acute weight loss 1 week after surgery occurred (26). The average weight of the animals for the two groups over the course of the experiment is shown in Figure 1. A statistically significant difference between the weights of the two groups was seen beginning at week 3 and continued until the end of the experiment indicating better animal health in the treated group.


Figure 1
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  		Fig. 1. The average weight of the animals in each group, control and experimental, throughout the course of the 12-week experiment is shown. The difference between groups is statistically significant starting on week 4.

 
Intraocular tumor studies
Fundoscopy.
During the 12-week experiment, fundoscopy examination revealed intraocular tumors in 8 of the 14 animals in the control group and in 4 of 14 in the experimental group. The first tumor detected was on the fourth week in both groups and after the seventh week, the total number of animals with detectable tumors remained unchanged. Observed tumors initially presented as flat patches of choroidal thickening that further progressed into subretinal masses of variable sizes and shapes as shown in Figure 2. As a consequence of tumor growth, some eyes developed retinal detachment and vitreous hemorrhage throughout the experiment.


Figure 2
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  		Fig. 2. Intraocular tumors. (a) A small tumor seen in a rabbit of the treated group through the dilated pupil (asterisk). (b) An eye from the control group with a large tumor and vitreous hemorrhage, completely filling the vitreous cavity. (c) Another flat discoid tumor is seen in a rabbit from the control group (asterisk). (d) Neovascular glaucoma was a complication due to tumor growth, seen as large vessels within the iris and corneal edema in this rabbit from the control group.

 
Gross and histopathological examination.
The enucleated eyes were examined post-mortem to determine the presence and morphology of the developed intraocular tumor's post-mortem (Figure 3). Macroscopically detectable masses were seen in five (35%) animals of the control group and three (21%) animals in the experimental group. Histopathological evaluation of the enucleated eyes revealed that tumors were present in 12 (86%) of the animals in the control group and in five (36%) of the experimental group. Melanomas were confirmed by positive staining with HMB-45 monoclonal antibody.


Figure 3
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  		Fig. 3. Intraocular tumors. (a) Macroscopy of an enucleated eye showing a large tumor (arrow heads). (b) HMB-45-positive immunostaining (red chromogen, original magnification x400). (c) High immunohistochemical expression of COX-2 in a rabbit in the control group (red chromogen, original magnification x400). (d) Lower immunohistochemical expression of COX-2 in a rabbit in the treated group (red chromogen, original magnification x400).

 
All intraocular tumors were comprised of epithelioid cells and presented with areas of necrosis. A high mitotic index was found, average of 34 in 10 high-power fields, with no statistical difference between the two groups (P = 0.12). Extraocular extension was seen in seven eyes (50%) of the control group and one eye (7%) of the experimental group, a statistically significant difference (P = 0.025).

COX-2 expression was diffuse throughout each intraocular tumor sample. In the control group, seven eyes had sufficient intraocular tumor size to perform immunohistochemical studies and showed a strong staining intensity for COX-2 expression. Of the five eyes from the experimental group that had intraocular tumors, one showed weak, two showed moderate and the remaining two tumors showed strong staining intensity. The cytospin of the original cell line did not stain for COX-2 expression in vitro.

Malignant cell dissemination.
CMCs were detectable in all rabbits from each group during at least one time point of the experiment. A general pattern of detection was seen for the two groups. In the control group, expression of Melan A was found in the blood samples starting from 1 week after inoculation of the cells into the eye of the rabbits. In contrast, expression of Melan A in the experimental group was not seen in any of the rabbit samples prior to week 3. This represents a delay of ~2 weeks for the appearance of detectable CMCs in the experimental group as compared with the control group.

Expression of Melan A in the control group generally followed a sinusoidal pattern, increasing from week 1 to week 4, then decreasing from week 5 to 7 and then beginning to increase again to week 12. This same sinusoidal pattern was seen in the blood samples from the experimental group. The overall expression of Melan A was on average 10- to a 100-fold lower in the experimental group, possibly indicating fewer CMCs in the treated animals (Figure 4).


Figure 4
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  		Fig. 4. Quantitative real-time PCR showing the detection of CMCs from the peripheral blood of rabbits that continued to the end of the 12-week experiment. CMCs were detected from week 1 in the control group and week 3 from the experimental group.

 
Metastasis.
Macrometastasis appeared as numerous hard white nodules usually located at the periphery of the inferior lobes of the lungs (Figure 5). Macroscopically detectable lung metastases were seen after the seventh week of the experiment in both groups. Lung masses were a consistent finding until the end of the experiment in the control group. In the experimental group, lung masses were only found in the 8th and 12th week. The end of the experiment found macroscopically detectable lung metastasis in six (43%) of the control rabbits and four (29%) of the experimental rabbits.


Figure 5
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  		Fig. 5. Metastatic disease. (a) Macroscopic view of a rabbit's lung where the metastases are seen as hard white nodules in the lower lobes (arrow). (b) Immunohistochemical expression of HMB-45 in a microscopic lung nodule (red chromogen, original magnification x400, arrow heads). (c) A microscopic liver nodule in a rabbit from the control group (hematoxylin and eosin, original magnification x400, arrow heads).

 
Histopathological and immunohistochemical examination of serial sections of the animal's lungs revealed metastatic disease in 10 animals in the control group and in eight animals in the experimental group (Figure 5). Metastatic disease was seen as clusters of HMB-45-positive malignant cells within the lung parenchyma (Figure 5). Liver metastasis was seen microscopically in one animal of the control group (Figure 5).

The cumulative incidence of metastases between the control and treatment group were significantly different (P = 0.02, Gray K-sample test), with the cumulative incidence of metastasis being higher in the control group. This was corroborated using the Kaplan–Meier curve, where metastasis occurred in the control group earlier than those in the treatment group (P = 0.02, log-rank test).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
To the best of our knowledge, this is the first time that local treatment of the eye with a COX-2 inhibitor in a xenograft animal model using human UM cells implanted into the eye has shown a systemic effect on metastatic formation. By killing one animal per week per group, we were able to histopathologically track the progression of the disease throughout the 12-week experiment.

This rabbit model of UM was previously published as the first to demonstrate micrometastases in the liver using human cell lines inoculated in the eye (26). We utilized the 92.1 cell line for this experiment as several studies have shown this cell line to be extremely aggressive in both in vitro and in vivo experiments (32,33). This aggressive phenotype was reflected in the model as with tumors expressed several histopathological indicators of worse prognosis including high mitotic index, vascular loops, areas of necrosis and epithelioid cell type (34). We have shown previously that the active metabolite of Nepafenac, Amfenac, can decrease the proliferation rate of human UM cell lines and increase the cytotoxic response of macrophages to these tumor cells (35). It is interesting to note that in the current model, we found liver metastasis only in a rabbit from the control group; none of the experimental group had detectable micrometastasis in the liver.

Although a percentage of the topically administered drug may be absorbed systemically, the comparatively low concentration of the active drug that is administered may result in a reduction of the risk of systemic side effects as compared with systemically administered COX-2 inhibitors (25). Previously published reports have shown the ability of Nepafenac to diffuse into the eye with little to no systemic effects (22–24). We also did not encounter any signs of ocular surface complications due to the repeated doses of the drug throughout the 12-week-long experiment.

By all veterinary standards of animal well being (animal weight, grooming and behavior), the control group did significantly worse than our treatment group. The animals were weighed weekly to assess potential signs of cyclosporin toxicity as well as to assess overall health. These weights showed a significant divergence between the two groups after the third week of the experiment with the experimental group demonstrating better general health. Animal weight is an accepted measurement of well being that is used by veterinarians to determine overall animal health. Detectable tumors by fundoscopy, macroscopic examination and microscopic immunohistochemical evaluations also showed a difference between the two groups. The experimental group had a total of five verifiable intraocular tumors, whereas the control group had a total of 11 tumors.

Staining for COX-2 expression was carried out on all intraocular tumors. Interestingly, the 92.1 cell line did not express COX-2 in culture prior to implantation into the eye of the animals, indicating that the microenvironment of the eye was necessary to induce COX-2 expression in the cells. All tumors expressed COX-2 to some degree; however, the tumors from the control group were all classified with high staining intensity. In contrast, only two tumors from the experimental group expressed high intensity staining, whereas the other three were classified as either moderate or weak. It is still unclear how Nepafenac functioned to inhibit the formation of more intraocular tumors in the experimental group as compared with the control group. Possible mechanisms that have previously been described include the inhibition of angiogenesis (36), proliferation (16), migration and invasive ability (37). All tumors expressed COX-2 staining to at least some degree. It is unclear why the experimental group showed lower expression of COX-2 staining as compared with the experimental group, although it may be possible that Nepafenac has an additional method of action. Several other COX-2 inhibitors have shown COX-2-independent methods of action, which may explain the differences in COX-2 expression in these groups (15,38). Alternatively, this reduction in COX-2 expression may reflect a less malignant phenotype of the intraocular tumor. Further investigation is required to address these questions.

Large nodular metastases in the lung were seen in five animals from the control group, starting at week seven of the experiment. In comparison, the experimental group developed nodular metastases in only two animals and these were not present until the 12th week of the model. UM cells were confirmed by immunohistochemical staining with HMB45 and Melan A from week 3 onwards in the animal lungs from both the control and experimental group. The cumulative incidence of micrometastasis was much higher in the control group, with the experimental group only beginning to present with micrometastases after the seventh week. This suggests that these cells took longer to reach the lungs in the treated group and took longer to establish large metastatic nodules as compared with our control group. To the best of our knowledge, this is the first time that a topically administered drug has delayed the development of metastatic disease. We used Melan A primers to detect CMCs in our samples based on previous work done by our laboratory which showed that Melan A was more specific and sensitive than tyrosinase for detecting CMCs using PCR (39). Using this specific methodology, we were able to detect CMCs to weeks earlier in the control group compared with the experimental group.

There are three different possible mechanisms for the aforementioned inhibition of large metastasis formation. The first may be the local effect of the COX-2 inhibitor on the primary tumor due to effects on proliferation, angiogenesis, and may prevent cells from leaving the eye by decreasing motility and invasive ability of these cells. While this probably plays a role in reducing the metastatic burden in the experimental group, we believe that there may be additional explanations for the differences between the two groups. Considering that we detected microscopic metastases and CMCs in both groups, it is apparent that cells were capable of exiting the eye irrespective of COX-2 inhibition. Thus, the second effect may be that the treatment with a COX-2 inhibitor altered the malignant cells that escaped from the eye in a fashion that inhibited these cells from giving rise to large metastatic nodules. It is also possible that the small amount of drug that becomes systemic after the topical administration is sufficient to affect the formation of these metastases. The attractive use of this topical therapeutic is the relatively low risk of systemic side effects. It is possible that a combination of two or all the proposed mechanisms of COX-2 inhibition that contributed to our presented results. Future studies are necessary to investigate what level of COX-2 inhibition and the mode of administration required to replicate the decrease in metastases seen in this model prior to clinical trials.


   Acknowledgments
 
We would like to thank the help and support provided for this animal model by the McGill University Animal Resource Center. In particular, we would like to thank Lori Burgess, Karen Stone and Dr Lynn Matsumiya. We would like to thank Dr. Martine Jager for the establishment of the 92-1 cell line.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 1, 2007; revised March 15, 2007; accepted March 30, 2007.


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