Currently accepted methods of humane endpoint determination for subcutaneous tumor growth models in mice include tumor size (generally maximum size of 1 cm), presence or absence of ulceration, and changes in body weight. Our laboratory has previously shown that body weight is not a reliable sole method of assessment for many reasons. There can be high between observer variability due to observer technique, time of day, and equipment errors. And, because the increasing tumor mass may mask pathologic weight loss, the use of weight loss to assess the well-being of animals with bulk producing tumors is problematic. This study explored the use of body condition (BCS) scoring and appearance and behavior scoring as additional criteria to better assess morbidity in these animals. To complete this study, 2 different tumor models were assessed: a skin papilloma model with multiple tumors produced after exposure to appropriate carcinogens, and a subcutaneous melanoma study where a single, bulk-producing tumor was produced after subcutaneous injection of tumor cells. Our data analysis revealed that BCS was significantly more reliable than body weight as an indicator of anorexia when correlated with a moribund clinical condition. However, our assessments also found that tumor growth in these models was found to be well tolerated. Tumors over 1 cm in diameter did not result in a decrease in BCS. Instead, we found that BCS consistently did not decline until the tumor became ulcerated or interfered with mobility because of location on or near a limb. Based on the conclusions of this study, our IACUC has updated its tumor evaluation guidelines to allow tumors of up to 2 cm in diameter, as long as mobility is not decreased, there is no ulceration, and the mouse maintains a BCS of 2. The results of this study will be of interest to investigators and animal care staff working with similar studies.

Our study evaluated tumor size, body weight, body condition score, appearance scores, natural behavior scores, provoked behavior scores, and tumor characteristics (e.g. ulceration) to determine which method of evaluation is the most sensitive for identifying animals who have compromised animal welfare when being used on subcutaneous tumor studies. Of all of these characteristics, the most sensitive indicators was presence of ulceration. As ulcers developed on tumors of any size, the body condition score, appearance scores, and behavior scores of the individual animal declined, but the decline in these parameters lagged behind the development of the ulcerations. As our results show that these parameters consistently decline after the development of an ulceration, we suggest that an appropriate endpoint criteria for subcutaneous tumor studies is the presence of an ulceration. This study also confirmed that monitoring of weight loss is largely unrewarding for the determination of endpoints for mice on subcutaneous tumor growth studies. There was no significant change in average weight loss between the beginning (~32 grams) and ending (~32.5 grams) weights. We hypothesized that this was because muscle loss was masked by increase in tumor size, but as body condition score remained relatively stable from beginning (2.8) to end (2.6) as well, significant muscle wasting does not appear to be a component of these two subcutaneous tumor animal models. Previous endpoint recommendations for subcutaneous tumors have included euthanasia when tumors reach 1 cm in diameter. Our study has shown that the growth of tumors up to 2 cm in diameter at their widest point is well tolerated by the mice. A body condition score of 1 is typically used as an indicator of severe emaciation and stimulus for euthanasia. In our study, no mouse with a tumor less than 2 cm in diameter at the widest point developed a body condition score of less than 2 – unless an ulceration developed on the tumor. This suggests that the tumor burden is well tolerated in these two animal models of subcutaneous tumor growth and that larger tumors can be allowed, if indicated by the experimental protocol.

The author would like to thank Ms. Rachel Luksic, Mr. Bryan Bustamante, and Ms. Carla Webb for their assistance in data collection. This study was supported in part by a grant from the John Hopkins Center for Alternatives to Animal Testing (CAAT) and the Merck Merial Summer Student Fellowship program.

IntroductionIntroduction ResultsResults

Acknowledgements Acknowledgements

DiscussionDiscussion

Endpoints for Mouse Subcutaneous Tumor Models: Refinement of Current CriteriaE. Paster,1 K. Villines,2 & D. Hickman1

1VA Medical Center, Portland, OR; 2CVM, Oregon State University, Corvallis, OR

Endpoints for Mouse Subcutaneous Tumor Models: Refinement of Current CriteriaE. Paster,1 K. Villines,2 & D. Hickman1

1VA Medical Center, Portland, OR; 2CVM, Oregon State University, Corvallis, OR

Materials & MethodsMaterials & Methods

Comparison of Weight and Body Condition Score: There was no significant difference in the beginning and end weights and body condition scores of all of the animals that we assessed. The data for the papilloma model is presented below:

Mice: For the skin papilloma model, the subjects were males and females, ages 4-6 months, from a transgenic strain that develops skin papillomas after application of carcinogens. The transgenic strain was designated as crossbred TGF- ß1/ ΔßRII-transgenic . For the subcutaneous melanoma model, the mice were C57BL/6J males, aged 8-12 weeks.

Environment: All mice were housed on ventilated racks in shoebox caging on corncob bedding. Block rodent chow and water were provided ad lib. The light cycle was 12:12 and temperature and humidity were maintained at 69-71 F and a minimum of 30%, respectively. The colonies of mice were screened for pathogens on a quarterly basis, using indirect sentinels. At the time of the experiment, all colonies were known to be free of mouse corona virus, Sendai virus, mouse parvovirus, minute virus of mice, ectromelia virus, reovirus type 3, pneumonia virus of mice, murine adenovirus, Mycoplasma pulmonis, lymphocytic choriomenigitis virus, mouse rotavirus, mouse encephalomyelitis virus, polyoma virus, murine cytomegalovirus, and rodent pinworms and mites.

Tumor Induction: For the skin papilloma model (Figure 1), the tumors were induced by the weekly topical application of dimethylbenzanthracene (DMBA) and 12-0-tetradecanoylphorbol-13-acetate (TPA) for 20 weeks. For the subcutaneous melanoma model, 3 x 105 MO5 cells were injected subcutaneously once. This cell line is a variant of the B16 melanoma tumor cell line that has been engineered to express obvalbumin.

Monitoring: Animals were evaluated by two observers at least 3 times per week for body weight, BCS, tumor number, and size of the largest tumor (if multiple). Animals were also assigned a numerical score corresponding to their presentation in appearance, unprovoked behavior, and provoked behavior. If animals lost 20% of body weight or if their BCS dropped to 1, the animals were euthanized. Originally, the protocols described euthanasia when tumors reached 1 cm diameter. However, the IACUC approved a maximum of 2 cm after presented with data that showed that mice with a 1 cm tumor still had good grooming and a BCS of at least 2 (Figure 2). If the tumor was ulcerated or interfered with the animal’s ability to ambulate, the animal was euthanized by carbon dioxide asphyxiation.

Figure 1: Mouse with papillomas

Comparison of Tumor Size and Body Condition Score: A total of 9 mice monitored on this study achieved tumor sizes that exceeded 1 cm diameter, but less than 2 cm diameter at the widest point. For all of these mice, the body condition score remained at 3 (unchanged from the initial BCS) or dropped to 2, with good appearance and behavior scores (Figure 2). In two of these mice, the tumors developed skin erosions that progressed to ulcers. As the ulcers progressed, the body condition score of these mice declined to 2 or 1 and the appearance and behavior scores also declined. The remainder of the mice were euthanized because they reached the experimental endpoint for our collaborator who needed to perform tissue collection and post-mortem analysis, not because of concerns regarding animal welfare.

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Figure 2: Mouse with subcutaneous melanoma tumor and BCS of 3, good appearance, and good behavior.

Comparison of General Clinical Presentation: There was no significant difference in the beginning and end appearance scores, natural behavior scores, and provoked behavior scores of all of the animals that we assessed, unless an ulceration developed on the tumor.