Tumor Models in Rodents

PURPOSE:

These veterinary recommendations are intended to assist the investigator in designing and conducting tumor model studies in rodents.  It is recognized that each study has certain unique characteristics and requirements and that the recommendations in this document are not universally applicable. The goal is to provide guidance on acceptable end-points and overall study design to assist the investigator in gaining successful IACUC approval of required experimental studies. 

BACKGROUND:

Tumor rodent models are critically important experimental model systems. Rodent tumor model systems are used in cancer biology where the goal is to study the growth and behavior of the tumors and in cancer treatment where the goal is to evaluate chemical, radiologic or immunologic therapies as potential cancer treatments.  Three different general rodent tumor model systems are in common use:

• Injection of tumor cells which can be used both for propagating a tumor cell line and to study various treatment regimens.  Transplantable tumors may be injected either orthotopically, in the tissue or site of origin, or ectopically, usually by subcutaneous injection in the flanks of the rodents, intraperitoneal injection, or intravenous injection.  To promote the engraftment of some experimental tumor lines, it may be necessary to modify the recipient’s immunologic or physiologic status. Low-level whole body irradiation or immunosuppressive agents may be used to further suppress the immune response of immunodeficient rodents (nude or SCID mice) before inoculation with human tumor xenografts.
• Spontaneous tumor development in specific inbred strains, spontaneous mutant strains, and genetically engineered mice.  In these cases the specific strain of rodent typically has a high and well-documented incidence of tumor development at specific age points.  These models are commonly used to study environmental and preventive therapeutic effects on tumor incidence and development, the specific genetically based mechanism resulting in tumor development, and cancer therapies.
• Tumors in rodents may be induced by chemicals, radiation (ultraviolet light), inflammation, infection, etc.  Of these, the chemically induced tumor model systems are the most common and result from exposing the animal to a known and well-documented carcinogen with a predictable level of tumor induction.

The importance of limiting the discomfort, pain and distress that the animals may experience during the conduct of biomedical research is well recognized and a primary force behind the animal welfare regulations governing the use of animals in research. Outcomes of tumor studies, including death as an endpoint, vary depending on the species and strain of animals, the tumor induction method, and the subsequent chemotherapy or other modality in cancer treatment studies. It is up to the investigator, who should be the most knowledgeable of the available models, to decide which model to use, to design the study to minimize animal pain and distress, and to present these to the IACUC for approval. It is the responsibility of the investigator to ensure that a complete and thorough search for alternatives to painful and distressful tumor studies is performed and documented.  At all times during this process, the well being of the research animals must be balanced against requirements of the study.

Tumor Guidelines for Mice and Rats

I. General Guidelines:

• All injectable and/or implantable materials used for establishing tumors in animals generally must first be evaluated for the presence of viral and bacterial contaminants.  This evaluation is essential to prevent the introduction of murine infectious agents into the animal facilities with the potential to seriously disrupt or destroy the research colonies of other investigators.  This evaluation may be made either by mouse antibody production (MAP) testing or by polymerase chain reaction testing (PCR) at an approved contracted organization.  Please contact and consult with a DLAR veterinarian to determine if the implantable tumor line must be tested and the most efficient and economical method of testing if it is required.  In rare cases it may be possible to house tumor-injected animals under biocontainment conditions, allowing the injection of tumor lines prior to testing.
• All injectable human tumor lines require IBC review and approval prior to use.  The use of human tumor lines also requires enrollment in the University of Kentucky Exposure Control Plan for Bloodborne Pathogens (http://ehs.uky.edu/ehs/ohs/bloodpathogens.html).

II. Tumor Burden Guidelines:

• The measured size of a tumor is only one criteria used to determine a humane endpoint. The overriding consideration for humane endpoints of oncological experiments as well as spontaneous tumors must be the overall health of the animal. The site of tumor implantation should be chosen to minimize damage to adjacent normal structures. Sites involving the special senses should be avoided. Clearly defined endpoints must be stated in the IACUC protocol.  The following guidelines for subcutaneous tumors are provided as recommendations:
  • For subcutaneous tumors the recommended maximum size is 20 mm in maximum diameter for a mouse or 40 mm maximum diameter for a rat. If the animal is host to more than one tumor, this size is the maximum allowable size for all tumors combined.
    • Subcutaneous tumors should be measured using calipers at least twice weekly after the tumor becomes visible.  Once the tumor reaches 10 mm in diameter in mice or 20 mm in diameter in rats, the tumors should be measured every other day. If a rapid growing tumor line is being evaluated then the frequency should be daily. 
      • All measurements should be documented and readily available to husbandry and veterinary personnel should health or welfare issues arise.
  • Ulceration of the skin overlying or surrounding tumors should result in removal of the animal from the study and euthanasia.  If ulceration is not to result in removal of the animal from the study, this should be clearly stated in the animal use protocol approved by the IACUC.
  • Tumors should be injected in locations that will not interfere with the normal ambulation or physiologic-social actions of the animal upon increase in tumor volume.  If ambulation, physiologic, or social actions of an animal are impaired by the tumor development it should be removed from the study and humanely euthanized. 

Suggested Wording for Animal Use Protocol (mice):

Suggested Wording for Animal Use Protocol (rats):

• Systemically injected tumors (intraperitoneal, intravenous, etc.) are more difficult to monitor. Intraperitoneal tumors may expand to a significant volume without great physiologic effect or they may result in various blockages or physiologic changes that impact animal health and well being.  Intravenously injected tumor cells tend to localize in capillary beds, primarily in the lungs, potentially resulting in pulmonary distress and respiratory failure.  Specific criteria for endpoint determinations will vary based on the tumor cell line, the expected site of implantation/metastasis, and the route of injection.
  • The principal investigator is typically the person most familiar with the likely complications and disease resulting from the specific tumor cell injection.
    • Criteria for intervention should be clearly delineated and specific whenever possible:
      • Quantitative criteria such as body weight loss and decrease in daily activity levels are the best criteria when possible or applicable. 
      • Behavioral observations of decreased activity, reluctance to move, or social isolation may be used.
      • Physiologic criteria such as labored respirations, cyanosis, anemia, ascites development, decreased feces production, inanition, or neurologic abnormalities may be appropriate criteria for systemic tumors
      • Body condition scoring (less than 2) is often the best overall method for assessing the animal’s overall condition and establishing the most appropriate time for intervention to minimize animal pain and distress.

Suggested Wording for Animal Use Protocol:

• All tumor-bearing animals must be observed on a scheduled basis and findings documented to assess the progress of tumor growth and/or metastasis, and the general condition of the animal. Records must be readily available and include all pertinent information including time and frequency of monitoring sessions, the name of the person monitoring the animals, identification of the animals, protocol number, the number of animals displaying symptoms, types of symptoms, and any treatments given to the animals (see IACUC Policies, Procedures, and Guidelines 116, "Research Medical Records" for more information). In circumstances where declining health status, morbidity, or unrelieved pain or discomfort results in a Medical Report to DLAR Veterinary services, every attempt will be made to contact the investigator and to reach a consensus regarding animal disposition in accordance with approved experimental endpoints. However, the final analysis and discharging of the University of Kentucky animal care and use regulatory responsibility rests with the Attending Veterinarian or his/her designated clinical veterinarian.

III. Cancer Treatment Studies:

In this class of study, not only must the tumor burden be considered, but the effect of the treatment modality must also be evaluated. The purpose of all cancer treatments, whether radiologic, immunologic or chemical, is to destroy or disable the cancer cells while minimizing damage to healthy tissues. The success of a treatment becomes a balance between cancer destruction and reduction of side effects.

Outcomes in cancer treatment studies and criteria for removal of animals from the study should be based upon both the anticipated complications due to the growth of the tumor and the potential complications related to the therapeutic modality.  The investigator should be familiar with the tumor’s behavior in the untreated animal while toxicity data and MSDS sheets can provide information regarding the toxicity of chemotherapeutic agents. End-point criteria should reflect both the complications related to tumor development and the complications reasonably anticipated due to the therapeutic treatment. 

Outcomes of cancer treatment studies, including death as an endpoint, vary depending on the species and strain of animals, the route of injection for transplantable tumors and the subsequent chemotherapy or other modality in cancer treatment studies. It is up to the investigator, who should be the most knowledgeable of the available models, to decide which alternatives to using live animals are necessary for a study and present these to IACUC for approval.  Death as an endpoint may be allowed by the IACUC only after full consideration of alternatives and a subsequent finding that none are scientifically acceptable for the proposed outcome. At all times during this process, the well being of the research animals must be balanced against requirements of the study and with due consideration of the relevance of the studies to human or animal health, the advancement of knowledge, or the good of society(1).

REFERENCES:

1. U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training. 1983. (http://www.grants.nih.gov/grants/olaw/references/phspol.htm#USGovPrinciples)
2. Ullman-Cullere' MH, Foltz CJ. 1999. Body condition scoring: a rapid and accurate method for assessing health status in mice. Laboratory Animal Science 49:319-323.