Guidelines for the welfare and use of animals in cancer research

Abstract

Animal experiments remain essential to understand the fundamental mechanisms underpinning malignancy and to discover improved methods to prevent, diagnose and treat cancer. Excellent standards of animal care are fully consistent with the conduct of high quality cancer research. Here we provide updated guidelines on the welfare and use of animals in cancer research. All experiments should incorporate the 3Rs: replacement, reduction and refinement. Focusing on animal welfare, we present recommendations on all aspects of cancer research, including: study design, statistics and pilot studies; choice of tumour models (e.g., genetically engineered, orthotopic and metastatic); therapy (including drugs and radiation); imaging (covering techniques, anaesthesia and restraint); humane endpoints (including tumour burden and site); and publication of best practice.

Figure 1

An illustrative process for tumour model selection and use. This representative schema provides an illustration of factors to be considered when designing an animal study. In this particular example, all the factors listed at a given stage (and potentially others) should be considered before moving down, stepwise, to the next stage. Here, an initial consideration is that the choice of model may be based on the relevant molecular status, clinical tumour type or in vitro studies. At the next stage, the animal host will be dictated by the need for, say, a human tumour xenograft versus a genetically engineered mouse model, which have advantages discussed in the text. Considerations of tumour environment and site then follow, after which, in therapy studies, are dosing and endpoint aspects. Note that this schema is illustrative and not prescriptive and that each study must be tailored to the specific scientific question and experimental objectives, with appropriate humane endpoints always applied and pilot studies carried out as needed.

Figure 2

Example of a drug discovery test cascade for identifying small-molecule antitumour drugs. A representative test cascade for identifying a potential small-molecule drug against a given target is shown. A subset of a compound library is initially screened vs the target in vitro, in recombinant protein or cellular assays, using high-throughput automation to identify ‘hits’. Subsequent leads are examined in more detail by assessing their effect on downstream molecular events in cells and their selectivity vs other proteins. A battery of additional in vitro tests is also used for measurement or prediction of physical properties and pharmacokinetic parameters. Only compounds with a promising balance of features are progressed to in vivo testing, usually in mice. Pharmacokinetic (PK) studies, used to understand drug exposure, may initially involve co-inoculation of low doses of compounds (‘cassette dosing’) to minimise animal usage. The tolerability of leads with favourable PK is then assessed at higher doses, before evaluating their pharmacodynamic (PD) effect on tumour and normal tissues at well-tolerated doses. Compounds that do not meet the anticipated level of performance at any stage may result in subsequent rounds of iterative medicinal chemistry to generate improved leads. Selected leads are progressed to efficacy testing to determine the link between target inhibition and the effect on tumour growth or spread (metastasis). Safety studies on late-stage leads are also required before a candidate drug can be selected for examination in cancer patients (not covered here). The application of the test cascade means that compounds are filtered by the earlier stage assays so that a smaller number of compounds, and only those of higher quality, are taken into later stage in vivo assays in animals.

Figure 3

Examples of technologies used in animals for therapeutic cancer research. In vivo tumour models have an essential role in the development of new cancer medicines, enabling the temporal and quantitative effects of treatment to be examined on tumour and normal tissues in the intact organism. Methods used include those to examine (clockwise from far left) molecular determinants of sensitivity to treatment (initially in vitro, corroborated in vivo) such as (a) gene mutations by sequencing, or (b) gene amplification by fluorescent in situ hybridisation; detection of target phospho-epitopes and their inhibition in tumour tissue as determined by: (c) immunohistochemistry or (d) western blotting of cell lysates; (e) tumour vascular density and maturation by fluorescent immunohistochemistry; (f) tumour mRNA expression by gene array analysis with hierarchical clustering of information; (g) imaging techniques such as dynamic contrast-enhanced MRI to measure tumour haemodynamics; and (h) pharmacokinetic analysis of drug concentrations in plasma by mass spectrometry.

Figure 4

Examples of in vivo imaging in pre-clinical cancer research. (A) Optical surface bioluminescence imaging of orthotopically xenografted human PC3 prostate carcinoma cells transfected with luciferase (PC3luc2a). Mice were imaged using a Charged Coupled Device (CCD) camera, which is super-cooled to enhance detection sensitivity and image resolution. The images shown were taken after systemic administration of luciferin, with ‘intensity of luminescence’ shown as ‘heat’ maps and red as maximum intensity. The scale shows the number of photons detected. Top panel: Untreated mice at day 8–41 after transplantation; bottom panel: before and after treatment with 5 mg kg⁻¹ taxotere on day 10. This technique is useful for monitoring treatment effects in deep-seated tumour sites. Light scattering through tissues makes precise quantitation difficult. (B) PET imaging of tumour cell proliferation using ¹⁸F-3′-fluoro-3′-deoxy-L-thymidine (FLT). Transverse and coronal (0.5 mm) images of HCT116 tumour-bearing mice 24 h before treatment and after 4 daily treatments with the histone deacetylase inhibitor LAQ842 at 25 mg kg⁻¹. 30- to 60-min summed images from a dynamic scan are presented. Numerous radiotracers are available for investigating specific biochemical pathways in vivo, if specialised facilities are available. The scale shows the intensity of radiotracer uptake. (C) Intravital imaging of tumour vasculature of the P22 rat sarcoma growing in a dorsal skin flap window chamber. The image was obtained by multi-photon fluorescence microscopy after i.v. administration of 70 kDa FITC–dextran. High spatial resolution is obtained but surgical intervention is required.