Environmental enrichment does not impact on tumor growth in mice

Cell lines

The mouse (C57BL/6) B16F10 melanoma tumor cell line⁵ (from NP Restifo, National Cancer Institute, Bethesda, USA) was maintained in complete medium consisting of DMEM (Gibco, Life Technologies, Grand Island, NY) with 10% heat-inactivated fetal calf serum (FCS; MultiSer, Thermo Trace, Melbourne) and additives (2 mM glutamine (Gibco), 100 μg/ml streptomycin (Sigma-Aldrich, St Louis, MO) and 100 U/ml penicillin (Sigma-Aldrich) in a humidified incubator at 37°C with 5% CO₂.

Environmental enrichment and standard housing

The black low density polyethylene plastic EE cage (Plastime, Castegnero, Italy) measured 81 cm (length) × 57 cm (width) × 34 cm (height) internally, and had a wire cage lid (Figure 1). It was stocked with the following stimulatory equipment: 2 exercise wheels, 3 PVC plumbing elbow pipes (2.54 cm diameter) bent at 90°, a 2.54 cm plumbing T-piece, 2 standard cages with holes drilled in their sides to allow mouse access and with tissues inside and pellet food provided on their wire lids, 2 cardboard boxes of tissues cut in half and inverted (refuges) and about 20 tissues scattered around. An extra EE cage was also purchased and when mice cages were cleaned fortnightly, all equipment and mice were transferred to the new cage which had clean bedding. Mice were allowed to acclimatize to their conditions for six weeks, prior to injection with tumors. The mice were not handled except for transferring during cage cleaning, and tumor measurement (on day 13 after tumor injection).

Figure 1. Environmental Enrichment cage setup.

Setup shows the refuges, exercise wheels, and tunnels in the environmental enrichment cage.

The four standard cages used measured 28 cm (length) × 14 cm (width) × 12 cm (height) internally and were made of polycarbonate plastic (Wiretainers, Melbourne, Australia).

Specific Pathogen Free 3 (SPF3) animal facility and conditions

Fortnightly cleaning of cages was as follows: The EE cage was scraped out manually, then cage and toys were soaked for 10 mins in hot water with 2–5% Decon 90 (Decon Laboratories Ltd, East Sussex, UK) solution, scrubbed with a brush, and rinsed in hot water. The cage was left for 2 weeks before being used again, as two cages were alternated. The standard cages were scraped out manually, washed in a tunnel washer using washing machine powder, before autoclaving.

Both EE and standard cages had a layer of FibreCycle (recycled paper pellets; FibreCycle P/L, Yatala, Qld, Australia) animal bedding pellets to a depth of approximately 2 cm on the bottom of the cages, and all mice were fed with irradiated Barastoc mouse food cubes (Ridley AgriProducts, Melbourne, Australia) based on wheat, wheat byproducts, oats, meat meal, canola oil, soyabean meal, skim milk powder, molasses, salt, vitamins, and minerals. Drinking water was filtered tap water adjusted to pH 2.5–3 with hydrochloric acid.

Both EE and standard cages were housed in the same room of the animal facility. Throughout the experiment mice were maintained on a 13-hour-on : 11-hour-off lighting schedule (lights on at 6.00 am and off at 7.00 pm) in a room thermostatically maintained at 20°C. Food and water were available ad libitum. The air in the facility was not HEPA filtered or humidity controlled, and there were 15 air changes per hour.

The Specific Pathogen Free (SPF) facility houses sentinel mice in each rack and these are monitored regularly for infectious agents with the aim of detecting any pathogenic agents. The facility was monitored for the microorganisms listed in Table 1, and none of these species were detected during the period of this study. The following microbiota are detected in the mice in this facility and are considered endemic in the SPF-3 rated animal facility: Mouse Norovirus, Rotavirus, Protozoa (Chilomastix bettencourti or Entamoeba muris, which are frequently found in intestinal tracts of normal rodents), Proteus spp. (probably P. mirabilis as this is a common inhabitant of the upper respiratory tract and faeces of normal mice), and Helicobacter spp.

Mouse tumor model

Ethics statement: This study was carried out in strict accordance with the recommendations of the Victorian Bureau of Animal Welfare, Department of Primary Industries, and the National Health and Medical Research Council’s Australian code of practice for the care and use of animals for scientific purposes. The protocol was approved by the Peter MacCallum Cancer Centre Animal Experimentation Ethics Committee under Permit number E396. All efforts were made to minimize suffering.

Wild type male C57BL/6 mice were purchased from the Walter and Eliza Hall Institute of Medical Research (Bundoora, Australia), at age three weeks, and randomly assigned in either the EE cage (20 mice) or in four standard cages with five mice each (20 mice in this group). Mice were habituated to their cages for 6 weeks prior to tumor injection. During the habituation period two mice died (one found dead and one was culled for hydrocephalus) in the standard housed group, so that this group consisted of 18 mice for tumor injection. After the six weeks habituation, mice were shaved on the flank and inoculated s.c. with 100 μl of a single-cell suspension of 1×10⁵ B16F10 melanoma cells in Ca²⁺- and Mg²⁺-free phosphate-buffered saline (Merck, Darmstadt, Germany) (day 0). The same person injected all mice for consistency. Tumor growth was monitored using calipers, and tumor area was calculated as the product of two perpendicular diameters. Mice were culled when tumors reached 200 mm² in size or at the first signs of stress.

Statistical analysis

Statistical significance in the experiment compared in vivo tumor growth and was determined by two-tailed Mann-Whitney test in Graphpad Prism (Graphpad Software, version 6.02) San Diego, California).

Environmental enrichment did not impact on B16F10 tumor growth

C57BL/6 male mice at three weeks of age were divided into two groups. One group of 20 mice was placed in an enriched environment (Figure 1), which consisted of a large cage (surface area of 231 cm² per mouse) with numerous pieces of stimulatory equipment (exercise wheels, tunnels, refuges, tissues), and the 18 mice in the other group were placed in four standard cages (4–5 mice/cage with surface area of 78 cm² per mouse) with tissues only.

After six weeks of habituation in their respective cages, all mice were injected subcutaneously with 1 × 10⁵ cells of B16F10 on their flank. Mice were not handled except for transfer to clean cages during routine fortnightly cleaning, until day 13 when all tumors were measured.

Figure 2 shows the tumor measurements on days 13 and 16 after tumor injection. Tumors on 33% (six of the standard group of 18 mice) and 30% (six of the EE group of 20 mice) of mice were ≥ 200 mm² on day 13 and these mice were culled on this day. The average size of tumors was 164.3 ± 25.4 (SEM) mm² (standard conditions) and 155.7 ± 29.8 (SEM) mm² (EE conditions) on day 13. Average tumor size between the two groups was not statistically significant (p=0.69). On day 16, 72% of standard housed mice (13 of the 18 mice) and 65% (13 of the 20 mice) of EE housed mice had been culled as tumors of these mice had reached the 200 mm² size threshold. All mice except one in each group had developed tumors. The experiment was terminated on day 16 as there was no significant difference between tumor sizes in the two groups.

Figure 2. No statistical difference between B16F10 tumor size between environmental enrichment housed mice and standard housed mice.

Tumor measurements shown on days 13 (first day of measurement) and 16 after tumor injection. Bar represents average measurement for the group. Error bar is ± SEM.

Differences between conditions in previous studies

Table 2 summarizes the conditions for previously published studies on cancer in mice housed in EE cages, compared with the current study, in an attempt to ascertain why the results between the studies were so different. Floor space area per mouse varied from 180 to 1250 cm², with the floor space largest in the study by Cao et al.², which was about five times that of our study. The number of mice per EE cage varied between five and 20, but was similar between the Cao et al. study and our study. Enrichment toys and equipment were similar between all studies, as was the age that the mice were introduced into the cages (3–4 weeks) and the time for habituation (6–9 weeks). Tumor lines were different between studies, as were the sites of s.c. injection and the frequency of handling the mice. Mice were male in all studies except one³ and of the C57BL/6 strain in all studies except one⁴. Other parameters that could have caused variability were not mentioned in all studies, such as food composition, cage material (chemical composition and emission of fumes may vary), cage cleaning (chemical residue may vary), lighting, temperature, whether standard and EE cages were housed in the same room, bedding composition, and microbiota detected in each animal facility. With so many unspecified variables it is difficult to determine what is causing three of the studies to find no durable statistically significant difference in tumor size between EE and standard housed mice, whilst one study found a significant difference.