Behaviour results.

<p>The mean frequency of rearing (±SEM) in the orthotopic (left axis; circles) and heterotopic study (right axis; triangles) in tumour bearing and control groups (filled versus open symbols); showing the significantly greater decline in activity in the tumour-bearing mice in the orthotopic study from CPP test 3 (P_3) to just prior to euthanasia (P_1).</p

Welfare Assessment following Heterotopic or Orthotopic Inoculation of Bladder Cancer in C57BL/6 Mice

<div><p>Few studies have assessed whether mice used as cancer models experience pain. Despite this possibility, the usual practice is to withhold analgesics as these are generally viewed as confounding. However, pain also alters cancer progression, so preventing it might not only be beneficial to welfare but also to study validity. Establishing the extent to which different cancer models result in pain is an important first step towards their refinement. We used conditioned place preference (CPP) testing and body-weight and behaviour analyses to evaluate the assumption that heterotopically implanted tumours result in less pain and fewer welfare concerns than those implanted orthotopically. C57Bl/6 mice received MB49^Luc luciferase expressing bladder cancer cells or saline implanted subcutaneously or into the bladder. These tumour-bearing or control groups underwent 2 daily 45 minute conditioning trials to saline or morphine (2mg/kg) and then a 15 minute drug-free preference test on day 3 of a 3 day cycle, continuing until the study ended. Tumours were imaged and behaviour data obtained following preference tests. Development of preference for the morphine-paired chamber (morphine-seeking) was determined over time. Heterotopic tumour development had no effect on morphine-seeking, and although the restraint used for heterotopic inoculation caused greater initial weight losses than anaesthesia, these mice steadily gained weight and behaved comparatively normally throughout the study. Orthotopic tumour inoculation caused no initial weight losses, but over the final 7 days these mice became less active and lost more body weight than cancer-free controls. This indicated orthotopic implantation probably caused a more negative impact on welfare or conceivably pain; but only according to the current test methods. Pain could not be confirmed because morphine-seeking in the tumour-bearing groups was similar to that seen in controls. Imaging was not found to be an effective method of monitoring tumour development surpassing manual tumour inspection.</p></div

Body weight results.

<p>Mean percentage changes from baseline (pre-inoculation) body weight (±SEM) in mice inoculated with MB49^luc bladder cancer (Tum) or DPBS (Ctrl) and conditioned to morphine (Mor) or saline (Sal). Panels respectively illustrate data spanning the first 18 days (6 CPP Tests) and the final 6 CPP tests prior to euthanasia (16 days) in mice inoculated orthotopically (a, b) or heterotopically (c, d).</p

Imaging results.

<p>(a) the mean total flux (TF ±SEM) of bioluminescent signals emanating from tumours implanted orthotopically (Orth; left axis, circles) or heterotopically (Het; right axis, triangles) in mice conditioned to morphine (Mor; closed symbols) or saline (Sal; open symbols) and imaged every 3 days (beginning on day 3) for 18 days (Day_CPP Test); (b) Mean TF (±SEM) during the final 6 CPP tests (P_6 to P_1; 18 days) before euthanasia; (c) Caliper measurements showing the mean tumour surface area (mm² ±SEM) of heterotopically implanted tumours over the first 18 post-inoculation days (6 CPP tests) and (d) last 16 days (6 CPP tests) in mice conditioned to morphine or saline.</p

CPP results.

<p>Proportionate S+ chamber residence time (mean ±SEM) over the final 6 CPP cycles (P_6 to P_1; 16 days) before euthanasia in mice inoculated orthotopically (a) or heterotopically (b) with MB49^luc bladder cancer (Tum) or DPBS (Ctrl) and conditioned over a repeated 3 day cycle to morphine (Mor) or saline (Sal).</p

Treatment groups in phase 2 of the study.

<p>Treatment groups in phase 2 of the study.</p

Ethogram used for behavioural observations in phase 1.

<p>Adapted from references [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163909#pone.0163909.ref036" target="_blank">36</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163909#pone.0163909.ref049" target="_blank">49</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163909#pone.0163909.ref051" target="_blank">51</a>].</p

Mean frequencies for Composite Pain Behaviour (CPB) scores across treatments and time points.

<p>Data presented ± 2 standard error of the mean. N = 28. Significant differences (*) are explained in the text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163909#pone.0163909.t005" target="_blank">Table 5</a>.</p

Treatment groups in phase 1 of the study.

<p>Treatment groups in phase 1 of the study.</p

Within-subjects comparison between post treatment time-points with baseline with associated <i>p</i>-values for differences where significance was found (phase 1 of the study).

<p>Differences are significant if p<0.05. AU: Action Unit; SC: Subcutaneous; IT: Intrathecal; ND: No significant difference.</p