Research ArticleExperimental Studies
Open Access

Wheel Running in Digital Ventilated Cages® Is Impaired in a Model of Cancer-induced Bone Pain

CHELSEA HOPKINS, IDA BUUR KANNEWORFF, BIRGITTE RAHBEK KORNUM and ANNE-MARIE HEEGAARD
In Vivo November 2025, 39 (6) 3205-3215; DOI: https://doi.org/10.21873/invivo.14120

Abstract

Background/Aim: Cancer-induced bone pain (CIBP) due to metastatic breast cancer is common and debilitating. Effective, long-term treatment options have side-effects that reduce patients’ quality of life. Preclinical models are valuable tools for testing novel analgesics, but new methods that are translationally and clinically relevant are necessary. This study aimed to assess spontaneous pain-like behavior of home cage activity and wheel running in Digital Ventilated Cages®.

Materials and Methods: Twenty BALB/cAnNHsd mice were housed in Digital Ventilated Cages® from Tecniplast® with GYM500 home cage running wheels. Ten mice underwent 4T1-Luc2 mammary gland adenocarcinoma cell inoculation into the right femur to establish CIBP and another ten mice underwent a sham procedure. Mice were assessed by limb use and static weight bearing to determine the development of CIBP and this was compared to the dark-phase home cage activity and wheel running in the Digital Ventilated Cages®.

Results: The 4T1-Luc2-inoculated mice displayed pain-like behavior in limb-use and weight-bearing tests, demonstrating a preference for the contralateral limb. The limb-use scores were compared with home cage activity and wheel running. Reduced wheel running distance corresponded to reduced limb-use scores, with the shortest wheel running distances corresponding to the lowest scores. However, this behavioral pattern was not observed in home-cage activity, which remained consistent throughout the study.

Conclusion: Wheel running behavior appears to be affected by the development of metastatic breast cancer. Wheel running in a Digital Ventilated Cage® may be a useful behavioral assessment of spontaneous pain-like behavior of CIBP and may be useful to assess analgesic efficacy.

Keywords:

Introduction

In patients with advanced breast cancer, 58% will experience bone minetastases (1) and of those, 68% will experience bone pain (2). These patients commonly experience worse pain and quality of life than patients with metastases in non-bone tissue. It has also been reported that of patients with painful bone metastases, 97% are taking analgesics, 55% opioids and 42% non-opioids (2). However, long-term pain relief therapies (e.g., non-steroidal anti-inflammatory drugs, opioids) lead to an increase in side-effects that impair quality of life (3). Preclinical in-vivo models are essential tools for investigating nociceptive mechanisms and novel analgesics (4).

Evoked pain tests are common in pre-clinical settings to assess mechanical hypersensitivity (e.g., Von Frey test) and thermal hypersensitivity (5, 6). However, spontaneous, ongoing pain is considered a more relevant issue for patients, and this is difficult to test in vivo (7). Pain is not only a sensory condition, but may also encompass stress, anxiety and depression, and limit social interactions. Sensory and affective conditions should be assessed in combination to develop a well-rounded model of pain. Furthermore, in vivo tests in models often occur over a short period during the day, when rodents are least active, which means that a measure of spontaneous pain fluctuations may be difficult to gauge (8).

Tecniplast SpA has developed Digital Ventilated Cages® (DVCs®) that can track home cage activity and monitor wheel running. The DVC® rack is equipped with an electromagnetic sensing board with capacitance-sensing technology to assess home cage activity; the board contains 12 electrodes that emit an electromagnetic field and when a mouse moves over these fields, it creates a disturbance that is recorded and measured. Readings are obtained continuously and unobtrusively when the cages are in the rack, allowing for continuous monitoring without handling interference during the 12-h dark phase when mice are more active (9). This provides the following benefits: evaluation of ethologically relevant behavior in a familiar home cage environment, minimizing researcher interference, and reducing handling stress, thus mitigating common confounding factors in pain behavioral studies. DVCs have been used to assess circadian rhythms (10-12), effect of standard procedures on cage activity (12, 13), severity monitoring (13-16), and, recently, a novel treatment for osteoarthritis pain (rest disturbance metric only) (17). More recently, our group has demonstrated that both DVC® wheel running and cage activity decreased in concert with a reduction in burrowing behavior and grid climbing in a model of fibrous dysplasia (18). Wheels with perpendicular magnets have been developed that can be placed in the cages, and their rotation, speed, and distance monitored automatically. Here we wanted to establish DVCs® in determining a behavioral outcome in a model of cancer-induced bone pain (CIBP).

Different systems to assess spontaneous behaviors have been designed that incorporate the ability to assess horizontal activity, vertical activity (e.g., rearing, climbing), and wheel running – all of which may be observed in home cage-monitoring systems as “home cage activity”. Horizontal activity more broadly describes movement in a two-dimensional testing area where a rodent is allowed to explore freely for a specified time. Vertical activity may be described as rearing, where the rodent stands only on its hind legs in order to extend vertically upwards. Reduced horizontal activity has been observed in fracture (19), arthritic (20), inflammatory (21, 22), neuropathic (22), and CIBP models (23). These experiments were conducted over short (60-120 min) (20-22) and 20-h periods (19, 23), but not in standard home cages. These experimental setups may introduce confounding stress and exploratory behaviors, and they require additional time to set up and carry out the experiment. Urban et al. (24) used a home cage-monitoring system (using LED units and cameras) to demonstrate reduced cage activity in a neuropathic pain model in a BALB/c mouse strain compared to naïve mice (25). Wheel running (during a short period) has been used in previous studies to assess the development of pain-like behavior in models of prostate CIBP (26), inflammation, and neuropathic pain (21, 22, 27).

The aim of this study was to determine if horizontal home cage activity and wheel running measured in the unperturbed environment of the home cage are affected by the development of breast CIBP. These metrics were compared to limb-use behavior, an established test of pain-like behavior in this mouse model.

Materials and Methods

Animals. All experiments were conducted according to the Danish Animal Experiments Inspectorate (Copenhagen, Denmark: 2020_15_0201_00439). Twenty 5-week-old female BALB/cAnNHsd mice were used in this study; 10 mice were inoculated with 4T1-Luc2 cells (ATCC, Manassas, VA, USA) and 10 mice underwent a sham procedure. Mice were purchased from Envigo (Lafayette, IN, USA) and housed in a certified specific pathogen-free facility at the University of Copenhagen where all experiments were conducted. The research unit maintained a temperature of 22±2°C and humidity of 55±10% in housing and experimental rooms. The light/dark cycle was 12-/12-h and light was kept at 60% intensity (7 am-7 pm). During the acclimatization period, mice were housed in individually ventilated GM500 cages with GYM500 cage wheels (Tecniplast®, Buguggiate, Italy) and during the testing period, mice were housed in the DVCs® within the DVC® rack (Tecniplast®). All cages contained wood-chip bedding material (Tapvei 2HV, Brogaarden, Denmark), one transparent red housing unit (Polycarbonate Mouse Tunnel; Datesand, Cheshire, UK), a running wheel (DVC® GYM500; Tecniplast®), nesting material (paper shavings; Brogaarden, Middelfart, Denmark), and a wooden gnawing block (Aspe små klodser, Brogaarden). Standard chow (Altromin 1324; Brogaarden) and tap water were provided ad libitum. Fresh food and water was provided once per week and cages were changed every 2 weeks. Severity monitoring (weight loss/gain, fur condition, facial grimace, etc.) was conducted during the baseline experiments, days 1-4 after surgical inoculation, and every experimental day thereafter. Severity monitoring included weight loss/gain, coat condition, aggression, mobility, etc. A predefined scoring system was implemented to ensure that mice did not surpass the humane endpoint. The humane endpoint was a mouse reaching a pre-defined severity score of 6 or a limb-use score 0. The experimental endpoint was defined as a 4T1-Luc2-inoculated mouse reaching a limb-use score of 0 and control mice reached the experimental endpoint when all 4T1-Luc2-inoculated mice had been euthanized. Mice were only handled using the tunnel handling method or by scooping them up with hands to minimize stress. All mice were euthanized by cervical dislocation.

Study design. Mice were stratified into a 4T1-Luc2-inoculated group and sham control group according to baseline weight-bearing data and body weight by the primary researcher (CH) to ensure that both groups had an equal average baseline behavior and weight. The experimenter (IBK) was blinded throughout the experiment to the group allocation. Blinding was not possible during data analysis due to the nature of the experiment, where group allocation is revealed during the course of the study. Each mouse acted as one experimental unit. Mice were excluded from the experiment if they demonstrated any indication of poor health prior to surgery, if they did not completely heal after surgery prior to day 5, if they demonstrated symptoms of tendon displacement (e.g., limping, inability to extend ipsilateral limb), or if they did not run on their cage wheel. All behavioral experiments outside the cage were conducted in the morning (9 am-12 pm) at the same time for each mouse.

Experimental timeline. Figure 1 demonstrates the timeline of the full experiment. Mice were introduced to the facilities at 5 weeks old. They acclimatized for 1 week prior to baseline experiments without interruption. At day −7 they were transferred to individually housed DVCs®. From days −7 to −5, mice were trained and habituated to the static weight-bearing procedure. On day −4, no behavioral or experimental procedures were conducted prior to the dark phase to obtain cage activity and wheel running data not influenced by experimenter handling. On days −3 and −2, mice underwent baseline limb-use and weight-bearing tests. The subsequent dark phases were used as baseline assessments to determine the effect of handling on cage activity and wheel running. Baseline X-ray images were collected on day −1 and surgery was conducted on day 0. Post-surgical care was conducted from days 1-4. From day 5 onwards, limb-use and weight-bearing tests were conducted every day during the light phase and cage activity and wheel running were only assessed in the dark phase. Imaging was conducted on day 8 prior to euthanasia when mice reached the experimental or humane endpoint.

Figure 1.
Figure 1.

Experimental timeline demonstrating animal behavior and cell culture procedures over the course of the experiment on bone pain induced by tumor developing from surgical implantation of 4T1-Luc2 breast cancer cells. DVC: Digital Ventilated Cage®; EEP: Experimental end-point.

Cell culture. Mouse mammary gland adenocarcinoma cells [Bioware Ultra Cell Line 4T1-Luc2; Caliper Life Sciences, Teralfene, Belgium; ATCC (CRL-2539™) parental line] were cultured as previously described (28, 29). Cells were cultured in vented sterile cell culture flasks (75 cm2; Cellstar®, Greiner, Austria) in a sterile environment. Cells were maintained in RPMI 1640 cell culture media without phenol red (Gibco®, Waltham, MA, USA), supplemented with 10% heat-inactivated fetal bovine serum (Gibco®) and 1% penicillin-streptomycin-glutamine (Gibco®). Cells were cultured for 2 weeks and then split 2 days before surgery to ensure log-phase growth. To split and harvest the cells, 0.5% trypsin-EDTA (Gibco®) was applied to the cells for 5 min. Trypsin was inactivated with regular cell medium, the solution was centrifuged, the supernatant discarded, and the cells were resuspended in Hank’s Balanced Salt Solution (Gibco®) to a density of 106 cells/ml. Cells were kept on ice thereafter.

Cancer cell inoculation in femur. Surgery was conducted as previously described, with minor modification (29). Mice were anesthetised with an intraperitoneal xylazine/ketamine cocktail (43 mg/kg ketamine – MSD Animal Health, AN Boxmeer, the Netherlands; 6 mg/kg xylazine – Rompun Vet, Bayer, Germany). Mice were maintained on 1-1.2% isoflurane (1,000 mg/g isoflurane; Attane Vet; ScanVet, Fredensborg, Denmark) throughout surgery. Eye ointment (Ophtha A/S, Activis Group, Gentofte, Denmark) was applied to the eyes, 0.9% saline was injected subcutaneously to prevent post-surgical dehydration, and 5 mg/kg carprofen (Carprosan Vet; Dechra, Northwich, UK) was injected subcutaneously. Mice were placed on a heated surgery table in a supine position. A small incision was made over the patella tendon and in the connective tissue on the medial side of the tendon. The tendon was positioned to the lateral side of the knee. A 30-G needle was used to manually drill a hole into the femoral epiphysis until the medullary cavity was reached. Using a 0.3 ml insulin syringe (BD, Franklin Lakes, NJ, USA), 10 μl of 4T1-Luc2 cells (4T1-Luc2-inoculated group) or Hank’s Balanced Salt Solution was injected into the cavity and incubated for 1 min. The hole was then filled with bone wax (Harvard Apparatus, Holliston, MA, USA) and the patella tendon was moved back to the central position. The region was thoroughly rinsed with 0.9% saline and the incision closed with two medical clips (Michel Suture Clips; Agnthos, Lidingö, Sweden). Mice were maintained in a prone position on a heat mat until they regained mobility. On day 1, mice were administered 5 mg/kg carprofen subcutaneously for post-surgical analgesia.

DVC® home cage activity and wheel running. Electromagnetic wheels (GYM500; Tecniplast) were secured to the inner side of each DVC®. When the wheel spins, the electromagnetic field produced by the magnets can be detected and tracked. The cumulative distance during the dark phase was assessed. Mice were acclimatized for 1 week with five mice per cage with a cage wheel in Individual Ventilated Cages®. This is an essential training and habituation step for the mice. Mice without wheel habituation may not use the running wheel during the testing period. After the acclimatization period, mice were single-housed in a DVC® and rack. Assessments outside of the cages were conducted during the same time in the morning, for the same period. This arrangement was practiced to ensure that dark-phase behavior was not influenced by behavior experiments and handling during the light phase. When mice were removed from their cages during the behavioral experiments, bedding material was moved away from the wheel as it can interfere with the wheel mechanism and magnets, preventing accurate readings. The rack was placed in the housing room away from the entrance door to prevent excess interference and in a position with minimal human traffic. The mice were housed with half the mice from each group at the top and bottom of the rack to determine if position in the rack was a confounding factor. Analysis was conducted on data collected from the dark phase (7 pm-7 am). Mice with a peak hourly distance of less than 500 m were excluded from the experiment. Home-cage activity metrics (Animal Locomotion Index) and the associated calculation have been described previously (9) and shown to be comparable to manually analyzed video analysis of mouse behavior in the cage. The Animal Locomotion Index was calculated every minute (four recordings per minute) and the average home-cage activity per hour was assessed.

When comparing data from different tests, cage-activity and wheel-running data collected from the dark phase prior to the limb-use and weight-bearing tests were matched.

Limb-use scoring. Mice were placed in an empty transparent box for 10 min to habituate. Thereafter, mice were transferred to an identical testing arena where they were monitored for 3 min. Mice were scored according to the following scale:

  • 4 – Normal gait

  • 3 – Insignificant limping

  • 2 – Significant limping and shift in bodyweight towards the healthy limb

  • 1 – Significant limping and partial lack of use of the ipsilateral leg

  • 0 – Total lack of use of the ipsilateral leg

Different boxes were used for each mouse.

Static weight bearing. Static weight bearing was assessed using the Incapacitance Tester (Version 5.2; Linton Instrumentation, Diss, UK). Mice were habituated and trained to maintain a testing position (hind paws individually placed on two weight plates) prior to baseline measurements. Triplicate measures of 3-s readings were obtained for every mouse, and the ratio of weight distribution was calculated.

X-ray and bioluminescent imaging. Mice underwent imaging on day 8 and when the mice reached their experimental or humane endpoint. Mice were injected intraperitoneally with 150 mg/kg D-Luciferin (PerkinElmer, Inc., Shelton, CT, USA) 9 min prior to imaging. Mice were sedated with 2.5% isoflurane (1,000 mg/g isoflurane; Attane Vet; ScanVet) and maintained throughout the imaging procedure. Lumina XR apparatus (Caliper Life Sciences) was used to obtain both X-ray and bioluminescent images in triplicate for 4T1-Luc2-inoculated mice. One X-ray image was obtained for each of the sham control mice.

Statistical analysis. The a-priori sample-size calculation was based on previous studies conducted on the same model (29), with power set at 80% and statistical significance set at p<0.05. Calculated group size was increased by 20% to account for possible exclusion.

Data collected by the DVC® system was extracted, with data summarized for every hour. Home cage-activity data are presented as previously described (9). Wheel running is presented as cumulative running distance per hour.

Due to the experimental design, with mice being euthanized during the course of the study, the last observation was carried forward to account for missing measures at later time points.

All graphs and plots presented were generated with GraphPad Prism 9.3.1 (GraphPad Software, La Jolla, CA, USA). The parametric data (weight bearing, home-cage activity, wheel running) were analyzed using the mixed-effects model, with one-way, or two-way analysis of variance with appropriate corrections for multiple comparison (see specific information in figure legends) in GraphPad Prism. The non-parametric data (limb use) was analyzed by the Friedman test in SAS® 9.4 (SAS Institute, Cary, NC, USA) with pairwise Mann-Whitney U-tests in Graphpad Prism.

Results

Mice in the sham group did not demonstrate significantly reduced behavior in any of the tests over the course of the experiment. Mice that were inoculated with 4T1-Luc2 cells in the right femur were evaluated daily by activity monitoring in DVCs®, limb use, and weight bearing. Presence of pain-like behavior in the 4T1-Luc2-inoculated mice was confirmed by a significant decrease in limb-use score compared to baseline from day 12 (Figure 2A) and in weight-bearing ratio from day 10 (Figure 2B) after inoculation.

Figure 2.
Figure 2.

Effect of intrafemoral inoculation of 4T1-Luc2 breast cancer cells on (A) limb use, (B) weight bearing, (C) wheel running, (D) home-cage activity, and (E) luciferase activity. Cumulative wheel-running distance and average Animal Locomotion Index were hourly DVC® data from the dark phase (7 pm-7 am) preceding a study day. Statistics were performed with Friedman test with pairwise Mann–Whitney U-tests (A) or mixed-effects model with Sidak multiple comparisons test (B-E). #Last observation carried forward to account for animals euthanized during the study and comparisons were made with baseline (BL) data. Significantly different from the sham group at: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. EEP: Experimental end-point.

4T1-Luc2-inoculated mice showed significantly decreased wheel running from day 12 (Figure 2C) compared to baseline. Horizontal activity did not change within the experimental timeframe (Figure 2D). Tumor growth in the ipsilateral femur was confirmed by in-vivo luciferase imaging on day 8 and upon reaching the humane and experimental endpoint (Figure 2E), which showed that all 4T1-Luc2-inoculated mice that developed pain-like behavior had indeed developed a tumor in their inoculated limb.

To evaluate the relationship between activity measures and an established measure of pain-like behavior, the DVC® data from the dark phases were matched to the limb-use score (pain-like development) obtained on the following study day (Figure 3A and B). 4T1-Luc2-inoculated mice reaching a limb-use score of 2 (significant limping) or 0 (complete lack of use of the ipsilateral limb) showed significantly lower wheel-running distance compared to baseline throughout the whole of the dark phase (Figure 3A and C). The horizontal activity did not differ by limb-use score (Figure 3B). Sham mice assessed during days that corresponded to a mean limb use score of 4, 2, and 0 in the 4T1-Luc2-inoculated group demonstrated no significant change in wheel running over time (Figure 3D).

Figure 3.
Figure 3.

Relationship between limb-use score and wheel running in a mouse model of breast cancer-induced bone pain. (A) Cumulative wheel running distance and (B) home cage activity in 4T1-Luc2-inoculated mice during the dark phase preceding the day the limb use (LU) score was acquired. (C) Diurnal pattern of wheel running in 4T1-Luc2-inoculated mice during the dark phase preceding the indicated limb use score. (D) Diurnal pattern of wheel running in sham mice during the dark phase on days corresponding to an average limb use score of 4, 2, 0 in the 4T1-Luc2-inoculated group. BL: Baseline; D#: day #. Statistical analysis was performed with one-way analysis of variance with Tukey’s test for multiple comparisons. Data are the mean±standard error of the mean.

Discussion

DVCs® were used to assess spontaneous pain-like behavior in a mouse model of breast CIBP. Wheel running was significantly reduced over time and in concert with the reduction of limb use and static weight bearing ability. However, horizontal cage activity did not demonstrate a significant change over time nor show any relationship with limb use or weight bearing.

Three measures of spontaneous pain-like development were reduced in this study as CIBP developed: limb use, weight bearing, and wheel running. Limb use and weight bearing are non-evoked behaviors but they are performed during the light phase when mice are less active over a short period of time (total of 15 minutes to perform both tests). Previous studies on this model using the same techniques have demonstrated that a decrease in limb use can be reversed with administration of 10 mg/kg morphine (30). The reduction of wheel running occurred in parallel with the reduction of the limb-use score. The first significant difference from baseline was observed on day 12 in both limb use and wheel running (Figure 2A and C); wheel running also significantly decreased comparably with limb-use scores, demonstrating lower wheel running when mice scored lower in gait analysis. This suggests that wheel running is affected by the development of a malignant tumor and the associated nociceptive pathology. However, wheel running within this system holds the significant advantage of occurring during the more active dark phase, over the entire 12-hour period.

We recently demonstrated that a model of fibrous dysplasia that developed pain-like behavior (reversed with morphine and carprofen in grid climbing and burrowing, respectively) also demonstrated deficits in wheel running and cage activity in the DVC® system (18). Our current study, along with the fibrous dysplasia study, indicate that different pain models may demonstrate different behaviors in DVC® cages that should be characterized before performing further experiments.

Wheel running has been utilized in other models (21, 22, 26, 27); however, those tests were performed during the light hours over a short period. Even so, similar to previous studies on prostate CIBP (26), inflammatory (21, 22), and chronic constriction injury models (22), this model also demonstrated significantly reduced wheel running behavior. However, there are no previous studies demonstrating long-period wheel running that was not in a home-cage environment; our data suggest that DVC® is a robust system capable of detecting the development of pain-like activity. There is evidence to suggest that these tests may be sex- (27) and model- dependent (22). Care should be taken to understand the sex and strain constraints in this system, prior to implementing further studies. There is a legitimate concern that wheel running constitutes a stereotypic behavior, defined as a repetitive behavior with no goal or function (31) and is to be avoided. However, a previous study demonstrated that wheel running is performed voluntarily by wild mice (32) and may be a rewarding and motivating activity (33).

In our study, there was no effect of CIBP on home-cage activity. The only other comparable studies on home-cage systems were performed by Urban et al. (24), where a reduction of home-cage activity was observed in BALB/c mice with a chronic constriction injury, and in our model of nociceptive fibrous dysplasia (18). Home-cage activity was not affected in an inflammatory or spared nerve-injury model (24). Horizontal activity (not in a home-cage environment) was affected in several models of bone pain (19-23). However, home-cage assessment offers an advantage of assessing pain-like behavior over an extended period, instead of in an experimental system confounded by external factors. Again, sex and the model used in the DVC® system should be taken into account prior to using the system for further studies.

Analgesics are necessary to assess whether change of behavior is due to pain-like development or other factors associated with stress or illness. However, the use of analgesics constrains spontaneous behavior due to their effects on overall behavior. For example, previous studies on burrowing (also a spontaneous behavior) demonstrated that most analgesics adversely affect the procedure. High doses of opioids (e.g., 10-30 mg/kg morphine) are necessary to treat mouse models of CIBP, as weak analgesics are ineffective (30, 34). Cobos et al. (21) tested the use of analgesics in short-term wheel-running assessments. In sham mice, a low dose of morphine (5 mg/kg) significantly reduced wheel running. In an inflammatory model (induced by Complete Freund’s Adjuvant), wheel running was partially reduced by morphine, but not significantly so, suggesting that morphine moderately restores wheel-running behavior in a pain-like model. This suggests that at least low doses of morphine might be used in intervention studies using wheel running as a behavioral outcome. However, an analgesic with a long half-life is required to span the 12-hour dark phase, or at least in this model, the first 4 hours when mice are most active. Morphine has a half-life of approximately 30 min, which would not work in this study design (35, 36), but buprenorphine hydrochloride (3-5 h half-life) or sustained-release buprenorphine which lasts longer, may be more suitable alternatives (37, 38).

The first limitation of this study is that it was not possible to directly test the effect of analgesics as described above, as morphine would likely reduce wheel running and weaker analgesics have poor efficacy in models of CIBP (34). Secondly, there was no difference observed in horizontal home-cage activity, and use of this system only for wheel running may not be a cost-effective behavioral tool.

This study demonstrates that the DVC® can be an exciting tool to assess pain-like behavior in different models of pain-like development. It facilitates the simultaneous study of two measures of spontaneous pain-like behavior over a long period, while reducing the introduction of confounding factors.

Conclusion

DVC® home-cage activity and wheel running were used to assess pain-like behavior in a model of breast CIBP. This method can be implemented to assess characteristics of other mouse models of painful diseases and perhaps test novel analgesics.

Footnotes

  • Authors’ Contributions

    Conceptualization: CH and AMH. Data curation: CH, IBK and AMH. Formal analysis: CH and IBK. Funding acquisition: AMH. Investigation: CH and IBK. methodology: CH and IBK. Project administration: CH and AMH. Resources: BRK and AMH. Software: BRK and AMH. Supervision: AMH. Validation: CH, IBK, BRK and AMH. Visualization: IBK. Writing – original draft preparation: CH. Writing – review and editing: CH, IBK, BRK and AMH.

  • Conflicts of Interest

    The Authors have no conflicts of interest to declare.

  • Funding

    Chelsea Hopkins and Anne-Marie Heegaard received funding for this project from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 814244.

  • Artificial Intelligence (AI) Disclosure

    No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.

  • Received September 8, 2025.
  • Revision received September 24, 2025.
  • Accepted October 1, 2025.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Wheel Running in Digital Ventilated Cages® Is Impaired in a Model of Cancer-induced Bone Pain
CHELSEA HOPKINS, IDA BUUR KANNEWORFF, BIRGITTE RAHBEK KORNUM, ANNE-MARIE HEEGAARD
In Vivo Nov 2025, 39 (6) 3205-3215; DOI: 10.21873/invivo.14120
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