Optimization and In Vivo Profiling of a Refined Rat Model of Walker 256 Breast Cancer Cell-Induced Bone Pain Using Behavioral, Radiological, Histological, Immunohistochemical and Pharmacological Methods

Abstract

In the majority of patients with advanced breast cancer, there is metastatic spread to bones resulting in pain. Clinically available drug treatments for alleviation of breast cancer-induced bone pain (BCIBP) often produce inadequate pain relief due to dose-limiting side-effects. A major impediment to the discovery of novel well-tolerated analgesic agents for the relief of pain due to bony metastases is the fact that most cancer-induced bone pain models in rodents relied on the systemic injection of cancer cells, causing widespread formation of cancer metastases and poor general animal health. Herein, we have established an optimized, clinically relevant Wistar Han female rat model of breast cancer induced bone pain which was characterized using behavioral assessments, radiology, histology, immunohistochemistry and pharmacological methods. In this model that is based on unilateral intra-tibial injection (ITI) of Walker 256 carcinoma cells, animals maintained good health for at least 66 days post-ITI. The temporal development of hindpaw hypersensitivity depended on the initial number of Walker 256 cells inoculated in the tibiae. Hindpaw hypersensitivity resolved after approximately 25 days, in the continued presence of bone tumors as evidenced by ex vivo histology, micro-computed tomography scans and immunohistochemical assessments of tibiae. A possible role for the endogenous opioid system as an internal factor mediating the self-resolving nature of BCIBP was identified based upon the observation that naloxone, a non-selective opioid antagonist, caused the re-emergence of hindpaw hypersensitivity. Bolus dose injections of morphine, gabapentin, amitriptyline and meloxicam all alleviated hindpaw hypersensitivity in a dose-dependent manner. This is a first systematic pharmacological profiling of this model by testing standard analgesic drugs from four important diverse classes, which are used to treat cancer induced bone pain in the clinical setting. Our refined rat model more closely mimics the pathophysiology of this condition in humans and hence is well-suited for probing the mechanisms underpinning breast cancer induced bone pain. In addition, the model may be suitable for efficacy profiling of new molecules from drug discovery programs with potential to be developed as novel agents for alleviation of intractable pain associated with disseminated breast cancer induced bony metastases.

Keywords: Walker 256 cells; Wistar Han female rat model; bone pain; bony metastases; breast cancer.

FIGURE 1

Timeline of assessments performed in individual experiments. AM, amitriptyline; C, clinical observations; GB, gabapentin; H, Hargreaves testing; HK, heat-killed; HT, histological assessment; I, immunohistochemical assessment; MP, morphine; MX, meloxicam; MP/GB/AM/MX-P, paw pressure testing after drug injection; MP/GB/AM/MX-V, von Frey testing after drug injection; N-H, Hargreaves testing after naloxone injection; N-P, paw pressure testing after naloxone injection; N-V, von Frey testing after naloxone injection; P, paw pressure testing; R, radiological assessment; V, von Frey testing; W256, Walker 256.

FIGURE 2

Body weight of rats from individual experiments. Panels in the figure show mean (±SEM) body weight of rats from (A) experiment 2, (B) experiment 3 and (C) experiment 5. HK, heat-killed; W256, Walker 256. There were no statistically significant differences in body weight between any treatment groups (p > 0.05; experiment 2, Two-way ANOVA, post hoc Bonferroni test; experiment 3 and 5, Mann–Whitney test).

FIGURE 3

Paw withdrawal thresholds (PWTs) of ipsilateral and contralateral hindpaws of rats. Panels in the figure show mean (±SEM) PWTs of rats from (A) experiment 2, (B) experiment 3 and (C) experiment 5. Rats with PWTs ≤6 g in the ipsilateral hindpaw were considered to have fully developed mechanical allodynia as indicated by the dotted line. HK, heat-killed; W256, Walker 256. ^∗p ≤ 0.05 (Two-way ANOVA, post hoc Bonferroni test) c.f. rats given an ITI of HK W256 cells.

FIGURE 4

Paw pressure thresholds (PPTs) of ipsilateral and contralateral hindpaws of rats. Panels in the figure show mean (±SEM) PPTs of rats from (A) experiment 2, (B) experiment 3 and (C) experiment 5. Rats with PPTs ≤80 g in the ipsilateral hindpaw were considered to have fully developed mechanical hyperalgesia as indicated by the dotted line. HK, heat-killed; W256, Walker 256. ^∗p ≤ 0.05 (Two-way ANOVA, post hoc Bonferroni test) c.f. rats given an ITI of HK W256 cells.

FIGURE 5

Paw thermal thresholds (PTTs) of ipsilateral and contralateral hindpaws of rats. Panels in the figure show mean (±SEM) PTTs of rats from (A) experiment 2, (B) experiment 3 and (C) experiment 6. HK, heat-killed; W256, Walker 256. There were no statistically significant differences in PTTs between the treatment groups in any of these experiments (p > 0.05; Two-way ANOVA, post hoc Bonferroni test).

FIGURE 6

Effect of naloxone on ipsilateral and contralateral PWTs of rats. Panels in the figure show mean (±SEM) PWT versus time curves from experiment 5 following naloxone or vehicle injection between (A) day 43–51 post-ITI and (B) day 53–66 post-ITI. Dotted line indicates the threshold PWT value at/below which the rats were considered to have fully developed mechanical allodynia. BCIBP (4 × 10⁵), group of rats given an ITI of 4 × 10⁵ W256 cells; HK, heat-killed; NAL, naloxone (15 mg/kg s.c.); Sham (4 × 10⁵), group of rats given an ITI of 4 × 10⁵ HK W256 cells; VEH, vehicle; W256, Walker 256. ^∗p ≤ 0.05 (Two-way ANOVA, post hoc Bonferroni test) c.f. rats given an ITI of HK W256 cells.

FIGURE 7

Temporal changes in PWTs of BCIBP rats in ipsilateral hindpaws following the administration of single bolus doses of analgesic and adjuvant drugs. Panels in the figure show temporal changes in mean (±SEM) PWT versus time curves following injection of (A) morphine, (B) gabapentin, (C) amitriptyline and (D) meloxicam.

FIGURE 8

Temporal changes in PPTs of BCIBP rats in ipsilateral hindpaws following administration of the single bolus doses of analgesic and adjuvant drugs. Panels in the figure show temporal changes in mean (±SEM) PPT versus time curves following injection of (A) morphine, (B) gabapentin, (C) amitriptyline and (D) meloxicam.

FIGURE 9

Radiological assessment of tibiae from BCIBP rats and corresponding sham rats. Panels in the figure show (A) 3D-μCT radiological image of a sham rat’s tibia at day 10 post-ITI, (B) trabecular bone of a sham rat’s tibia at day 10 post-ITI, (C) 3D-μCT radiological image of a BCIBP rat’s tibia at day 10 post-ITI, (D) trabecular bone of a BCIBP rat’s tibia at day 10 post-ITI, (E–H) morphometric changes in BCIBP rats’ tibiae relative to sham rats’ tibiae at day 10 post-ITI, (I) 3D-μCT radiological image of a sham rat’s tibia at day 48 post-ITI, (J) trabecular bone of a sham rat’s tibia at day 48 post-ITI, (K) 3D-μCT radiological image of a BCIBP rat’s tibia at day 48 post-ITI, (L) trabecular bone of a BCIBP rat’s tibia at day 48 post-ITI, (M–P) morphometric changes in BCIBP rats’ tibiae relative to sham rats’ tibiae at day 48 post-ITI. ^∗p ≤ 0.05 (Two-way ANOVA, post hoc Bonferroni test). Scale bar – 5 mm.

FIGURE 10

Histological assessment of tibiae from BCIBP rats and corresponding sham rats. Panels in the figure show representative images of H&E staining of tibial sections of (A) sham rat at day 10 post-ITI, (B) BCIBP rat at day 10 post-ITI, (C) sham rat at day 48 post-ITI and (D) BCIBP rat at day 48 post-ITI. Black arrowheads show destruction of cortical bone of tibiae. Scale bar – 1 mm.

FIGURE 11

Immunocytochemical staining of Walker 256 cell line for Cytokeratin 18 using ab187573 (Abcam) antibody. Panels in the figure show (A) cytokeratin 18 (B) DAPI and (C) A and B merged.

FIGURE 12

Immunohistochemical staining of Cytokeratin 18 in tibial sections of BCIBP rats and corresponding sham rats using ab187573 (Abcam) antibody. Panels in the figure show immunofluorescence imaging of tibial sections of (A) sham rat at day 7 post-ITI, (B) BCIBP rat at day 7 post-ITI, (C) sham rat at day 38 post-ITI and (D) BCIBP rat at day 38 post-ITI.