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.

Transcriptomic characterisation of the optimised rat model of Walker 256 breast cancer cell‐induced bone pain

“…This mechanical hypersensitivity was spontaneously reversed at day 46 (resolved pain behaviours). These results were in agreement with our previous report describing the apparently spontaneous resolution of pain hypersensitivities in this cancer‐induced bone pain model in rats, despite the ongoing presence of cancer cells in the ipsilateral tibiae …”

“…Using μCT scans, histological assessment with H&E staining, and immunohistochemical staining against Cytokeratin 18 in tibial bones, we have demonstrated that cancer cells were present in the bones and pathological changes associated with metastatic lesions such as osteolytic bone degradation was evident. This was the case both during the period when pain behaviour (< day 21) was evident and also during the period when pain behaviour was apparently resolved (> day 21), as reported in detail in our previous studies . Group of rats inoculated with live W256 cells developed significant ( P ≤ .05) mechanical hypersensitivities in the bilateral hindpaws at days 7–10 post‐inoculation (pain behaviours present), when compared to group of rats inoculated with HK W256 cells (Figure ).…”

“…As per the detailed analysis of clinical observations parameters as reported in our study published earlier, no significant health issues were identified in these animals in the cancer‐induced bone pain model, indicating localization of cancer in the tibiae. Although the cancer was present in the tibial bones, the cancer did not metastasize to vital organs as assessed during the model establishment phase.…”

“…However, there is insufficient information on global gene expression changes at the level of dorsal root ganglia (DRGs) and the spinal cord, occurring in the pathophysiology of BCIBP. Similar to the clinical situation, despite the ongoing presence of cancer cells in the bones, the pain hypersensitivities in the Walker 256 BCIBP model in rats appear to spontaneously resolve in the medium term, due to endogenous opioid‐sensitive mechanisms . However, genetic changes in the lumbar DRGs that underpin the apparent spontaneous resolution of pain hypersensitivities in this model despite the ongoing presence of cancer in the tibiae, are yet to be elucidated and hence the rationale for our study herein.…”

“…It is necessary that we understand the gene level changes occurring in the neural tissues that underpin the pathophysiology of pain in the preclinical models used in the drug discovery processes. Previous works from our laboratory have established that the Walker 256 breast cancer cell‐induced bone pain model in rats is a research tool that mimics key features of the human BCIBP condition and can be very useful in investigating the mechanisms of BCIBP and in discovery of novel analgesics against this debilitating pain condition . However, there is insufficient information on global gene expression changes at the level of dorsal root ganglia (DRGs) and the spinal cord, occurring in the pathophysiology of BCIBP.…”