Establishment, optimization and characterization of a rat model of breast cancer induced bone pain

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 rodent models of cancer induced bone pain were induced by systemic injection of cancer cells, resulting in widespread formation of cancer metastases and poor general animal health necessitating early euthanasia on welfare grounds.In my thesis, Chapter 1 explores briefly the literature on concepts involving pain processing, pathophysiology of breast cancer and Walker 256 breast cancer cell- induced bone pain model in rats.My first aim was to establish and comprehensively characterise a clinically relevant and optimised Wistar Han female rat model of BCIBP involving unilateral intra-tibial injection (ITI) of Walker 256 breast carcinoma cells. The results described in Chapter 2 portray the establishment and optimisation of this model. I found the optimum number of Walker 256 cells producing bilateral hypersensitivity in the hind paws of rats whilst maintaining satisfactory animal health to be 4.5 x 105 cells / 10 µL. The rats unilaterally injected with cancer cells developed bilateral mechanical allodynia and mechanical hyperalgesia, but not thermal hyperalgesia. I further characterised this model using tibial bone histology and micro- omputed tomography (µCT) scans, which confirmed the presence of osteolytic lesions due to cancer induced bone destruction. Interestingly, the pain hypersensitivity in the hind paws of rats given an ITI of Walker 256 cells resolved after approximately 25 days, which was reversed by administration of the opioid receptor antagonist, naloxone, suggesting a possible role of endogenous opioid system. Further, I fully validated the model pharmacologically by testing the clinically used standard analgesic drugs viz. morphine, gabapentin, amitriptyline and meloxicam and found their corresponding ED50-Ipsilateral values for anti-allodynia to be 1.3 mg/kg, 47.1 mg/kg, 20.1 mg/kg and 3.9 mg/kg, respectively.Although, Walker 256 cell line is one of the most commonly used rat breast cancer cell lines in experimental research, the molecular genetic profile of this cell line is not known. One of the aims of Chapter 3 was therefore to perform gene expression characterisation of Walker 256 cell line using next-generation RNA sequencing. The gene expression profile of Walker 256 cell line resembled to Basal-B subtype of human breast cancer cell lines. Specifically, Walker 256 cells express the marker Her2 (Erbb2), but not estrogen or androgen receptors, and also lack progesterone receptor typically found in human Her2-positive breast cancers. To gain additional insight into the pathophysiological mechanisms contributing to the development of BCIBP, in this Chapter, I also assessed gene expression changes in dorsal root ganglia (DRGs) and spinal cord of rats using next-generation sequencing. In the spinal cord of BCIBP rats during the pain state (day 10 post-ITI), 294 genes were differentially expressed in the spinal cord compared to sham rats, including several genes known to have roles in pain processing pathway. In addition, 25 genes were differentially expressed in the DRGs of BCIBP rats at the resolved-pain state (day 48 post-ITI) compared to BCIBP rats in pain state.The last aim of my project, described in Chapter 4, was to assess the analgesic efficacy of J-2156, a somatostatin receptor– 4 (SST4) agonist, in the optimized rat model of BCIBP. J-2156 elicited antiallodynia and anti-hyperalgesia in the BCIBP model in a dose-dependent manner. In BCIBP rats, the vast majority of cell bodies of small peptidergic (77 %) and non-peptidergic C fibre neurons (86 %) as well as medium-large diameter neurons (92 %) in the DRGs expressed the SST4 receptor. This distribution in BCIBP rats did not significantly differ from that in the sham rats. Consistent with a peripheral mechanism of action, treatment with J-2156 caused a decrease in phosphorylated extracellular signal-regulated kinase (pERK) expression in the spinal dorsal horn. In summary, in my PhD project, I have successfully established, optimized and characterized a rat model of BCIBP, which is well-suited for probing the mechanisms underpinning BCIBP and for efficacy profiling of new molecules. I have performed transcriptomic characterization of Walker 256 rat breast cancer cell line, which gave detailed insights into the potentials and limitations of Walker 256 as a breast cancer cell line. I have also performed transcriptomic characterization of the DRGs and spinal cord of BCIBP rats compared to sham rats, which has given important information on gene-level changes associated with BCIBP. Lastly, I have found that J- 156 has potential to alleviate BCIBP and hence, this provides valuable insight into the role of SST4 receptor as an analgesic drug target in the pathophysiological state of BCIBP.