Isolation and characterisation of canine osteosarcoma cells to investigate mechanisms of chemoresistance

Background: Osteosarcoma (OSA) is the most prevalent primary bone cancer in humans and is 27 times more prevalent in dogs. Unfortunately, over the past 2 decades, the rate of 5-year survival for metastatic OSA has not improved in humans or dogs. It is thought that this is mainly due to chemoresistance and relapse, and our research group has recently identified autophagy as a potential mechanism for chemoresistance in human OSA, although the role of autophagy in canine OSA chemoresistance is still unknown. Due to the fact that pet dogs share both environmental and lifestyle attributes with their human owners, there is an increased awareness that ‘One Medicine’, the mutual co-study of animals and humans, could be beneficial for both humans and dogs in OSA research. Aims: In this study, the aim was to optimise the protocol for isolating primary canine OSA cells and to compare chemoresistance and the role of autophagy in these cells with an immortalised canine osteosarcoma cell line (OSCA) and canine osteoblasts isolated from trabecular bone. Methods: OSA tumour biopsies were isolated during limb amputation surgery in canine patients diagnosed with OSA. For optimisation of OSA cell isolation from biopsies, three different protocols were tested, by varying serum sources, introducing collagenase digest or adding a Rho Kinase Inhibitor to encourage cell attachment. Alizarin Red Staining, alkaline phosphatase (ALP) assay and qPCR on differentiation markers were performed to determine the osteoblastic characteristics of the cells. To determine the chemosensitivity of the three cell types to doxorubicin and carboplatin, drug response assays were carried out. qPCR was carried out to investigate the role of autophagy-related genes in chemoresistant properties of canine OSA cells. Results: Inclusion of a RhoKinase inhibitor and pre-coating cell culture plastic with FBS was determined as essential for OSA cell isolation by collagenase digest. Cells from canine OSA biopsies and immortalised OSCA cells displayed a higher capacity to differentiate into osteoblast-like cells, producing ALP and alizarin red positive mineral nodules, compared to primary canine osteoblasts, and this was confirmed with qPCR analysis of osteoblastic differentiation markers. Results from drug dose-response and wound migration assays suggested that canine OSA cells were more resistant to chemotherapy drugs than primary canine osteoblasts. qPCR of autophagy-related genes was inconclusive and requires further investigation. Conclusions: We have successfully developed a protocol to isolate primary canine OSA cells from tumour biopsies and have identified the ability of these cells to differentiate towards the osteoblastic lineage. When compared to normal canine osteoblasts, these cells have inherent chemoresistant properties, however, the role of autophagy requires further investigation. By further characterising the chemoresistant and metastatic properties of these primary canine OSA cells, we will be one step closer to identifying novel targets to treat both canine and human OSA