Characterisation of three novel canine osteosarcoma cell lines producing high levels of matrix metalloproteinases

Abstract

Three canine osteosarcoma cell lines were established from spontaneous pelvic and radial osteosarcomas. The cell populations cultured exhibited characteristics of malignancy and consisted of adherent, pleomorphic, mostly large spindle-shaped or polyhedral cells, characterised by the presence of numerous cytoplasmic granules and vacuoles. The main ultrastructural features included the presence of abundant rough endoplasmic reticulum and numerous cytoplasmic vesicles, deposit vacuoles and small cytoplasmic protrusions. Zymography showed that the cell lines produce high levels of MMP-2 and MMP-9, enzymes directly involved in crucial aspects of the metastatic process. Consistent with their osteoblastic lineage and malignant phenotype, all cell lines were immunoreactive to vimentin, osteopontin, PCNA, p53, MMP-2 and MMP-9, while they were negative for cytokeratin, desmin, SMA, Factor VIII, NSE, GFAP, Rb and p21 protein. No retroviral particles or RNA were detected ultrastructurally or with RT-PCR, although the possibility of viral involvement in osteosarcoma cannot be excluded. The new cell lines provide excellent in vitro models that may allow further studies on the pathobiology of canine osteosarcoma to be undertaken.

Introduction

Osteosarcoma is the most frequently diagnosed and reported canine bone tumour (Tjalma, 1966). It is considered one of the most malignant and aggressive tumours, with a predilection in large and giant breeds (Tjalma, 1966; Palmer, 1993). Osteosarcoma in dogs bears a striking resemblance, both clinically and histopathologically, to osteosarcoma in people (Misdorp, 1980), and is, therefore, a readily available and highly comparable spontaneously occurring cancer that can and has been used as a model for understanding human osteosarcoma and for establishing improved treatment regimes in people. Despite significant advances on many fronts with regard to osteosarcoma in recent years, including in imaging, surgery and chemotherapy, there remains the need for the identification of new therapeutic targets that will lead to treatment strategies which will improve the current prognosis, alone or in conjunction with current regimes.

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes implicated in the degradation and remodeling of extracellular matrix and in vascularisation (Matrisian, 1992). A growing body of evidence indicates that MMPs are also involved in a number of pathologic processes, including tumour invasion and metastasis in experimental cancer models (Sawaya et al., 1998) and in human malignancies. MMP-2 and -9 (gelatinases), a group within the MMP family (Matrisian, 1992), have been shown to be capable of degradation of critical, in terms of their importance in the process of tumour invasion and metastasis, basement membrane and extracellular matrix components (Senior et al., 1991). Increased concentrations of MMPs, and gelatinases in particular, are associated with invasion, metastasis and poor prognosis in numerous human malignancies (Sugiura et al., 1998). Although the literature on MMPs in tumours of domestic animals is sparse, MMP-2 and -9 have been detected in canine osteosarcoma (Lana et al., 2000; Loukopoulos et al., 2003a) and cutaneous mast cell tumours (Leibman et al., 2000) in levels higher than in unaffected stromal tissue. They were more recently shown to be present in a wide range of canine tumours and normal tissues (Loukopoulos et al., 2003a); their link to tumour type and grade was demonstrated and, importantly, MMP production was shown to correlate with clinicopathological parameters of prognostic importance, particularly with regard to osteosarcoma (Loukopoulos et al., 2003a).

The demonstration of MMP involvement in canine tumours makes MMPs a promising therapeutic target; the use of MMP inhibitors, in particular, shows enormous potential for clinical application in the treatment of canine tumours (Brown and Giavazzi, 1995). A necessary first step in the strategic approach to the design of clinical trials on canine osteosarcoma patients using MMP inhibitors, or any other candidate drugs, is their evaluation in in vitro and in vivo models of canine osteosarcoma. MMPs have been shown to be produced by human osteosarcoma cell lines (Kawashima et al., 1994) and normal human osteoblastic cells in culture (Johansen et al., 1992), as well as various other cell lines, such as human bronchial epithelial, monocytic leukaemia, fibrosarcoma (Morodomi et al., 1992), and breast cancer cell lines. However, there are no available canine osteosarcoma or other tumour cell lines that are known to produce MMPs. According to one study, canine osteosarcoma cells did not produce any endogenous collagenase (MMP-1) in the culture medium (Cakir and Hahn, 1999).

A small number of canine osteosarcoma cell lines have been produced and partially characterised to date, originating from various sites, including the radius (Bostock and Owen, 1970; Nieves et al., 1998), the humerus (Nieves et al., 1998), the femur (Norrdin et al., 1989), the tibia (Norrdin et al., 1989), the mandible (Hong et al., 1998) and the scapula (Hong et al., 1998), and some of them have been used to further understand aspects of the pathobiology of osteosarcoma (Hong et al., 2000). Six clonal cells were also established, characterised, classified using a limiting dilution technique and further cultured to produce a highly metastasising cell line (Barroga et al., 1999).

Furthermore, although the clinical and pathological features of osteosarcoma vary according to its location (Palmer, 1993), with, for example, osteosarcomas arising in the appendicular skeleton generally exhibiting a more malignant phenotype than axial ones, there is only one cell line that has originated from the axial skeleton (mandible).

The aim of the study was to produce and characterise canine osteosarcoma cell lines from both appendicular and axial locations, and to examine whether they produce MMPs, in order to establish an in vitro model that will allow further studies on the pathobiology of canine osteosarcoma to be undertaken, and will also allow the preclinical evaluation of potential therapeutic agents.