The role of cancer stem cells in osteosarcoma

Osteosarcoma (OS) is the most common bone malignancy often producing aggressive tumours in adolescents. OS aetiology is poorly understood, however, recent studies suggest OS cancers contain a small population of cancer stem cells (CSC) which initiate tumour growth. The cancer stem cell hypothesis describes cancers as a hierarchical population of heterogeneous cells. It has been proposed that CSC are at the base of this hierarchy and are responsible for the initiation, growth and spread of the tumour and pose a therapeutic challenge due to enhanced chemotherapy resistance. This project had three aims: identifying whether OS cell lines contain subpopulations of putative CSC, identifying if CSC contribute to chemotherapeutic resistance and to elucidate paracrine cell signals controlling OS tumour growth. Eight OS cell lines (143B, Cal72, G292, HOS, MG63, MNNG-HOS, U2OS and SaOS-2) along with the breast cancer cell line MCF7 have been analysed for the presence of sub-population of cells expressing putative CSC markers (aldehyde dehydrogenase and CD117). The intracellular enzyme aldehyde dehydrogenase (ALDH) and tyrosine kinase receptor CD117 were found to be heterogeneously expressed amongst the cell lines. All cell lines when plated at low density could recapitulate the colony hierarchies based on variation in colony morphology (holoclones, meroclones and paraclones), this was originally observed in carcinoma cell lines and is further putative evidence a CSC hierarchy exists within these cell lines. ALDH expressing cells were found to be confined to the holoclones (in cell lines with ALDH populations comprising less than 10 % of the total population) indicating that putative CSCs reside within this population. All OS cell lines also expressed mesenchymal markers (high vimentin and CD44 expression and low e-cadherin expression) suggesting they are a progenitor of mesenchymal stem cells. The expression of CD117 was found to negatively correlate with cisplatin chemotherapy resistance whereas ALDH inhibition using the specific antagonist diethylaminobenzalde sensitised different cells lines to opposing chemotherapeutics, suggesting a heterogenous response of OS ALDH cells to cytotoxic compounds. All OS cells lines, except 143B and HOS, were found to secrete a paracine growth factor which was capable of significantly enhancing their own growth. U2OS conditioned media was also able to enhance the growth of the breast cancer cell line MCF7 and a fibrosarcoma cell line (HT1080). Analysis of the cytokine expression profile of OS cell lines (HOS, MG63 and U2OS) demonstrated these cells secrete a broad range of cytokines associated with inflammation. The cytokine CCL-2 was identified as the putative OS paracrine growth factor as determined by the response to recombinant CCL-2, receptor antagonism and CCL-2 RNA interference. Genes with altered expression in response to CCL-2 were associated with transcription, suggesting that CCL-2 enhances proliferation through its downstream effect on transcription. Overall this thesis has contributed to the field of oncology by further defining the populations of putative CSC within a panel of OS cell lines. In addition the identification of a novel OS growth factor provides a possible adjuvant therapeutic target, which could aid in the reduction of OS proliferation