Using two-way ANOVA, Mann Whitney and t-test (as indicated in the figure legends), data were assessed for statistical significance, where P 0

Using two-way ANOVA, Mann Whitney and t-test (as indicated in the figure legends), data were assessed for statistical significance, where P 0.05 was deemed the minimum statistical significance upon comparison of samples. Results Comparison of biological characteristics of the paired SW480 and SW620 cancer cell lines The morphology of SW480 and SW620 cells were analysed by phase contrast microscopy to reveal two cell morphologies (Fig. model of cancer progression. Their chemosensitivity and CSC properties were investigated. A range of assays were performed, including the side population assay, ALDEFLUOR assay, tumoursphere assay and assessment of CSC-associated surface phenotypes. It was determined that the SW480 and SW620 cells exhibited similar growth rates, although the SW480 cells were more migratory in wound healing assays on collagen and fibronectin matrices. SW480 and SW620 cells displayed similar CSC profiles, however, SW480 cells demosntrated significantly greater tumoursphere forming efficiency over SW620 cells. Tumourspheres derived from SW480 and SW620 cells also displayed differential sensitivity to 5-fluorouracil, oxaliplatin, geldanamycin and novobiocin that was not apparent when cells were grown under adherent conditions. Taken together, these results suggest that although the two cell lines have similar levels of putative CSC populations, there are differences in their biology that cannot be explained by these CSC levels. To the best of our knowledge, this is the first study to conduct a detailed analysis of the CSC populations using multiple assays in a paired cell line model. These results have clinical relevance for the understanding of the differences between primary tumours and their metastatic counterparts. assays have been used to identify putative CSC populations. These include the side population (SP) and ALDEFLUOR assays, the detection of specific cell surface markers, and the assessment of the ability of cells to grow as tumourspheres (TS) in suspension (13C15). The SP assay identifies putative CSCs based on the high activity of the ATP-binding cassette transporter protein (ABC)G2, which is also implicated in drug resistance due to its role in the efflux of chemotherapeutics from the cell (16,17). On the other hand, the ALDEFLUOR assay identifies CSCs using another unique CSC marker, aldehyde dehydrogenase (ALDH). The detoxifying effect of ALDH is thought to protect stem cells against oxidative damage and may modulate Splitomicin the proliferative capacity of stem cells (18). As a functional assay, the generation of three-dimensional spheres using serum-free culture methods takes advantage of the stem-like nature of CSC by allowing survival from anoikis and this method has been utilised for the identification and expansion of CSC populations (19) In colon cancer, putative CSCs have been identified using a range of the aforementioned techniques, in particular according to the expression of the cell surface protein markers, CD44 and CD133, and to the expression of ALDH and ABCG2 (4,14,19C21). The most appropriate Splitomicin and accurate method of identifying of CSCs remains a subject of intense debate, Furthermore, many researchers remain sceptical as to the role of this subpopulation in cancer initiation and progression. In particular, whether or not the presence of CSCs determines the metastatic potential of the tumour has yet to be fully elucidated (3). In Splitomicin this study, we used a paired colon cancer cell line model derived from a single patient, representative of the primary tumour (SW480) and its lymph node metastisis (SW620) (22). As the SW480 and SW620 cell lines developed from the same genetic background, Splitomicin they provide an model to study the cellular changes that occur during cancer progression and development of a metastatic phenotype. Our analysis focussed on the comparative analysis of putative CSC populations in these paired lines. We hypothesised that if CSC are responsible for metastasis, then the SW620 cell line may be enriched in CSC compared to the SW480 cell line and therefore should be the more chemoresistant of the two cell lines. Materials and methods Adherent cell lines and culture conditions Paired human colon adenocarcinoma cell lines SW480 (primary/pre-metastatic tumour; cat. no. 87092801) and SW620 (lymph node metastasis; cat. no. 87051203) were purchased from the European Collection of Animal Cell Cultures (Salisbury, UK). SW480 and SW620 cells Rabbit Polyclonal to TPD54 were maintained in Leibovitz’s L-15 medium with GlutaMAX?-I (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 10% (v/v) fetal bovine serum (Biowest, Nuaill, France) and 100 U/ml PSA (Gibco; Invitrogen) at 37C. Cell lines used in experiments exhibited passage.