Affiliations: Uro-oncology Research Group, Centre for Cancer Research & Cell Biology, Queen's University Belfast, Belfast, Northern Ireland, UK | Bio-imaging & Informatics Research Group, Centre for Cancer Research & Cell Biology, Queen's University Belfast, Belfast, Northern Ireland, UK | Belfast Health and Social Care Trust, Belfast, Northern Ireland, UK | The UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin, Ireland
Note: [] Corresponding author: Dr. Kate Williamson, Uro-oncology Group, Centre for Cancer Research & Cell Biology, Queen's University Belfast, Room G014, 97 Lisburn Road, Belfast, BT9 7BL, Northern Ireland, UK. Tel.: +44 289097 2790; Fax: +44 289097 2776; E-mail: [email protected].
Abstract: Background: Two novel assays quantifying Epithelial to Mesenchymal Transition (EMT) were compared to traditional motility and migration assays. TGF-β1 treatment of AY-27 rat bladder cancer cells acted as a model of EMT in tumourigenesis. Methods: AY-27 rat bladder cancer cells incubated with 3 ng/ml TGF-β1 or control media for 24 or 48 h were assessed using novel and traditional assays. The Spindle Index, a novel measure of spindle phenotype, was derived from the ratio of maximum length to maximum width of cells. The area covered by cells which migrated from a fixed coverslip towards supplemented agarose was measured in a novel chemoattractant assay. Motility, migration and immunoreactivity for E-cadherin, Vimentin and cytokeratin were assessed. Results: TGF-β1 treated cells had increased “spindle” phenotype together with decreased E-cadherin, decreased Cytokeratin-18 and increased Vimentin immunoreactivity. After 48 h, the mean Spindle Index of TGF-β1 treated cells was significantly higher than Mock (p=0.02, Bonferroni test) and there were significant differences in migration across treatment groups measured using the novel chemoattractant assay (p=0.02, Chi-square). TGF-β1 significantly increased matrigel invasion. Conclusion: The Spindle Index and the novel chemoattractant assay are valuable adjunctive assays for objective characterization of EMT changes during tumourigenesis.
