BACKGROUND: Two novel assays quantifying Epithelial to Mesenchymal Transition (EMT) were compared to traditional motility and migration assays. TGF-beta1 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-beta1 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-beta1 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-beta1 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-beta1 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.
BACKGROUND: Two novel assays quantifying Epithelial to Mesenchymal Transition (EMT) were compared to traditional motility and migration assays. TGF-beta1 treatment of AY-27 ratbladder cancer cells acted as a model of EMT in tumourigenesis. METHODS: AY-27 ratbladder cancer cells incubated with 3 ng/ml TGF-beta1 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-beta1 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-beta1 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-beta1 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.
Authors: Akshita B Bhatt; Mohit Gupta; Van T Hoang; Suravi Chakrabarty; Thomas D Wright; Steven Elliot; Ishveen K Chopra; Darlene Monlish; Katie Anna; Matthew E Burow; Jane E Cavanaugh; Patrick T Flaherty Journal: Front Oncol Date: 2019-07-30 Impact factor: 6.244