Novel in vitro assays for the characterization of EMT in tumourigenesis

Cell Oncol. 2010;32(1-2):67-76. doi: 10.3233/CLO-2009-0501.

Abstract

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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Dedifferentiation*
  • Cell Line, Tumor
  • Cytological Techniques / methods*
  • Epithelial Cells / cytology*
  • Epithelial Cells / metabolism
  • Neoplasms / metabolism
  • Neoplasms / pathology*
  • Neoplasms / physiopathology*
  • Neoplastic Processes*
  • Rats
  • Transforming Growth Factor beta1 / metabolism

Substances

  • Transforming Growth Factor beta1