Novel in vitro assays for the characterization of EMT in 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 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.


CONCLUSIONS
The Spindle Index and the novel chemoattractant assay are valuable adjunctive assays for objective characterization of EMT changes during tumourigenesis.
Koo
Vincent
V
Uro-oncology Research Group, Centre for Cancer Research & Cell Biology, Queen's University Belfast, Belfast, Northern Ireland, UK.
El Mekabaty
Amgad
A
Hamilton
Peter
P
Maxwell
Perry
P
Sharaf
Osama
O
Diamond
Jim
J
Watson
Jenny
J
Williamson
Kathleen
K
D000818:Animals; D054337:Cell Dedifferentiation; D045744:Cell Line, Tumor; D003584:Cytological Techniques / Q000379:methods*; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D009369:Neoplasms / Q000378:metabolism / Q000473:pathology* / Q000503:physiopathology*; D009385:Neoplastic Processes; D051381:Rats; D053773:Transforming Growth Factor beta1 / Q000378:metabolism
D053773:Transforming Growth Factor beta1