Open AccessNovel In Vitro Assays for the Characterization of EMT in Tumourigenesis
Author(s) -
Vincent Koo,
Amgad El Mekabaty,
Peter Hamilton,
Perry Maxwell,
Osama Sharaf,
Jim Diamond,
Jenny Watson,
Kathleen Williamson
Publication year - 2010
Publication title -
analytical cellular pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.576
H-Index - 24
eISSN - 2210-7185
pISSN - 2210-7177
DOI - 10.1155/2010/985981
Subject(s) - vimentin , matrigel , cytokeratin , epithelial–mesenchymal transition , motility , in vitro , chemistry , microbiology and biotechnology , pathology , cancer research , biology , cancer , immunohistochemistry , medicine , metastasis , biochemistry
Background : Two novel assays quantifying Epithelial to Mesenchymal Transition (EMT) were compared to traditional motility and migration assays. TGF- β 1 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- β 1 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- β 1 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- β 1 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- β 1 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.