Novel In Vitro Assays for the Characterization of EMT in Tumourigenesis

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.Conclusions: The Spindle Index and the novel chemoattractant assay are valuable adjunctive assays for objective characterization of EMT changes during tumourigenesis.

Cancer Mechanobiology: Microenvironmental Sensing and Metastasis

“…Elevation of specific ECMs including laminin-332, periostin, and fibronectin has been observed in EMT as mentioned above. Although these phenomena with 3D in vitro studies − provide robust mechanistic insights to EMT of cancer cells, promising results are not simply derived or matched with in vivo models. , …”

Tumor microenvironment and epithelial-mesenchymal transition in bladder cancer: Cytokines in the game?

“…A change in the actin cytoskeletal architecture during this phase results in phenotypical changes where front-rear polarity replaces apical-basal polarity. ( Koo et al, 2010 ; Lu and Kang, 2019 ). Molecularly, it occurs a decrease in epithelial-related genes, such as CDH1 , TJP1 , CLDN1 and specific cytokeratin genes, and an increase in mesenchymal-related genes, such as VIM , CDH2 , ITGB1 and ITGB2 ( Koo et al, 2010 ; Lu and Kang, 2019 ).…”

“…( Koo et al, 2010 ; Lu and Kang, 2019 ). Molecularly, it occurs a decrease in epithelial-related genes, such as CDH1 , TJP1 , CLDN1 and specific cytokeratin genes, and an increase in mesenchymal-related genes, such as VIM , CDH2 , ITGB1 and ITGB2 ( Koo et al, 2010 ; Lu and Kang, 2019 ). Additionally, cells exhibiting EMT characteristics can degrade the extracellular matrix by MMPs ( Xu et al, 2009 ; Lu and Kang, 2019 ).…”

“…Additionally, cells exhibiting EMT characteristics can degrade the extracellular matrix by MMPs ( Xu et al, 2009 ; Lu and Kang, 2019 ). As a result, these cells increase motility, develop resistance to apoptosis, and become isolated, which culminates in cell invasion and migration ( Xu et al, 2009 ; Koo et al, 2010 ). According to in silico analysis, EMT signaling pathways were shown to be significantly activated from NMIBCs to MIBCs ( Cao et al, 2020 ).…”

“…TGF-β1 treatment of T24 and BIU87 BlCa cell lines resulted in increased fascin1 levels, an important molecule in tumor migration and invasion ( Zhang et al, 2016 ). Finally, AY-27, a rat cell line, treated with TGF-β1 resulted in alterations in morphology, with the increase of spindle shaped cells, while the polygonal shaped cells decreased, as well as cell-to-cell contact ( Koo et al, 2010 ). In Smad-dependent signaling, it occurs recruitment and phosphorylation of SMAD2 and SMAD3 ( Heldin et al, 2012 ; Gonzalez and Medici, 2014 ; Papageorgis, 2015 ; Gupta et al, 2016 ).…”