Abstract 1235: Quantitative high-content imaging reveals novel modulators of the metastatic hybrid state during epithelial-mesenchymal transition
Authors: Purcell Daniel J. , Wheeler Christina E. , Deaver Stacie E. , Judson-Torres Robert L. , Lamouille Samy 
Published: April 2023

Epithelial-mesenchymal transition (EMT) is a normal developmental and healing process that can be pathologically reactivated during cancer progression. Through EMT, neoplastic cells obtain metastatic capabilities like enhanced motility, invasion, and resistance to apoptosis. Recent studies have highlighted dynamic cell plasticity during EMT, including a highly tumorigenic population of cells within the full spectrum of transitions that adopt a partial EMT state. These hybrid populations retain a higher degree of metastatic ability due to the dual nature of their phenotype; however, concomitant expression of epithelial and mesenchymal traits makes characterization and analysis difficult. Additionally, the total expression of certain markers can remain unchanged during EMT while their subcellular localization and function is altered. Therefore, there is a need to improve analytical methods to better characterize hybrid EMT populations and elucidate the genetic modulators that govern advancement along the EMT spectrum. In this study, we provide an optimized method of automated whole-well imaging and analysis for characterization of the EMT spectrum and hybrid phenotypes. We utilized three different TGF-β-induced EMT models. Cell lines cultured in 96-well plates were treated with TGF-β and/or a specific inhibitor of the TGF-β receptor, SB431542, at different times between 30 minutes and 48 hours before fixing and labeling by immunofluorescence. Expression and subcellular localization of TGF-β effectors Smad2/3; epithelial markers E-cadherin, ZO-1, and β-catenin; and mesenchymal markers N-cadherin, fibronectin, and vimentin, were analyzed and quantified using an automated cell imager system and associated analysis software. In addition, nuclei and cell perimeters were identified to analyze protein nuclear translocation and cell size and shape, respectively. Through these studies, we have successfully isolated a hybrid cell subpopulation emerging at 18 hours of TGF-β treatment. These cells display a partial loss of E-cadherin and upregulation of fibronectin that constitutes a unique proteome distinct from that of fully epithelial and mesenchymal populations. Using RNA-interference, we are currently testing 15 candidate genes we previously identified from a microRNA screen on EMT progression and the hybrid state. In summary, we developed and optimized a novel imaging and analysis method for rapid assessment of EMT cell populations, and identification of novel modulators of the EMT spectrum. This method can be more widely applied to screen for small molecules or therapeutic peptides affecting EMT-driven malignancy. Given that the hybrid phenotype is now considered to have higher metastatic characteristics overall, we believe this approach will facilitate identification and testing of potential targets for treatment of metastatic disease.

Citation Format: Daniel J. Purcell, Christina E. Wheeler, Stacie E. Deaver, Robert L. Judson-Torres, Samy Lamouille. Quantitative high-content imaging reveals novel modulators of the metastatic hybrid state during epithelial-mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1235.