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Cellular plasticity and transitional phenotypes add to complexities of cancer metastasis that can be initiated by single cell epithelial to mesenchymal transition (EMT) or cooperative cell migration (CCM). Our study identifies novel regulatory cross-talks between Tcf21 and Slug in mediating phenotypic and migration plasticity in high-grade serous ovarian adenocarcinoma (HGSC). Differential expression and subcellular localization associate Tcf21, Slug with epithelial, mesenchymal phenotypes, respectively; however, gene manipulation approaches identify their association with additional intermediate phenotypic states, implying the existence of a multistep epithelial-mesenchymal transition program. Live imaging further associated distinct migratory modalities with the Tcf21/Slug status of cell systems and discerned proliferative/passive CCM, active CCM and EMT modes of migration. Tcf21–Slug balance identified across a phenotypic spectrum in HGSC cell lines, associated with microenvironment-induced transitions and the emergence of an epithelial phenotype following drug exposure. Phenotypic transitions and associated functionalities following drug exposure were affirmed to ensue from occupancy of Slug promoter E-box sequences by Tcf21. Our study effectively provides a framework for understanding the relevance of ovarian cancer plasticity as a function of two transcription factors.

Metastatic dissemination generates an aggressive disease facilitated by enhanced migratory and invasive properties. Experimental approaches employ several in vitro and in vivo assays toward quantification of these functionalities. In vitro assessments of cell motility often employ endpoint assays that rely on the global efficacy of wound closure and thwart quantification of migratory phenotypes observed during metastatic dissemination. Recent studies highlight the distinct signatures associated with individual vs. collective cell migration and necessitate the incorporation of these modalities into routine analyses. Advances in live cell imaging that permit real-time visualization of pathophysiological processes can be employed toward elucidating phenotypic plasticity associated with cell migration to overcome caveats inherent to end-point assays. Herein, we corroborate live cell imaging with the in vitro scratch assay toward quantification of migratory modalities in transformed cells. Our protocol describes a step-by-step approach for live cell setup of the scratch assay, and details analyses employed toward definition of three quantitative metrics viz., displacement, velocity and number of nearest neighbors. The current protocol (from scratch induction to data acquisition) is implemented for ~30 h and provides global/single-cell resolution of migratory phenotypes as opposed to the endpoint assays. Routine application of this protocol in cancer biology can aid the design of therapeutic regimes targeting specific migratory modalities and significantly contribute to the dissection of associated molecular networks.