A new approach to epithelial-mesenchymal transition diagnostics in epithelial tumors: double immunofluorescent staining and flow cytometry

Method summary Double immunofluorescent staining approach for flow cytometry has been developed to study the quantitative vimentin expression in whichde novoexpression is observed only when an epithelium cell enters the EMT. The level of the marker's expression in epithelial cells was determined as the ratio (%) of the number of the cells coexpressing cytokeratins and vimentin to the total number of tumor cells expressing cytokeratins. Clinical validation of the new method was conducted in tumors of narrowly defined cohort of 32 patients: stage III high-grade serous ovarian cancer, suboptimal surgical cytoreduction and platinum- and taxane-based chemotherapy. A new method of double immunofluorescent staining for flow cytometry has been created to evaluate quantitative expression of mesenchymal protein vimentin only in epithelial cells of a solid tumor that is a mix of different origin cells.De novo vimentin expression is strongly associated with epithelial-mesenchymal transition and therefore is a metastatic potential marker of epithelial tumor cells. In comparison with semiquantitative available methods, the proposed one has several advantages, such as the accurate measurement of the marker's expression, and minimization of spatial and temporal tumor heterogeneity. Clinical validation of the method has revealed inverse correlation between the quantitative index of epithelial-mesenchymal transition level and progression-free survival using Kaplan-Meier curves and the COX proportional hazards ratio in 32 ovarian cancer patients.