Reconstruction of single cell lineage trajectories and identification of diversity in fates during the epithelial-to-mesenchymal transition

Abstract

AbstractThe epithelial-to-mesenchymal transition (EMT) gives rise to a variety of different cell states, yet it remains unclear whether these distinct states represent varying degrees of progression along a single trajectory or serve as endpoints of multiple potential trajectories that individual cells might pursue. Understanding the intricacies of these diverse EMT trajectories and cell fates, especially using lineage tracing at the single cell level using single cell RNA sequencing (scRNAseq) data, remains challenging. In this study, we employed optimal-transport analysis to reconstruct the most probable past trajectories of different cell fates during TGF-beta-induced EMT in the MCF10A cell line. Our analysis revealed three distinct temporal processes: those resulting in a low EMT, partial EMT, and high EMT state. The partial EMT trajectory exhibited substantial variations in the EMT signature and displayed unique cellular characteristics, including high stemness, low proliferation scores, and a potent cellular response to hypoxia. Along the partial EMT trajectory, we observed downregulation of the EED and EZH2 genes together with early high expression of TGFBI, KRT8, and CDH1—a finding that was validated by recent CRISPR-associated knock-out screening studies. Through our early differential gene expression analysis, we comprehensively explored genes linked to proliferation and stemness, identifying ITGB4, LAMA3, and LAMB3 as differentially expressed in the early stages of the partial versus high EMT trajectory. Our analysis thus elucidates novel potential targets for modulating EMT trajectories.