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

Exploring the complexity of the epithelial-to-mesenchymal transition (EMT) unveils a diversity of potential cell fates; however, the exact timing and intricate mechanisms by which early cell states diverge into distinct EMT trajectories remain unclear. Studying these EMT trajectories through single cell RNA sequencing is challenging due to the necessity of sacrificing cells for each measurement. In this study, we employed optimal-transport (OT) analysis to reconstruct the past trajectories of different cell fates during TGF-beta-induced EMT in the MCF10A cell line. Our analysis revealed three distinct trajectories leading to low EMT, partial EMT, and high EMT states. Cells along partial EMT trajectory showed substantial variations in the EMT signature and exhibited pronounced stemness. Throughout this EMT trajectory, we observed a consistent downregulation of the EED and EZH2 genes. This finding was validated by recent inhibitor screens of EMT regulators and CRISPR screen studies. Moreover, we applied our analysis of early-phase differential gene expression to gene sets associated with stemness and proliferation, pinpointing ITGB4 , LAMA3 , and LAMB3 as genes differentially expressed in the initial stages of the partial versus high EMT trajectories. We also found that CENPF , CKS1B , and MKI67 showed significant upregulation in the high EMT trajectory. While the first group of genes aligns with findings from previous studies, our work uniquely pinpoints the precise timing of these upregulations. Finally, the latter group of genes represents newly identified regulators, shedding light on potential targets for modulating EMT trajectories. Significance Statement In our study, we investigated cellular trajectories during EMT using a time-series scRNAseq dataset. OT analysis was used to infer cell-to-cell connections from scRNAseq data, allowing us to predict cell linkages and overcome limitations of sequencing such as the need to sacrifice cells for each measurement. This approach allowed us to identify diverse EMT responses under uniform treatment, a significant advancement over previous studies limited by the static nature of scRNAseq data. Our analysis identified a broad set of genes involved in the EMT process, uncovering novel insights such as the upregulation of cell cycle genes in cells predisposed to a high EMT state and the enhancement of cell adhesion marker genes in cells veering towards a partial EMT state. This work enriches our understanding of the dynamic processes of EMT, showcasing the varied cellular fates within the same experimental setup. ### Competing Interest Statement F.M. is a co-founder of and has equity in Harbinger Health, has equity in Zephyr AI, and serves as a consultant for both companies. She is also on the board of directors of Exscientia Plc. F.M. declares that none of these relationships are directly or indirectly related to the content of this manuscript. All other authors declare no conflicts.