Deciphering functional intra-tumoral heterogeneity in BRAF-driven mouse thyroid cancer reveals EMT trajectory and metabolic remodeling.

A better understanding of functional heterogeneity of tumor cells may lead to the improvement of the diagnosis and development of effective therapies for patients with papillary thyroid cancer (PTC). Here, the transcriptional heterogeneity and hierarchical trajectory of malignant thyrocytes were investigated by single-cell RNA sequencing analysis in an adult-onset autochthonous mouse model of PTC driven by a BRAF. Within BRAF thyrocytes, we identified distinct subpopulations exhibiting varying degrees of mesenchymal transformation and stability. Further analysis uncovered an epithelial-mesenchymal transition (EMT) trajectory, where malignant subpopulations progress from an intermediate to a more mesenchymal and malignant state. The EMT-related phenotype of each subpopulation was validated with an organoid culture system. Further analysis demonstrated that major malignant subpopulations maintain distinct EMT states through subpopulation-specific pathway usage, whereas transitioning cells undergo a dynamic shift in molecular strategy by using different pathway combinations or distinct gene sets within the same EMT-related pathways along the EMT trajectory. Moreover, metformin diminished the most malignant subpopulation in the PTC mouse model and inhibited EMT potential in both regular and organoid cultures. Importantly, the functional heterogeneity of tumor cells identified in PTC mouse model have clinical relevance. Furthermore, transcriptome dynamics analysis revealed an EMT trajectory-related gene module with substantial predictive value for human PTCs with BRAF. Lastly, our analysis reveals that p53 deficiency lowers the tumorigenic threshold for BRAF and promotes cell state transition towards a more malignant state, rather than altering cellular heterogeneity or the transition trajectory. Subsequent analysis in vitro demonstrated that p53 loss might confer more malignant potential to BRAF thyrocytes through modulating metabolic plasticity. Collectively, our work not only deciphers the functional intra-tumoral heterogeneity of BRAF-drieven PTC but also thereby provides a theoretical foundation for developing EMT-targeted treatment.