TGF-beta generates multiple groups of cells during the progression of partial endothelial-mesenchymal transition (EndoMT)

Tumor progression and metastasis are regulated by endothelial cells (ECs) which modify their characteristics through a process known as endothelial-mesenchymal transition (EndoMT). EndoMT is a cellular differentiation process in which ECs lose their properties and differentiate into mesenchymal cells. The cells undergoing EndoMT differentiate through a spectrum of intermediary phases, suggesting that some cells remain in partial EndoMT state and exhibit endothelial/mesenchymal hybrid phenotype. However, because of the lack of specific markers, detailed analysis of partial EndoMT has been hampered. Transforming growth factor-beta (TGF-beta) plays a central role in the induction of EndoMT. In the present study, we showed that inhibition of TGF-beta signaling suppressed EndoMT in the tumors of oral cancer cells. We have also established a novel EndoMT reporter cell system, EndoMT reporter endothelial cells (EMRECs). EMRECs underwent TGF-beta-induced EndoMT enabling the visualization of sequential changes during EndoMT. Using the EMRECs, we characterized the gene profiles of the multiple EndoMT stages and identified CD40 as a novel partial EndoMT-specific marker. CD40 expression was upregulated in the TGF-beta-treated ECs and the partial EndoMT cells but decreased in the full EndoMT cells. Furthermore, single-cell RNA sequencing analyses revealed that CD40 expression was highly enriched in human tumors, in the population of cells expressing both endothelial and mesenchymal cell markers. The present findings provide a better understanding of the mechanisms underlying TGF-beta-induced EndoMT and will allow the development of novel therapeutic strategies targeting the EndoMT-driven cancer progression and metastasis.