CD40 is expressed in the subsets of endothelial cells undergoing partial endothelial-mesenchymal transition in tumor microenvironment

Tumor progression and metastasis are regulated by endothelial cells undergoing endothelial-mesenchymal transition (EndoMT), a cellular differentiation process in which endothelial cells lose their properties and differentiate into mesenchymal cells. The cells undergoing EndoMT differentiate through a spectrum of intermediate phases, suggesting that some cells remain in a partial EndoMT state and exhibit an endothelial/mesenchymal phenotype. However, detailed analysis of partial EndoMT has been hampered by the lack of specific markers. Transforming growth factor-beta (TGF-beta) plays a central role in the induction of EndoMT. Here, we showed that inhibition of TGF-beta signaling suppressed EndoMT in a human oral cancer cell xenograft mouse model. By using genetic labeling of endothelial cell lineage, we also established a novel EndoMT reporter cell system, the EndoMT reporter endothelial cells (EMRECs), which allow visualization of sequential changes during TGF-beta-induced EndoMT. Using EMRECs, we characterized the gene profiles of multiple EndoMT stages and identified CD40 as a novel partial EndoMT-specific marker. CD40 expression was upregulated in the cells undergoing partial EndoMT, but decreased in the full EndoMT cells. Furthermore, single-cell RNA sequencing analysis of human tumors revealed that CD40 expression was enriched in the population of cells expressing both endothelial and mesenchymal cell markers. Moreover, decreased expression of CD40 in EMRECs enhanced TGF-beta-induced EndoMT, suggesting that CD40 expressed during partial EndoMT inhibits transition to full EndoMT. The present findings provide a better understanding of the mechanisms underlying TGF-beta-induced EndoMT and will facilitate the development of novel therapeutic strategies targeting EndoMT-driven cancer progression and metastasis.