1240 Endovascular progenitors initiate and drive de novo vascularisation in melanoma

Abstract

The development of new vascular structures is a pre-requisite for melanoma growth and spread. We aimed to better identify and characterize the source of de novo endothelium in melanoma. Among Lin-CD34+ cells, expression levels of VEGFR2 and CD31 defined three distinct endothelial sub-populations. Lineage tracing of endothelial cells (Cdh5CreER/RosaYFP) demonstrated a maturation sequence from endovascular progenitor (EVP) via transit amplifying (TA) to fully differentiated (D) cells in B16 melanoma. Transplantation and clonal culture at limiting dilution demonstrated EVP cells were the only endothelial sub-population with self-renewal and engraftment capacity. Clonal analyses of multicolour lineage tracing (Cdh5CreER/Rainbow3) further showed the contribution of different progenitors to venous and arterial structures within tumours. RNA-seq demonstrated significant differences between populations and pointed to Sox18, JAK/STAT and Notch signalling to be significantly upregulated in the EVP. In Sox18CreER/Rosa-YFP reporter mice, EVP activated Sox18 expression as early as 3 days after tumour inoculation. We next sought to specifically target EVP activity. Anti-IL6Rα and gp-130 antibodies blocked JAK/STAT signalling, significantly reducing EVP infiltrate into the tumour. Importantly, this caused a significant reduction of the vascular network and a reduction in tumour size. To validate the activity on Notch signalling within EVP, we used the Notch conditional knockout model RBPJlox/lox/Cdh5CreER/Rosa-YFP. Deletion of Notch signalling significantly reduced EVP and vessel formation in tumours. Importantly, a significant reduction in size and weight of the tumour was also observed. In conclusion, we demonstrated the importance of EVP initiating the de novo vascular network in melanoma and blocking JAK/STAT or Notch signalling prevented EVP infiltration and vessel formation.