Oral Presentation 32nd Lorne Cancer 2020

Endovascular progenitors initiate and drive de novo vascularisation in melanoma (#15)

James JD Dight 1 , Jatin JP Patel 1 , Ghazaleh GH Hashemi 1 , Kiarash KK Khosrotehrani 1
  1. University of Queensland - Diamantina Institute, Indooroopilly, QLD, Australia

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 and HCmel12 melanoma. In lineage tracing experiments utilising Sox18CreER/Rosa-YFP and Cdh5CreER/Rosa-YFP reporter mice, EVPs activated Sox18 expression as early as 3 days after tumour inoculation and formed mature vascular networks. EVP cells also were the only endothelial sub-population with self-renewal and engraftment capacity. Clonal analyses of multicolour lineage tracing (Cdh5CreER/Rainbow3) further showed different progenitors contributing to venous and arterial structures within tumours. RNA-seq demonstrated significant differences between populations and pointed to Sox9 and IL-6/JAK/STAT signalling to be significantly upregulated in the EVP. We next sought to specifically target EVP activity. Anti-IL6Rα and Ruxolitinib 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 of Sox9 signalling within EVP, we used Sox9 conditional knockout model Sox9lox/lox/Cdh5CreER/Rosa-YFP. Deletion of Sox9 signalling significantly reduced EVP infiltrate and subsequent vessel formation in tumours. Importantly, a significant reduction in size and weight of the tumour was also observed. Notably, human Endothelial Colony Forming Cells (ECFCs) upregulate Sox9 and IL-6 signalling in the presence of human melanoma co-culture and these pathways are negatively regulated after Ruxolitinib treatment. This is the first description of Sox9 in the endothelium and its subsequent regulation by IL-6/JAK-STAT signalling.