SOX9 reactivation in cancer vessels shapes the tumor micro-environment through hypoxia and immune depletion promoting tumor growth and metastasis

The development of new vascular structures is a pre-requisite for tumor growth and spread. This process is often disorganised and produces immature and leaky vessels and relies at least in part on the activity of endovascular progenitor cells (EVPs), residing in vessel walls and giving rise to mature endothelial cells in de novo blood vessel networks in the tumor. Sox9 is a transcription factor that is playing an important role in stem cell self-renewal and fate choice and is highly upregulated in EVPs. In this study, we aimed to explore how Sox9 activity in the endothelium affects tumor vascularisation, microenvironment, and metastasis. Indeed, Sox9 expression was upregulated in tumor endothelial cells of mice harbouring melanomas. Similarly, we observed the up regulation of SOX9 in human endothelial cells exposed to melanoma cell co-culture or conditioned medium resulting in increased colony formation and reduced maturity as revealed in tube formation assays. Endothelial-specific conditional knockout of Sox9 (Sox9fl/fl/Cdh5CreERt2/Rosa-YFP) resulted in a significant reduction in total endothelial cells in B16-F0 or HcMel12 melanoma tumors inoculated intradermally in both flow-cytometry, lineage tracing and immunostaining of tumor sections. Functionally, there was a significant reduction in tumour size and lung metastases after Sox9 deletion in the endothelium. Importantly, despite a major reduction in the number and area of CD31 ⁺ vessels there was a significant increase in pericyte coverage suggesting increased maturity of the remaining vessels upon Sox9 deletion in the endothelium. These changes in the endothelium translated into a reduction in hypoxia as demonstrated by decreased GLUT1 expression and reduced nuclear localisation of HIF1α. RNA sequencing of sorted tumor cells as well as spatial transcriptomics of tumor sections with endothelial-specific deletion of Sox9 versus controls confirmed the reduction in hypoxia and showed dramatic increases in CD4 and CD8 immune T cell infiltration in the centre of tumors as confirmed by immunostaining. In summary, endothelial-specific Sox9 deletion resulted in fewer and more mature de novo vessels in the centre of the tumor and reduced metastatic dissemination, suggesting strategies that target this pathway may restore the normal function of blood vessels in tumors and prevent disease progression.