For in vivo administration, FLT1 and ctrl plasmid DNA was complexed with in vivo-jetPEI. 15 μg of DNA per one injection was complexed with in vivo-JetPEI at an N/P ratio of 6 in 5% glucose solution for intratumoural injection. The mixture was incubated for at least 30 min at room temperature. Randomised cohorts of the tumour-bearing WT and HIF-1α KO mice received intratumoural injections of 15 μg of DNA in 100 μl of 5% glucose solution every 2 days starting on day 6 until endpoint at day 14. Control mice were injected with control plasmid.

Productive angiogenesis, a prerequisite for tumour growth, depends on the balanced release of angiogenic and angiostatic factors by different cell types within hypoxic tumours. Natural killer (NK) cells kill cancer cells and infiltrate hypoxic tumour areas. Cellular adaptation to low oxygen is mediated by Hypoxia-inducible factors (HIFs). We found that deletion of HIF-1alpha in NK cells inhibited tumour growth despite impaired tumour cell killing. Tumours developing in these conditions were characterised by a high-density network of immature vessels, severe haemorrhage, increased hypoxia, and facilitated metastasis due to non-productive angiogenesis. Loss of HIF-1alpha in NK cells increased the bioavailability of the major angiogenic cytokine vascular endothelial growth factor (VEGF) by decreasing the infiltration of NK cells that express angiostatic soluble VEGFR-1. In summary, this identifies the hypoxic response in NK cells as an inhibitor of VEGF-driven angiogenesis, yet, this promotes tumour growth by allowing the formation of functionally improved vessels.