Characterisation of the in vivo role of Eltd1 in physiological and tumour angiogenesis using knockout mouse models.


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Dian Wang1,Koon Hwee Ang2,Massimo Masiero3,Esther Bridges2,Alison Banham3,Adrian Harris2
1University of Oxford,2Department of Oncology, University of Oxford, John Radcliffe Hospital, Headington, Oxford, Oxfordshire, UK,3Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, Oxfordshire, UK

Abstract

Background

ELTD1 (Epidermal growth factor, latrophilin and seven transmembrane domain-containing protein 1) is an orphan adhesion G protein-coupled receptor. Our group discovered that ELTD1 could promote angiogenesis and in clinical samples (head and neck, ovarian, renal and colorectal cancers), ELTD1 was up-regulated in endothelial cells (EC) within tumours. In vivo, Eltd1 siRNA in ovarian and colorectal cancer xenograft models resulted in reduced tumour growth and improved survival. In the current study, Eltd1 floxed mice were generated to investigate its normal and pathological roles in vivo and its potential as a therapeutic target.

Method

The Eltd1 knockout strategy deleted exons 4 and 5, introducing a frameshift mutation and premature translational stop codon, to eliminate Eltd1 expression. Tamoxifen-inducible knockout of Eltd1 was performed in all tissues (Rosa26-Cre) or specifically in EC (PDGFβ-Cre), and Eltd1 was also constitutively knocked out in EC (Tie2-Cre). Eltd1 expression was analysed by IHC, q-PCR and RNAscope, EC sprouting ability was assessed ex vivo and the growth and histology of syngeneic cancer models were analysed.

Results

Eltd1 knockout was validated at the transcript and protein level and was particularly evident in normal lung and kidney tissues. Body weight, organ weight and organ structure exhibited no significant differences after Eltd1 deletion. Aortic ring assays indicated that ECs from tamoxifen-inducible knockout mice had less sprouting ability compared with control mice and constitutive EC Eltd1 knockout mice. Syngeneic breast (E0771) and colorectal (MC38) tumours grown subcutaneously in the three Eltd1 knockout mice models showed no difference in tumour volume to control mice, but exhibited significantly reduced vasculature density and increased necrosis.

Conclusion

Eltd1 deletion did not exhibit any toxicity in normal tissues but reduced the density of the tumour vasculature, particularly in inducible models, supporting further therapeutic development.