Blocking CLEC14A-MMRN2 binding inhibits sprouting angiogenesis and tumour growth.
We previously identified CLEC14A as a tumour endothelial marker. Here we show that CLEC14A is a regulator of sprouting angiogenesis in vitro and in vivo. Using a human umbilical vein endothelial cell spheroid-sprouting assay, we found CLEC14A to be a regulator of sprout initiation. Analysis of endothelial sprouting in aortic ring and in vivo subcutaneous sponge assays from clec14a(+/+) and clec14a(-/-) mice revealed defects in sprouting angiogenesis in CLEC14A-deficient animals. Tumour growth was retarded and vascularity reduced in clec14a(-/-) mice. Pull-down and co-immunoprecipitation experiments confirmed that MMRN2 binds to the extracellular region of CLEC14A. The CLEC14A-MMRN2 interaction was interrogated using mouse monoclonal antibodies. Monoclonal antibodies were screened for their ability to block this interaction. Clone C4, but not C2, blocked CLEC14A-MMRN2 binding. C4 antibody perturbed tube formation and endothelial sprouting in vitro and in vivo, with a similar phenotype to loss of CLEC14A. Significantly, tumour growth was impaired in C4-treated animals and vascular density was also reduced in the C4-treated group. We conclude that CLEC14A-MMRN2 binding has a role in inducing sprouting angiogenesis during tumour growth, which has the potential to be manipulated in future antiangiogenic therapy design.
Angiogenesis Laboratory, Institute for Biomedical Research, Schools of Immunity and Infection and Cancer Sciences, College of Medical and Dental Sciences, The Medical School, University of Birmingham, Birmingham, UK.
P J
PJ
Noy
Angiogenesis Laboratory, Institute for Biomedical Research, Schools of Immunity and Infection and Cancer Sciences, College of Medical and Dental Sciences, The Medical School, University of Birmingham, Birmingham, UK.
P
P
Lodhia
Angiogenesis Laboratory, Institute for Biomedical Research, Schools of Immunity and Infection and Cancer Sciences, College of Medical and Dental Sciences, The Medical School, University of Birmingham, Birmingham, UK.
K
K
Khan
Angiogenesis Laboratory, Institute for Biomedical Research, Schools of Immunity and Infection and Cancer Sciences, College of Medical and Dental Sciences, The Medical School, University of Birmingham, Birmingham, UK.
X
X
Zhuang
Institute of Cancer Studies, School of Cancer Sciencies, University of Birmingham, Birmingham, UK.
D G
DG
Ward
Angiogenesis Laboratory, Institute for Biomedical Research, Schools of Immunity and Infection and Cancer Sciences, College of Medical and Dental Sciences, The Medical School, University of Birmingham, Birmingham, UK.
A R
AR
Verissimo
School of Immunity and Infection, Institute for Biomedical Research, The Medical School, University of Birmingham, Birmingham, UK.
A
A
Bacon
Angiogenesis Laboratory, Institute for Biomedical Research, Schools of Immunity and Infection and Cancer Sciences, College of Medical and Dental Sciences, The Medical School, University of Birmingham, Birmingham, UK.
R
R
Bicknell
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000954:Antigens, Surface / Q000378:metabolism*; D001011:Aorta / Q000166:cytology*; D015815:Cell Adhesion Molecules / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018920:Coculture Techniques; D055785:Gene Knockdown Techniques; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D037181:Lectins, C-Type / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D009389:Neovascularization, Pathologic; D011485:Protein Binding / Q000187:drug effects; D034741:RNA, Small Interfering / Q000494:pharmacology*; D018874:Spheroids, Cellular / Q000166:cytology
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; C570898:CLEC14A protein, human; D015815:Cell Adhesion Molecules; C089826:EMILIN3 protein, human; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins; D034741:RNA, Small Interfering