Role of human cripto-1 in tumor angiogenesis.
BACKGROUND
Human cripto-1 (CR-1) promotes cell transformation and increases migration and invasion of various mouse and human epithelial cell lines. We investigated whether CR-1 also stimulates angiogenesis.


METHODS
We used human umbilical vein endothelial cells (HUVECs) to measure in vitro migration with fibronectin-coated Boyden chambers, invasion with Matrigel-coated Boyden chambers, proliferation with a tetrazolium salt, and differentiation with an in vitro Matrigel assay. We investigated new blood vessel formation in vivo by use of Matrigel-filled silicone cylinders implanted under the skin of nude mice and by use of a breast cancer xenograft model with CR-1-transfected or control Neo-transfected MCF-7 human breast cancer cells. We also used a blocking anti-CR-1 monoclonal antibody to investigate the role of CR-1 in angiogenesis in vivo and in vitro. All statistical tests were two-sided.


RESULTS
CR-1 stimulated HUVEC proliferation, migration, and invasion and induced HUVEC differentiation into vascular-like structures on Matrigel. In vivo, recombinant CR-1 protein induced microvessel formation in Matrigel-filled silicone cylinders, and microvessel formation was statistically significantly inhibited with a blocking anti-CR-1 monoclonal antibody (CR-1 and antibody = 127% of microvessel formation compared with that in untreated control cylinders and CR-1 alone = 259%; difference = 132%, 95% confidence interval [CI] = 123% to 140%; P<.001). Tumors formed by CR-1-transfected MCF-7 cells in the cleared mammary fat pad of nude mice had higher microvessel density than tumors formed by control Neo-transfected MCF-7 cells (CR-1-transfected cells = 4.66 vessels per field and Neo-transfected cells = 2.33 vessels per field; difference = 2.33 vessels per field, 95% CI = 1.2 to 2.8; P = .004).


CONCLUSIONS
CR-1 appears to have an important role in the multistep process of angiogenesis.
Bianco
Caterina
C
Tumor Growth Factor Section, Mammary Biology and Tumorigenesis Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Strizzi
Luigi
L
Ebert
Andreas
A
Chang
Cindy
C
Rehman
Aasia
A
Normanno
Nicola
N
Guedez
Liliana
L
Salloum
Rita
R
Ginsburg
Erika
E
Sun
Youping
Y
Khan
Nadia
N
Hirota
Morihisa
M
Wallace-Jones
Brenda
B
Wechselberger
Christian
C
Vonderhaar
Barbara K
BK
Tosato
Giovanna
G
Stetler-Stevenson
William G
WG
Sanicola
Michele
M
Salomon
David S
DS
D000818:Animals; D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor / Q000378:metabolism; D001943:Breast Neoplasms / Q000098:blood supply*; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D003094:Collagen; D004338:Drug Combinations; D042783:Endothelial Cells / Q000378:metabolism*; D004815:Epidermal Growth Factor / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D005260:Female; D016222:Fibroblast Growth Factor 2 / Q000032:analysis; D058851:GPI-Linked Proteins; D006133:Growth Substances / Q000378:metabolism; D006801:Humans; D007118:Immunoassay; D036341:Intercellular Signaling Peptides and Proteins; D007797:Laminin; D008562:Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D051379:Mice; D008819:Mice, Nude; D009363:Neoplasm Proteins / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D009389:Neovascularization, Pathologic / Q000378:metabolism*; D011509:Proteoglycans; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014183:Transplantation, Heterologous; D014471:Umbilical Veins; D042461:Vascular Endothelial Growth Factor A / Q000032:analysis
D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D004338:Drug Combinations; D058851:GPI-Linked Proteins; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D007797:Laminin; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D011509:Proteoglycans; D011994:Recombinant Proteins; C070636:TDGF1 protein, human; D042461:Vascular Endothelial Growth Factor A; D016222:Fibroblast Growth Factor 2; C059485:matrigel; D004815:Epidermal Growth Factor; D003094:Collagen