The Role Of CD73 In Tumor Angiogenesis

Ecto-5'-nucleotidase(EC3.1.3.5,CD73,ecto-5'-NT) is a glycosyl phosphatidylinositol(GPI)-linked,membrane-bound glycoprotein which hydrolyzes extracellular nucleoside monophosphates into bioactivenucleoside intermediates. Recent studies showed that CD73 was highly up-regulated in metastatic tumors,such as breast cancer and colon cancer.Angiogenesis is necessary for tumor metastasis. The angiogenesis in human breast cancer xenograft in nude mice was decreased by inhibiting the activity of CD73.Our experiments also suggested angiogenesis of the mice breast cancer xenograft in CD73^+/+ C57BL/6 mice were more obvious than CD73^-/- mice.Therefore,CD73 plays an important role in the progress of tumor angiogenesis.However,it is remained unknown how CD73 affects it.In this study, firstly,we isolated and cultured the primary pulmonary microvessel endothelial cells from CD73^+/+ and CD73^-/- mice,then studied the effects of CD73 on tumor angiogenesis by formation of capillary-like test in vitro;secondly,we observed tumor angiogenesis using tumor bearing model in CD73^+/+ and CD73^-/- C57BL/6 mice respectively;thirdly,to investigate the mechanism of CD73 on tumor angiogenesis, we screened angiogenesis-associated proteins regulated by CD73 by proteomics.Our project studied the relationship between CD73 and tumor blood metastasis,tried to put forward a new molecule correlated with tumor angiogenesis.This study includes four parts:1.Identification of CD73^+/+ and CD73^-/- C57BL/6 mice.We detected the genetype of mice by PCR and confirmed CD73 activity by enzyme histochemistry. 188bp band is the product for CD73^+/+ C57BL/6 mice with positive stainings in the liver,heart and kidney;280bp band is the product for CD73^-/- C57BL/6 mice without staining in above tissues.2.Isolation,culture and identification of primary pulmonary microvessel endothelial cells(pPMEC) of CD73^+/+ and CD73^-/- C57BL/6 mice.Then adhesion, migration and angiogenesis assays were done using pPMEC.The biological behavior of the primary pulmonary microvessel endothelial cells of CD73^+/+ and CD73^-/- C57BL/6 mice were markedly different.The ability of CD73^-/- pPMEC adhering to CollagenⅣwas much stronger than CD73^+/+ pPMEC.However,the migration test showed that the mobility of CD73^+/+ pPMEC was dramatically higher than CD73^-/- pPMEC.Thus,CD73 probably promoted endothelial cells migration.Since motility of endothelial cells was pivotal in angiogenesis,we considered that CD73 might promote angiogenesis.Cancer conditioned medium made by mice prostatic cancer RM-1 cell line accelerated migration of CD73^+/+ pPMEC further.In vitro tube formation assay suggested that the tubes formed by CD73^+/+ pPMEC in Matrigel were more intensive and integrate than CD73^-/- pPMEC.Compared with CD73^+/+ pPMEC cultured with normal medium,tube formation in CD73^+/+ pPMEC cultured with cancer conditioned medium was earlier.So substances secreted by cancer cells may promote endothelial cells,especially CD73^+/+ pPMEC,form tubes easily and quickly.3.Study of tumor angiogenesis in CD73^+/+ and CD73^-/- C57BL/6 mice in vivo. We set up the mice breast cancer and prostatic cancer bearing models and observed tumor angiogenesis in CD73^+/+ and CD73^-/- C57BL/6 mice.The results showed that tumor angiogenesis in CD73^+/+ mice was more prominent than CD73^-/- mice in the earlier stage(2 weeks),the size of tumors in CD73^+/+ mice was larger than that of CD73^-/- mice from 2 weeks to 4 weeks.However,there was no big difference in angiogenesis,size of tumor and weight after five weeks.Hereby,CD73 probably associated with early tumor angiogenesis.4.Investigation on the mechanism of CD73 on tumor angiogenesis by proteomics to screen angiogenesis-associated proteins regulated by CD73. Two-dimensional gels electrophoresis with silver staining of total protein of pPMEC form CD73^+/+ and CD73^-/- mice suggested four different spots,which were characterized by MALDI-TOF-MS/MS.We found thatβ-actin was down-regulated after CD73 knocked out.β-actin is one of skeleton proteins in cells,which participates in motility in most cells.Higher expression ofβ-actin can explain that migration and tube-formation of CD73^+/+ pPMEC were much stronger than CD73^-/- pPMEC.Other different proteins in Two-dimensional gels are still unknown,which may relate to CD73 expression.In conclusion,CD73 can promote endothelial cells forming new vessels under cancer condition,thereby facilitate tumor grow and blood metastasis.This study offers new thought for early treatment of cancer.