Deletion of thrombospondin leads to accelerated growth of breast cancers that arise spontaneously in neutransgenic mice.This resistance to a second tumor challenge is inversely proportional to the size of the tumor inoculum and directly proportional to the size of thefirst tumor.Once it was demonstrated that a tumor could generate a negative regulator of angiogenesis, then it became clear that a primary tumor, while stimulating angiogenesis in its own vascular bed, could possibly inhibit angiogenesis in the vascular bed of a distant metastasis.However, at least two conditions would be necessary:first, the primary tumor would need to generate an angiogenic promoter in excess of an inhibitor in its own vascular bed and, second, the putative inhibitor would need to have a longer halflife in the circulation than the angiogenic promoter.They were not neovascularized and usually formed a microcylinder around a single microvessel.In these dormant metastases, of the tumor cells were proliferating and to were undergoing apoptosis.Fur ther, serum from these tumorbearing mice specif ically inhibited alanine capillary endothelial cell proliferation in vitro by more than. The tumorbearing serum also inhibited angiogenesis on the chick chorioallantoic membrane compared to serum from mice without tumors. There was no change in tumor cell proliferation, but tumor cell apoptosis fell to. Systemic Flumazenil administration of angiostatin purif ied from mouse urine signif icantly inhibited angiogenesis in lung metastases and restricted their growth to a microscopic dormant size, and recombinant angiostatin potently inhibitedgrowth of other tumor types.Lower panel: lungs removed at the same time from animals in which the tumor had been removed and the animals killed days later.This dormant metastasis is approximately microns in its longest diameter.Right panel: lung metastasis from an animal killed days after removal of the primary tumor, showing or new vessels in an enlarging metastasis.D, A human prostate carcinoma growing on the dorsum of a SCID immunodef icient mouse almost completely inhibits cornea neovascularization induced by an implanted sustained release pellet of bFGF. At least one of these tumorderived enzymes, urokinase plasminogen activator, converts plasminogen to plasmin, while a phosphoglycerate kinase from hypoxic tumor cells then reduces the plasmin so that it can be converted to angiostatin by one of several different metalloproteinases. Other types of tumors have since been reported to generate angiostatin. Human prostate carcinoma cells express enzymatic activity that conver ts human plasminogen to the angiogenesis inhibitor angiostatin.In these tumors, the total angiogenic output of the primary tumor was decreased by transfected angiostatin, which opposed in a dosedependent manner the activity of the tumors secreted angiogenic promoter, but never completely counteracted it.It should be emphasized that the rate of tumorgrowth was directly proportional to total angiogenic output of the tumor, inversely proportional to angiostatin production and to tumor cell apoptosis, and virtually independent of tumor cell proliferation.Gene transfer with a different angiogenesis inhibitor, thrombospondin, gave similar results.A provocative recentfinding is that proliferation of circulating precursor endothelial cells derived from bone marrow is inhibited at signif icantly lower concentrations of angiostatin than is proliferation of endothelial cells isolated from tissues.This aff inity appears to be specif ic for endothelial cells and does not occur withfibroblasts.