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Tumors depend on the formation of a functioning microvascular network in order to grow and metastasise. The anti-angiogenic agent Angiostatin4.5 (AS4.5) is the naturally occurring isoform of Angiostatin in human plasma. AS4.5 is a distinct compound from the highly publicised Angiostatin owned at the time by Entremed. AS4.5 is a 55kD protein comprising kringles 1 to 4 plus 85% of kringle 5 of plasminogen. In vitro, AS4.5 induces endothelial cell (EC) apoptosis, inhibits EC proliferation and migration but has no direct effect on a range of tumor cell types. In vivo administration of AS4.5 results in inhibited tumor growth, presumably as a result of changes to the tumor vasculature. In this in vivo study we have investigated the effects of AS4.5 on the developing tumor vasculature. Dorsal skin-fold window chambers were surgically implanted in SCID mice under Hypnorm/midazolam anaesthesia. A small (≈1 mm) tumor fragment of human endometrial adenocarcinoma (EA) was transplanted into the chamber. Mice were treated with AS4.5 2mg/kg/day s.c. for 10 consecutive days starting 24hr after surgery (day 1). Controls received saline only. Control n = 3, AS4.5 treated n = 3. Shortly before intravital imaging, each mouse was injected i.v. with fluorescently (DiI) labelled erythrocytes. Video and still images were captured on days 1 to 11 at a range of objective magnifications (2.5x-20x) using both transmitted and fluorescent light. Vascular morphometry was analysed using in-house developed computer software. EA tumors treated with AS4.5 grew significantly slower than controls (by day 11, they were 156±13% and 282±46% of their initial area, respectively). Vascularization commenced on day 3 in controls and on day 4 in treated tumors. On days 6 to 8, treated tumors had fewer small calibre capillaries (length < 80 μm; diameter < 12 μm) and, on days 7 and 8, greater interstitial distance (average distance of a tumor cell from the nearest vessel; day 7, treated: 83±29; control: 20±3, day 8, treated: 43±6; control: 19±3 μm). Also during that period erythrocyte velocity was lower in treated tumors in comparison to controls (day 8, treated: 221±23; control: 384±31, day 9, treated: 210±14; control: 337±29 μm/min). However, when tumors were fully vascularized, treated tumors appeared more efficiently vascularized, in terms of both number of small calibre vessels and erythrocyte velocity, then the equivalent sized controls on an earlier day. Here we show that administration of AS4.5 to developing tumors slows the vascularization process, as manifested by a reduced rate of development of small calibre vessels, higher interstitial distance and lower erythrocyte velocity. These effects are sufficient to produce substantial tumor growth inhibition. The possibility that blood flow of treated tumours, once fully vascularised, is higher than in untreated tumours requires further investigation.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]