Intratumoral administration of endostatin plasmid inhibits vascular growthand perfusion in MCa-4 murine mammary carcinomas

Endostatin, a fragment of the COOH-terminal domain of mouse collagen XVIII is a recently demonstrated endogenous inhibitor of tumor angiogenesis and e ndothelial cell growth. Antiangiogenic therapy with endostatin in animals r equires multiple and prolonged administration of the protein. Gene therapy could provide an alternative approach to continuous local delivery of this antiangiogenic factor ill vivo, Established MCa-4 murine mammary carcinomas , grown in immunodeficient mice, were treated with intratumoral injection o f endostatin plasmid at 7-day intervals. At the time of sacrifice, 14 days after the first injection, endostatin-treated tumor weights were 51% of con trols (P < 0.01), Tumor growth inhibition was accompanied by a marked reduc tion in total vascular density. Specifically, computerized image analysis s howed a 18-21% increase in the median distances between tumor cells and bot h the nearest anatomical (CD31-stained) vessel [48.1 +/- 3.8 versus 38.3 +/ - 1.6 <mu>m (P < 0.05)] and the nearest tumor-specific (CD105-stained) vess el [48.5 +/- 1.5 versus 39.8 +/- 1.5 <mu>m (P < 0.01)]. An increased apopto tic index of tumor cells in endostatin-treated tumors 13.2 +/- 0.5% versus 1.9 +/- 0.3% (P < 0.05)] was observed in conjunction with a significant dec rease in tumor perfused vessels (DiOC(7) staining), and an increase in tumo r cell hypoxia (EF5 staining). Hypoxia resulting from endostatin therapy mo st likely caused a compensatory increase of in situ vascular endothelial gr owth factor (VEGF) and VEGF receptor mRNA expression, Increased immunoreact ivity of endostatin staining in endostatin-treated tumors was also associat ed with an increased thrombospondin-l staining [1.12 +/- 0.16 versus 2.44 /- 0.35]. Our data suggest that intratumoral delivery of the endostatin gen e efficiently suppresses murine mammary carcinoma growth and support the po tential utility of the endostatin gene for cancer therapy.