Effect of Retroviral Endostatin Gene Transfer on Subcutaneous and Intraperitoneal Growth of Murine Tumors

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Abstract
Background: Inhibiting tumor angiogenesis is a promising new strategy for treating cancer. Difficulties with the stability, manufacture, and long-term administration of recombinant antiangiogenic proteins have prompted investigators to use gene therapy to generate these proteins in vivo. We investigated whether transfer of the gene encoding the angiogenesis inhibitor endostatin into the murine liver cell line NMuLi could inhibit tumor growth in vivo. Methods: NMuLi cells were transduced with retroviral vectors containing the murine endostatin gene. The presence and function of endostatin in transduced cell supernatants were confirmed by competitive enzyme immunoassay and endothelial cell proliferation assays. Nude mice were given a subcutaneous or intraperitoneal injection with NMuLi cells, control transduced cells (NEF-null), or endostatin-transduced clones (NEF-Endo1 to 4) and were monitored for tumor growth. All statistical tests were two-sided. Results: Supernatants from the clone secreting the lowest amount of endostatin (NEF-Endo4, 28 ng/mL) inhibited endothelial cell proliferation by 6% (95% confidence interval [CI] = 0% to 12%), and those from the clone secreting the highest amount (NEF-Endo1, 223 ng/mL) inhibited endothelial cell proliferation by 20% (95% CI = 13% to 27%). Increased levels of endostatin were detected in tumor lysates, but not serum, of mice given a subcutaneous injection of NEF-Endo1 cells. After 63 days, mice given a subcutaneous injection of parental NMuLi or NEF-null cells had tumor volumes of 2400 mm3 (95% CI = 1478 mm3 to 3300 mm3) and 2700 mm3 (95% CI = 2241 mm3 to 3144 mm3), respectively, compared with mean tumor volumes of less than 30 mm3 in mice given an injection of NEF-Endo clones, a statistically significant difference (P<.001). After 123 days, all 16 mice given an intraperitoneal injection of parental NMuLi or NEF-null cells had died, compared with only three (9%) of 32 mice given an injection of NEF-Endo clones. Conclusions: Retroviral endostatin gene transfer leads to secretion of functional endostatin that is sufficiently active to inhibit tumor growth. Further studies of retroviral endostatin gene transfer for the treatment of cancer are warranted.