Role of Thymosin 4 in Tumor Metastasis and Angiogenesis

Background: Expression of the small peptide thymosin 4 is associated with angiogenesis induction, accelerated wound healing, and the metastatic potential of tumor cells. However, little is known about the mechanism(s) by which thymosin 4 promotes metastasis. Methods: Northern blot analysis and immunohistochemistry were used to examine thymosin 4 expression in mouse melanoma B16 cell lines and in B16-F10 cells derived from metastatic mouse lung tumors, respectively. B16-F10 cells infected with adenoviruses containing a thymosin 4 expression vector or an empty vector were injected subcutaneously and intravenously into C57BL/6 mice to evaluate tumor growth and metastatic potential, respectively. In vitro assays were used to study cell migration, invasion, matrix metalloproteinase activity, cell proliferation, and angiogenic activity of adenovirus-infected B16-F10 cells. Statistical significance of all results was analyzed by two-tailed Student’s t tests. Results: Thymosin 4 mRNA was expressed in primary cultured B16-F10 cells derived from lung metastases and in B16-F10 cells that had formed lung tumors after being injected into mice but not in the B16-F1, B16-F10, or B16-BL6 cell lines. The mean tumor sizes in mice 20 days after injection with B16-F10 cells infected with thymosin 4–expressing adenovirus and with control adenovirus were 21.7 mm (95% confidence interval [CI] 17.7 to 25.7 mm) and 13.3 mm (95% CI 11.1 to 15.3 mm), respectively (difference 8.4 mm; P .036). The mean numbers of metastatic lung nodules in mice (n 20) 2 weeks after intravenous injection with thymosin 4–expressing adenovirus and with control adenovirus were 46.7 (95% CI 35.0 to 57.7) and 10.9 (95% CI 6.2 to 15.6), respectively (difference 35.8 metastatic lung nodules, P<.001). Thymosin 4 overexpression was associated with a mean 2.3-fold increase (95% CI 1.9to 2.7-fold increase; P<.001) in B16-F10 cell migration and a mean 4.4-fold increase (95% CI 3.3to 5.5-fold increase; P<.001) in the number of blood vessels in solid tumors derived from injected B16-F10 cells but had no effect on cell invasion, proliferation, or matrix metalloproteinase activity. This induction of angiogenesis by thymosin 4 was associated with induction of vascular endothelial growth factor expression. Conclusion: Thymosin 4 may stimulate tumor metastasis by activating cell migration and angiogenesis. [J Natl Cancer Inst 2003;95:1674–80]