Production of VEGF165 by Ewing's sarcoma cells induces vasculogenesis and the incorporation of CD34⁺ stem cells into the expanding tumor vasculature

The Ewing's sarcoma cell line TC71 overexpresses vascular endothelial growth factor isoform 165 (VEGF₁₆₅), a potent proangiogenic molecule that induces endothelial cell proliferation, migration, and chemotaxis. CD34⁺ bone marrow stem cells can differentiate into endothelial and hematopoietic cells. We used a transplant model to determine whether CD34 ⁺ cells migrate from the bone marrow to Ewing's sarcoma tumors and participate in the neovascularization process that supports tumor growth. We also examined the role of VEGF₁₆₅ in CD34₊ cell migration. Human umbilical cord CD34⁺ cells were transplanted into sublethally irradiated severe combined immunodeficient mice. Seven days later, the mice were injected subcutaneously with TC71 tumor cells. Tumors were excised 2 weeks later and analyzed by immunohistochemistry. The tumor sections expressed both human VE-cadherin and mouse CD31, indicating involvement of donor-derived human cells in the tumor vessels. To determine the role of VEGF₁₆₅ in the chemoattraction of CD34₊ cells, we generated two VEGF ₁₆₅-deficient TC71 clones, a stable anti-sense VEGF₁₆₅ cell line (Clone 17) and a VEGF₁₆₅ siRNA-inhibited clone (TC/siVEGF_7-1). The resulting VEGF₁₆₅-deficient tumor cells had normal growth rates in vitro, but had delayed growth when implanted into mice. Immunohistochemical analysis revealed decreased infiltration of CD34⁺ cells into both VEGF₁₆₅-deficient tumors. These data show that bone marrow stem cells contribute to the growing tumor vasculature in Ewing's sarcoma and that VEGF₁₆₅ is critical for the migration of CD34₊ cells from the bone marrow into the tumor.