We have previously demonstrated that bone marrow stem cells participate in the tumor vessel expansion that supports the growth of TC71 Ewing’s sarcoma in vivo and that VEGF165 is critical for tumor growth. The purpose of these studies was to determine whether these stem cells proliferate following migration and if VEGF165 plays a role in the chemoattraction of bone marrow cells to the tumor vasculature. An MHC-mismatch bone marrow transplant model was used. Donor bone marrow cells from CB6F1 mice (MHC H-2b/d) were injected into lethally irradiated BALB/cAnN mice (MHC H-2d). TC71 human Ewing’s sarcoma cells were injected subcutaneously into recipient mice following engraftment. The mice were sacrificed at different time points, tumors excised and analyzed by immunohistochemistry (IHC). CD31 and H2-Kb double fluorescent staining in tumor tissue indicated that the number of donor bone marrow stem cells (H2-Kb) increased in the tumor vessels with tumor growth. Using Ki67 as a proliferation marker, we demonstrated that these donor bone marrow cells were proliferating following migration to the tumor area. To determine whether VEGF165 was involved in the chemotactic process, VEGF165-siRNA-transfected cells (TC-si-7-1) or TC-control transfected cells (TC-control) were injected subcutaneously into mice. The tumors were then treated with Ad-VEGF165, Ad-VEGF189, Ad-β-gal or PBS 5 days after tumor cell injection. Bone marrow cells labeled with CM-Dil were injected into the tumor bearing mice in 3 weeks. Tumors were then harvested 2 days later and analyzed by immunohistochemistry. Migration of bone marrow cells was significantly decreased in the TC-si-7-1 tumors compared to the TC-control tumors. TC-si-7-1 tumors also had significantly fewer vessels. Injection of Ad-VEGF165 but not Ad-VEGF189 or Ad-β-gal increased bone marrow cell migration and tumor vascularity. CD31 analysis indicated that the bone marrow cells contributed to the tumor vascularity. Together these data indicate that bone marrow cells migrate to the tumor area, proliferate following migration and that VEGF165 plays a critical role in the recruitment of these cells to the growing tumor.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]