Delta-like ligand 4 (DLL4) is essential for the formation of mature vasculature. However, the role of DLL4-Notch signaling in pericyte/vascular smooth muscle cell (vSMC) development is poorly understood. We sought to determine whether DLL4-Notch signaling is involved in pericyte/vSMC formation in vitro and during vasculogenesis in vivo using 2 Ewing sarcoma mouse models. Inhibition of DLL4 with the antibody YW152F inhibited pericyte/vSMC marker expression by bone marrow (BM) cells in vitro. Conversely, transfection of 10T1/2 cells with the active domains of Notch receptors led to increased expression of pericyte/vSMC markers. Furthermore, the blood vessels of Ewing sarcoma tumors from mice treated with YW152F had reduced numbers of BM-derived pericytes/vSMCs, fewer open lumens, and were less functional than the vessels in tumors of control-treated mice. Tumor growth was also inhibited. These data demonstrate a specific role for DLL4 in the formation of BM-derived pericytes/vSMCs and indicate that DLL4 may be a novel therapeutic target for the inhibition of vasculogenesis.
Ewing's sarcoma accounts for a disproportionately high portion of the overall pediatric mortality rate compared to its rare incidence in the pediatric population. Little progress has been made since the introduction of traditional chemotherapies, and understanding the biology of the tumor is critical for developing new therapies. Ewing's sarcomas rely on a functional vascular supply, which is formed by a combination of angiogenesis and vasculogenesis. Recent insights into the molecular regulation of bone marrow (BM) cell participation in vascular development have identified VEGF, SDF-1α, and DLL4 as critical players in the vasculogenesis process. Clinical trials using vascular targeting agents, specifically targeting VEGF or DLL4, are underway.
An MHC-mismatch bone marrow (BM) transplant Ewing's sarcoma mouse model was used to investigate whether BM cells participate in the vessel formation that support Ewing's sarcoma lung metastasis. BM cells from H-2K b/d donor mice were transplanted into sublethally irradiated H-2K d recipient mice. Donor BM cells were identified using the H-2K b marker. Engraftment was confirmed by identifying the H-2K b IL-1β-type specific polymorphism. After engraftment highly lung metastatic TC71-PM4 cells were injected intravenously. Mice were sacrificed 10 weeks after tumor cell injection. Hematoxylin-and-eosin staining was performed to identify lung metastatic foci. These tumors were then evaluated using immunohistochemical analysis. H-2K b -positive cells were found in lung metastases but not in normal lung, liver or spleen tissues. Injection of CM-Dillabeled BM cells into tumor bearing and control mice showed that nonspecific organ migration occurred at 24 h, but that these cells were absent 1 week later in control mice. These data suggest that the migration of the H-2K b BM cells to lung nodules was specific because these cells were observed 14 weeks after transplantation. Co-localization of H-2K b and CD31 or VE-Cadherin demonstrated that some endothelial cells were BM-derived. Co-localization of H-2K b and Desmin, smooth muscle actin (α-SMA) or PDGFR-β indicated that a fraction of pericytes was also BMderived. These results suggest that BM cells participate in the vascular formation that supports the growth of Ewing's sarcoma lung metastases. BM cells migrated to the metastatic tumor and differentiated into endothelial cells and pericytes. These data indicated that targeting this process may have therapeutic potential.
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