Despite multiple changes in chemotherapy over the past 15-20 years, the 2-year metastasis- free survival for Ewing's sarcoma remains at 40% with a 3-year overall survival of 50%. Understanding the mechanisms that contribute to the growth of Ewing's sarcoma may assist in the development of new therapeutic approaches. We demonstrated that Ewing's tumor cell lines and primary patient tumor specimens overexpress VEGF with a shift from the membrane- bound 189 isoform to the soluble 165 isoform. VEGF165 has been shown to chemoattract stem cells. Using our Ewing's sarcoma nude mouse model in a transplant setting we demonstrated that bone marrow (BM) cells migrate into the tumor, differentiate into endothelial cells and contribute to the formation of the tumor vasculature. We demonstrated that there are 2 distinct patterns of stem cell infiltration and 2 different phenotypes that migrate into the tumor. Murine Sca1+Gr1+ stem cells and human CD34+ cells co-localized to tumor vessels and differentiate into endothelial cells and pericytes. Murine Sca1-Gr1+ and human CD34- cells migrated deep in the tumor tissue away from tumor vessels and differentiated into macrophages. We demonstrated that pericytes and VEGF165 is responsible for the chemoattraction of these stem cells. This activity was not duplicated by VEGF189. Further investigations using VEGF165-siRNA support our hypothesis that these migrated stem cells play an important role in tumor growth and tumor vessel development. Taken together our data indicate that vasculogenesis, in addition to angiogenesis is involved in the expansion of the tumor vasculature contributing to about 10% of the vessels during the first week of tumor growth. We wish to continue investigating the vasculogenesis process, determine the importance of this process in both primary and metastatic tumor growth and investigate the mechanism(s) by which stem cells are attracted to specific tumor vascular areas. We propose to (1) determine whether BM-derived cells are integrated together with local endothelial cells to form tumor vessels and whether this is modified in the absence of VEGF165;(2) determine the importance of vasculogenesis to tumor growth and whether vasculogenesis can restore growth and vessel development in the absence of VEGF165;(3) determine whether NOTCH signaling, in specific DLL4, plays a role in the chemoattraction of stem cells to the Ewing's tumor;(4) determine whether BM stem cells participate in the growth of Ewing's metastases in the lung and bone. Understanding how vasculogenesis contributes to the growth and metastasis of Ewing's sarcoma and ascertaining the chemotactic signals involved in this process may open up new therapeutic approaches to treat patients with this disease. This application will investigate how bone marrow (BM) stem cells contribute to the vasculature expansion that supports the growth of Ewing's sarcoma and the mechanisms that control the migration of BM cells into the tumor. We will also determine whether BM stem cells contribute to the growth of Ewing's metastases in the lung and bone. The survival rates for Ewing's sarcoma have not improved for >20 years. Understanding how tumor vessels are formed may identify new therapeutic approaches for this type of cancer.

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National Cancer Institute (NCI)
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Cancer Immunopathology and Immunotherapy Study Section (CII)
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Ault, Grace S
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University of Texas MD Anderson Cancer Center
Other Domestic Higher Education
United States
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Hamdan, Randala; Zhou, Zhichao; Kleinerman, Eugenie S (2014) Blocking SDF-1?/CXCR4 downregulates PDGF-B and inhibits bone marrow-derived pericyte differentiation and tumor vascular expansion in Ewing tumors. Mol Cancer Ther 13:483-91
Zhou, Zhichao; Yu, Ling; Kleinerman, Eugenie S (2014) EWS-FLI-1 regulates the neuronal repressor gene REST, which controls Ewing sarcoma growth and vascular morphology. Cancer 120:579-88
Huang, Gangxiong; Zhou, Zhichao; Wang, Hua et al. (2012) CAPER-? alternative splicing regulates the expression of vascular endothelial growth factor??? in Ewing sarcoma cells. Cancer 118:2106-16
Hamdan, Randala; Zhou, Zhichao; Kleinerman, Eugenie S (2011) SDF-1? induces PDGF-B expression and the differentiation of bone marrow cells into pericytes. Mol Cancer Res 9:1462-70
Stewart, Keri Schadler; Zhou, Zhichao; Zweidler-McKay, Patrick et al. (2011) Delta-like ligand 4-Notch signaling regulates bone marrow-derived pericyte/vascular smooth muscle cell formation. Blood 117:719-26
Stewart, Keri S; Kleinerman, Eugenie S (2011) Tumor Vessel Development and Expansion in Ewing's Sarcoma: A Review of the Vasculogenesis Process and Clinical Trials with Vascular-Targeting Agents. Sarcoma 2011:165837
Zhou, Zhichao; Stewart, Keri Schadler; Yu, Ling et al. (2011) Bone marrow cells participate in tumor vessel formation that supports the growth of Ewing's sarcoma in the lung. Angiogenesis 14:125-33
Yu, Ling; Su, Bing; Hollomon, Mario et al. (2010) Vasculogenesis driven by bone marrow-derived cells is essential for growth of Ewing's sarcomas. Cancer Res 70:1334-43
Schadler, Keri L; Zweidler-McKay, Patrick A; Guan, Hui et al. (2010) Delta-like ligand 4 plays a critical role in pericyte/vascular smooth muscle cell formation during vasculogenesis and tumor vessel expansion in Ewing's sarcoma. Clin Cancer Res 16:848-56
Guan, Hui; Zhou, Zhichao; Cao, Ying et al. (2009) VEGF165 promotes the osteolytic bone destruction of ewing's sarcoma tumors by upregulating RANKL. Oncol Res 18:117-25

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