TITLE: Estrogen Mobilizes Circulating Bone Marrow Progenitor Cells to Promote Tumor Neovasculature: Lessions from Ischemic Model Provide a Novel Breast Cancer Target PRINCIPAL INVESTIGATOR:

submitted to the 2008 Era of Hope meeting in the Baltimore Convention Center Neo-vascularization or angiogenesis is critical in sustaining growth of tumors as well as subsequent metastases. Circulating bone marrow derived endothelial progenitor cells (BM-EPC) have been observed to contribute to neo-vascularization of tumors and the identification of cellular and molecular mediators will impact clinical care. In this pilot program we focused on developing an animal model that could examine the contribution of bone marrow derived progenitor endothelial cells (BM-EPC) on neovascularization. Further, since estrogen is a major modulator of breast cancer initiation and progression and that estrogen affects BM-EPC migration, we examined the contribution of the BM-EPC induced neo-vascularization. Our animal model was designed to test the hypothesis “Estrogen mobilizes circulating BM-EPC which home to the implanted breast carcinomas and promote tumor neo-vascularization.” We utilized Tie2/GFP-Balb/c ± ovariectomized ± estrogen supplementation. These mice were transplanted with bone marrow derived from Tie2/GFP mice that were used as donors. Tumors were induced in these mice by surgical implantation of Tg1 or 4T1 (ATCC) murine mammary adenocarcinoma cells (derived from syngeneic BALB/c mice; 2 × 10 cells/0.3 ml PBS) into the fourth inguinal mammary gland after clearing the fat pad region of BMT mice. At the end of the experimental period tumor incidence and progression was monitored by immunohistochemical analysis and the BM-EPCs mobilization at the tumor site was measured and correlated with capillary density. We observed the concomitant mobilization of GFP and CD133 (marker of EPC) double-positive cells at the tumor site. Comparison of estrogen supplemented and non-supplemented group, revealed that E2 supplementation enhances both mobilization of GFP-CD133+ cells (EPCs) in the tumors as well as mobilized EPCs physically integrate into neo-vasculature resulting in significantly higher capillary density. The contribution of estrogen in angiogenesis and tissue remodeling, which are two processes indispensable for the growth of tumors, was also observed through Q-RT-PCR experiments on excised tumor-inoculated mammary tissues, in which the transcripts of various angiogenic cytokines involved in these two critical processes were significantly increased. E2 stimulated EPCs were also observed to secrete paracrine factors which increased the proliferation and migration of 4T1 tumor cells. Lastly, EPCs isolated from BM were observed to form tube like structures (tubulogenesis) upon addition of media obtained from E2 stimulated tumor cells. In conclusion we have discovered a novel role of estrogen stimulated BM-EPCs in tissue remodeling in a breast cancer animal model and presumably a novel therapeutic