Hypoxia Stimulates Vascular Mimicry by Inducing Ewing`s Sarcoma Stem Cells to Differentiate into Vascular Pericytes that Express EWS-FLI-1

Vasculogenesis and angiogenesis are required for expansion of the Ewing’s sarcoma vasculature. We demonstrated that pericytes and the DLL4 Notch signaling pathway are critical to the formation of these new tumor vessels. Using double fluorescent staining, we discovered that a subset of tumor vascular pericytes (Desmin⁺, NG2⁺) expressed EWS in TC-71 tumor tissue and patient tumor samples suggesting that these pericytes originated from Ewing’s tumor cells. These cells were in areas of hypoxia defined by HIF-1α staining. This area also had increased CD133⁺ cells. Culturing TC-71 cells under hypoxic conditions induced sphere formation, and expression of the stem cell markers CD133, Sox-2, Oct 3/4 and Nanog. Hypoxia also led to the upregulation of DLL4 and the pericyte markers Desmin, SMA and PDGF-BB. To determine whether TC-71 stem cells can contribute to the pericyte pool, CD133⁺ and CD133^- TC-71 cells were isolated following hypoxic culture and incubated with 5μg/ml DDL4. Desmin and NG2 expression were upregulated by DLL4 in the CD133⁺ cells but not the CD133^- cells. This up-regulation was blocked by the ɣ-secretase inhibitor DAPT. To confirm that TC-71 cells had differentiated into pericytes, TC-71 cells were transduced with a Desmin-driven promoter vector linked to GFP. Culturing these transduced cells under hypoxic conditions resulted in GFP expression confirming differentiation into a pericyte lineage. While solid tumors are known to contain subsets of undifferentiated embryonic-like cells with plasticity to serve an endothelial function, this is the first demonstration that the hypoxic tumor microenvironment can trigger conversion of Ewing’s sarcoma stem cells into pericytes that contribute to the expansion of the tumor vascular network.