Acute Myeloid Leukemia Cells Fuse with Vascular Endothelium

Abstract 504

The hematopoietic niche supports both normal and malignant hematopoiesis. We have evaluated the functional interactions between leukemic cells and the endothelial cell component of the hematopoietic niche. Highly enriched populations of circulating human acute myeloid leukemia (AML) cells were obtained by therapeutic leukapheresis. These primary AML cells rapidly homed to and associated with pre-existing vascular tubes in vitro. Co-culture of human lung microvascular endothelial cells (HLMVEC) with AML cells both inhibited AML apoptosis by ∼50% (p <0.005) and significantly increased AML cell number (p<0.01), indicating an important supportive effect of the endothelium on AML growth. A direct pro-angiogenic effect of the AML cells was revealed by the induction of vascular tube formation by HLMVECs in co-culture. Screening of known angiogenic factors showed an induction of GCSF expression and an increase in IL-6 levels in AML and HLMVEC co-cultures. Together, these findings indicate a mutually supportive, functional interaction between AML cells and endothelium in vitro. To evaluate the interactions of AML and vascular endothelium in vivo, primary human AML cells were transplanted into NOD/SCID IL2Rγ null mice. In highly engrafted mice (>50% human AML cells in the bone marrow) we observed leukemic infiltration into the perivascular region of both large and small vessels of the mouse liver, and AML cells were frequently integrated into the vascular endothelium. Confocal microscopy surprisingly revealed the co-expression of human CD45 and the mouse endothelial cell marker CD31. To evaluate these apparently fused leukemia/endothelial cells further, single cell suspensions from the livers of mice engrafted with AML cells were FACS sorted based on the co-expression of human CD33 and mouse CD31. Multiplex PCR analysis of single hCD33+/mCD31+ cells verified the presence of both mouse genes (fah and tspy or D2Mit206) and human genes (flt3 and tty-15). The presence of the patient specific, leukemia-associated Flt3-ITD mutation confirmed that fusion occurs between malignant cells and vascular endothelium. AML and endothelial cell fusion was also confirmed by interphase fluorescent in situ hybridization (FISH) with species specific centromeric probes. In conclusion, the results from this study indicate that the functionally important cross-talk between normal hematopoietic stem/progenitor cells and vascular endothelium extends to malignant hematopoiesis. The fusion of primary AML cells with normal endothelial cells raises the possibility that vascular endothelium may serve as a reservoir for occult leukemia.