SUMMARY How disseminated tumor cells (DTCs) engage specific stromal components in distant organs for survival and outgrowth is a critical but poorly understood step of the metastatic cascade. Previous studies have demonstrated the importance of the epithelial-mesenchymal transition (EMT) in promoting the cancer stem cell properties needed for metastasis initiation, while the reverse process of mesenchymal-epithelial transition (MET) is required for metastatic outgrowth. Here we report that this paradoxical requirement for simultaneous induction of both MET and cancer stem cell traits in DTCs is provided by bone vascular niche E-selectin, whose direct binding to cancer cells promotes bone metastasis by inducing MET and activating Wnt signaling. E-selectin binding activity mediated by α1–3 Fucosyltransferases Fut3/Fut6 and Glg1 are instrumental to the formation of bone metastasis. These findings provide unique insights into the functional role of E-selectin as a component of the vascular niche critical for metastatic colonization in bone.

Background: During inflammation, the selectins engage glycosylated macromolecules expressed on blood leukocytes under fluid shear conditions. Results: Although all three myeloid ␣1,3-fucosyltransferases FUT9, FUT7, and FUT4 regulate human E-selectin ligand biosynthesis, FUT7 and FUT4 are sufficient to form L/P-selectin ligands. Conclusion: FUT9 plays a significant role during human, but not mouse, leukocyte-endothelial interactions. Significance: This study identifies potential ␣(1,3)FUTs regulating inflammation in humans.

• A single a(2,3) sialyltransferase, ST3Gal-4, controls sLe X biosynthesis on N-and O-glycans in cells of human myeloid lineage.• Blocking this enzyme activity prevents human neutrophil adhesion to E-, P-, and L-selectin.The precise glycosyltransferase enzymes that mediate selectin-ligand biosynthesis in human leukocytes are unknown. This knowledge is important because selectin-mediated cell tethering and rolling is a critical component of both normal immune response and various vascular disorders. We evaluated the role of 3 a(2,3)sialyltransferases, ST3Gal-3, -4, and -6, which act on the type II N-Acetyllactosamine structure (Galb1,4GlcNAc) to create sialyl Lewis-X (sLe X ) and related sialofucosylated glycans on human leukocytes of myeloid lineage. These genes were either silenced using lentiviral short hairpin RNA (shRNA) or functionally ablated using the clustered regularly interspaced short palindromic repeat/ Cas9 technology. The results show that ST3Gal-4, but not ST3Gal-3 or -6, is the major sialyltransferase regulating the biosynthesis of E-, P-, and L-selectin ligands in humans. Reduction in ST3Gal-4 activity lowered cell-surface HECA-452 epitope expression by 75% to 95%. Glycomics profiling of knockouts demonstrate an almost complete loss of the sLe X epitope on both leukocyte N-and O-glycans. In cell-adhesion studies, ST3Gal-4 knockdown/knockout cells displayed 90% to 100% reduction in tethering and rolling density on all selectins. ST3Gal-4 silencing in neutrophils derived from human CD34 1 hematopoietic stem cells also resulted in 80% to 90% reduction in cell adhesion to all selectins. Overall, a single sialyltransferase regulates selectin-ligand biosynthesis in human leukocytes, unlike mice where multiple enzymes contribute to this function. (Blood. 2015;125(4):687-696)

The function of the mechanosensitive, multi-meric blood protein von Willebrand factor (VWF) is dependent on its size. We tested the hypothesis that VWF may self-associate on the platelet glycoprotein Ib (GpIb) receptor under hydrodynamic shear. Consistent with this proposition, whereas Alexa-488-conjugated VWF (VWF-488) bound platelets at modest levels, addition of unla-beled VWF enhanced the extent of VWF-488 binding. Recombinant VWF lacking the A1-domain was conjugated with Alexa-488 to produce A1-488. Although A1-488 alone did not bind platelets under shear, this protein bound GpIb on addition of either purified plasma VWF or recombinant full-length VWF. The extent of self-association increased with applied shear stress more than 60 to 70 dyne/cm 2. A1-488 bound plate-lets in the milieu of plasma. On application of fluid shear to whole blood, half of the activated platelets had A1-488 bound, suggesting that VWF self-association may be necessary for cell activation. Shearing plate-lets with 6-m beads bearing either immobilized VWF or anti-GpIb mAb resulted in cell activation at shear stress down to 2 to 5 dyne/cm 2. Taken together, the data suggest that fluid shear in circulation can increase the effective size of VWF bound to platelet GpIb via protein self-association. This can trigger mechanotransduction and cell activation by enhancing the drag force applied on the cell-surface receptor. (Blood. 2010;116(19):3990-3998) Introduction von Willebrand factor (VWF) is a large, multidomain glycoprotein found in normal blood at concentrations of approximately 10 g/mL. 1 The protein plays an important role in hemostasis by both carrying the coagulation protein factor VIII (FVIII) in circulation and by regulating the adhesion of platelets to sites of vascular injury. Whereas the DD3 domain of VWF binds FVIII, the A1 and C1 domains engage platelet receptors glycoprotein Ib (GPIb) and IIb 3 (GPIIb-IIIa), respectively. Monomeric VWF has a molecular mass of approximately 250 kDa. This unit further polymerizes, via disulfide linkage formation in the endoplasmic reticulum and Golgi of endothelial cells and megakaryocytes. Multimeric VWF size ranges from 0.5 to 20 MDa. 2 Ultra/unusually-large VWF is secreted from the Weibel-Palade bodies of endothe-lial cells on stimulation with a variety of agonists associated with inflammation and thrombosis, including thrombin, histamine, and tumor necrosis factor-. The hemostatic potential of VWF increases with protein size and the magnitude of the applied hydrodynamic shear. 3,4 Ultra/ unusually-large VWF secreted from endothelial cells under shear is extended in the form of strings or bundles on the vessel wall. 5,6 Shear-mediated extension enhances cleavage of the cryptic Y 1605-M 1606 bond within the VWF-A2 domain by the constitutively active blood metalloprotease, ADAMTS13. In addition to cleavage when immobilized on the endothelium, VWF subjected to fluid shear in flowing blood 7 and on platelets 8 is also susceptible to proteolysis by ADAMTS13. Together, these mechanisms reduce a...