How cancer cells bind to vascular surfaces and extravasate into target organs is an underappreciated, yet essential step in metastasis. We postulate that the metastatic process involves discrete adhesive interactions between circulating cancer cells and microvascular endothelial cells. Sialyl Lewis X (sLe X ) on prostate cancer (PCa) cells is thought to promote metastasis by mediating PCa cell binding to microvascular endothelial (E)-selectin. Yet, regulation of sLe X and related E-selectin ligand expression in PCa cells is a poorly understood factor in PCa metastasis. Here, we describe a glycobiological mechanism regulating E-selectin-mediated adhesion and metastatic potential of PCa cells. We demonstrate that ␣1,3 fucosyltransferases (FT) 3, 6, and 7 are markedly elevated in bone-and liver-metastatic PCa and dictate synthesis of sLe X and E-selectin ligands on metastatic PCa cells. (1). Delineating mediators of PCa metastasis to target organs may lead to identification of prognostic biomarkers and anti-metastatic therapeutics. Recently, our lab has advanced a scenario to account for organ metastasis involving PCa cell adhesion to surface receptors expressed on microvascular endothelial cells of the target organ (2-4). Bone-metastatic PCa cells are known to attach more avidly to bone marrow endothelial cells (BMEC) compared with endothelial linings of nontarget organs (5, 6). For example, human bone-metastatic PCa MDA PCa 2b (MDA) cells roll and adhere on BMEC by binding endothelial (E)-selectin, raising the possibility that PCa metastasis could be conferred through E-selectin Ϫ E-selectin ligand adhesive interactions (2-4). In fact, BM microvessels express E-selectin constitutively, while E-selectin is inducible on endothelial linings of inflamed tissues and of bronchial mucosa, a common PCa target tissue (7-9). Mechanistically, an identical traffic control axis involving selectins is well-known in the extravasation of hematopoietic progenitor cells (HPC) into BM (10, 11). HPC rolling on E-and platelet (P)-selectin is regulated by sLe X -bearing glycoforms of CD44 (HCELL), PSGL-1 and glycolipids (10,12,13). Synthesis of sLe X in HPC is catalyzed in the Golgi compartment by members of the glycosyltransferase gene family (14). The final step involves the transfer of fucose to N-acetylglucosamine at the terminal ␣2,3 sialo-lactosamine unit by ␣1,3 fucosyltransferases (FT) 3, 4, 5, 6, and/or 7, depending on cell type (13, 15). That metastatic PCa cells traverse the vasculature through 'hematopoietic mimicry' is consistent with several observations, including the association of sLe X with PCa grade and progression (2, 16, 17), cancer cell E-selectin ligand activity is a direct correlate with metastatic potential (18,19) and cancer cells can trigger E-selectin expression on liver sinusoidal microvasculature to steer circulating cancer cells to the liver (20,21). Thus, mapping glyco-metabolic pathways regulating E-selectin ligand synthesis may be vital for understanding PCa metastasis.In this report, we identify E-s...

Galectin-1 (Gal-1), aβ-galactoside-binding protein, can alter fate and effector function of T helper (Th) cells; however, little is known about how Gal-1 induces Th cell differentiation. Here, we show that both uncommitted and polarized Th cells bound by Gal-1 expressed an immunoregulatory signature defined by IL-10. IL-10synthesis was stimulated by direct Gal-1 engagement to cell surface glycoproteins, principally CD45, on activated Th cells and enhanced by IL-21 expression through the c-Maf/aryl hydrocarbon receptor (AhR) pathway, independent of antigen-presenting cells. Gal-1-induced IL-10+ Tcells efficiently suppressed T cell proliferation and T-cell-mediated inflammation and promoted the establishment of cancer immune-privileged sites. Collectively, these findings show how Gal-1 functions as a major glycome determinant regulating Th cell development, inflammation and tumor immunity.

Galectin-1 (Gal-1), a β-galactoside–binding lectin, plays a profound role in modulating adaptive immune responses by altering the phenotype and fate of T cells. Experimental data showing recombinant Gal-1 (rGal-1) efficacy on T cell viability and cytokine production, nevertheless, is controversial due to the necessity of using stabilizing chemicals to help retain Gal-1 structure and function. To address this drawback, we developed a mouse Gal-1 human Ig chimera (Gal-1hFc) that did not need chemical stabilization for Gal-1 ligand recognition, apoptosis induction, and cytokine modulation in a variety of leukocyte models. At high concentrations, Gal-1hFc induced apoptosis in Gal-1 ligand+ Th1 and Th17 cells, leukemic cells, and granulocytes from synovial fluids of patients with rheumatoid arthritis. Importantly, at low, more physiologic concentrations, Gal-1hFc retained its homodimeric form without losing functionality. Not only did Gal-1hFc–binding trigger IL-10 and Th2 cytokine expression in activated T cells, but members of the CD28 family and several other immunomodulatory molecules were upregulated. In a mouse model of contact hypersensitivity, we found that a non-Fc receptor-binding isoform of Gal-1hFc, Gal-1hFc2, alleviated T cell-dependent inflammation by increasing IL-4+, IL-10+, TGF-β+, and CD25high/FoxP3+ T cells, and by decreasing IFN-γ+ and IL-17+ T cells. Moreover, in human skin-resident T cell cultures, Gal-1hFc diminished IL-17+ T cells and increased IL-4+ and IL-10+ T cells. Gal-1hFc will not only be a useful new tool for investigating the role of Gal-1 ligands in leukocyte death and cytokine stimulation, but for studying how Gal-1–Gal-1 ligand binding shapes the intensity of immune responses.

Galectin-1 (Gal-1) has been shown to play a major role in tumor immune escape by inducing apoptosis of effector leukocytes and correlating with tumor aggressiveness and disease progression. Targeting the Gal-1 – Gal-1 ligand axis, thus, represents a promising cancer therapeutic approach. Here, to test the Gal-1-mediated tumor immune evasion hypothesis and demonstrate the importance of Gal-1-binding N-acetyllactosamines in controlling the fate and function of anti-tumor immune cells, we treated melanoma- or lymphoma-bearing mice with peracetylated 4-fluoro-glucosamine (4-F-GlcNAc), a metabolic inhibitor of N-acetyllactosamine biosynthesis, and analyzed tumor growth and immune profiles. We found that 4-F-GlcNAc spared Gal-1-mediated apoptosis of T and NK cells by decreasing their expression of Gal-1-binding determinants. 4-F-GlcNAc enhanced tumor lymphocytic infiltration and promoted elevations in tumor-specific cytotoxic T cells and IFN-γ levels, while lowering IL-10 production. Collectively, our data suggest that metabolic lowering of Gal-1-binding N-acetyllactosamines may attenuate tumor growth by boosting anti-tumor immune cell levels, representing a promising approach for cancer immunotherapy.

Advanced prostate cancer (PCa) commonly metastasizes to bone, but transit of malignant cells across the bone marrow endothelium (BMEC) remains a poorly understood step in metastasis. PCa cells roll on E-selectin+ BMEC through E-selectin ligand-binding interactions under shear flow, and PCa cells exhibit firm adhesion to BMEC via β1, β4 and αVβ3 integrins in static assays. However, whether these discrete PCa cell-BMEC adhesive contacts culminate in cooperative, step-wise transendothelial migration into bone is not known. Herein, we describe how metastatic PCa cells breach BMEC monolayers in a step-wise fashion under physiologic hemodynamic flow. PCa cells tethered and rolled on BMEC and then firmly adhered to and traversed BMEC via sequential dependence on E-selectin ligands and β1 and αVβ3 integrins. Expression analysis in human metastatic PCa tissue revealed that β1 was markedly upregulated compared with expression of other β subunits. PCa cell breaching was regulated by Rac1 and Rap1 GTPases and, notably, did not require exogenous chemokines as β1, αVβ3, Rac1 and Rap1 were constitutively active. In homing studies, PCa cell trafficking to murine femurs was dependent on E-selectin ligand, β1 integrin and Rac1. Moreover, eliminating E-selectin ligand-synthesizing α1,3 fucosyltransferases (α1,3 FT) in transgenic adenoma of mouse prostate (TRAMP) mice dramatically reduced PCa incidence. These results unify the requirement for E-selectin ligands, α1,3 fucosyltransferases, β1 and αVβ3 integrins and Rac/Rap1 GTPases in mediating PCa cell homing and entry into bone and offer new insight on the role of α1,3 fucosylation in PCa development.