The initial selectin-dependent events that mediate tumor cell tethering to platelets, leukocytes, and vascular endothelium can regulate the extravasation and colonization of metastatic cells into distant tissues. Little is known, however, about the identity of selectin counter-receptors on tumor cells, which facilitate the metastatic process. To address this issue, we performed SDS-PAGE analysis of membrane proteins, metabolic inhibition studies, blot rolling assays, and cell-free flow-based adhesion experiments using microbeads coated with CD44 immunoprecipitated from carcinomas and purified selectins as substrate. Here, we demonstrate that variant isoforms of CD44 (CD44v) on LS174T colon carcinoma cells possess P-/L-/E-selectin binding activity, in contrast to the standard isoform of CD44 (CD44s) on hematopoietic-progenitor cells (HPCs), which is primarily an L-/E-selectin ligand. Moreover, the selectin-binding determinants on CD44v from LS174T cells are sialofucosylated structures displayed on O-linked glycans, akin to those on P-selectin glycoprotein ligand-1, but distinct from the HECA-452-reactive N-glycans on CD44s expressed on HPCs. Using flow-based adhesion assays, we systematically characterize shear-dependent LS174T CD44 vs. HL60 CD44s adhesion to E-/P-/L-selectin. The novel finding that CD44v are selectin ligands offers a unifying perspective on the apparent enhanced metastatic potential associated with tumor cell CD44v overexpression and the critical role of selectins in metastasis.

Metastasis of circulating tumor cells requires a multistep cascade of events initiated by adhesion of tumor cells to the vascular endothelium of involved tissues. This process occurs under the forces of blood flow and is promoted by adhesion molecules specialized to interact under shear conditions. The endothelial molecule E-selectin is a major mediator of these adhesive events, and there is strong evidence that E-selectin receptor-ligand interactions contribute to the formation of metastasis. However, little is known about the identity of E-selectin ligand(s) expressed on cancer cells. To address this issue, we did SDS-PAGE analysis of membrane proteins, metabolic inhibition studies, and blot rolling assays of LS174T, a colon carcinoma cell line known to interact with E-selectin under physiologic flow conditions. Our studies show that LS174T cells express the hematopoietic cell E/L-selectin (HCELL) glycoform of CD44, which functions as a high-affinity E-selectin glycoprotein ligand on these cells. However, in contrast to the HCELL glycoform on human hematopoietic progenitor cells, which expresses carbohydrate-binding determinant(s) for E-selectin primarily on N-glycans of standard CD44, the relevant determinant(s) on LS174T cells is expressed on O-glycans and is predominantly found on variant isoforms of CD44 (CD44v). Our finding that tumor-associated CD44 splice variant(s) express E-selectin ligand activity provides novel perspectives on the biology of CD44 in cancer metastasis. (Cancer Res 2005; 65(13): 5812-7)

Although well recognized that expression of E-selectin on marrow microvessels mediates osteotropism of hematopoietic stem/progenitor cells (HSPCs), our knowledge regarding the cognate E-selectin ligand(s) on HSPCs is incomplete. Flow cytometry using E-selectin-Ig chimera (E-Ig) shows that human marrow cells enriched for HSPCs (CD34 ؉ cells) display greater E-selectin binding than those obtained from mouse (lin ؊ /Sca-1 ؉ / c-kit ؉ [LSK] cells). To define the relevant glycoprotein E-selectin ligands, lysates from human CD34 ؉ and KG1a cells and from mouse LSK cells were immunoprecipitated using E-Ig and resolved by Western blot using E-Ig. In both human and mouse cells, E-selectin ligand reactivity was observed at ϳ 120-to 130-kDa region, which contained two E-selectin ligands, the P-selectin glycoprotein ligand-1 glycoform "CLA," and CD43. Human, but not mouse, cells displayed a prominent ϳ 100-kDa band, exclusively comprising the CD44 glycoform "HCELL." E-Ig reactivity was most prominent on CLA in mouse cells and on HCELL in human cells. To further assess HCELL's contribution to E-selectin adherence, complementary studies were performed to silence (via CD44 siRNA) or enforce its expression (via exoglycosylation). Under physiologic shear conditions, CD44/ HCELL-silenced human cells showed striking decreases (> 50%) in E-selectin binding. Conversely, enforced HCELL expression of LSK cells profoundly increased E-selectin adherence, yielding > 3-fold more marrow homing in vivo. These data define the key glycoprotein E-selectin ligands of human and mouse HSPCs, unveiling critical species-intrinsic differences in both the identity and activity of these structures. (Blood. 2011; 118(7):1774-1783) IntroductionA greater understanding of the molecular effector(s) directing trafficking and engraftment of hematopoietic stem/progenitor cells (HSPCs) into bone marrow is critical for optimizing therapeutic outcomes of hematopoietic stem cell transplantation. It is well established that homing of HSPCs into marrow is a conspicuously nonrandom process regulated by discrete adhesive interactions between HSPCs in blood flow and target bone marrow endothelium. [1][2][3][4] Studies in both humans and mice indicate that E-selectin is constitutively expressed on marrow endothelial cells, 5,6 and intravital studies have revealed that HSPC migration to marrow occurs at specialized microvascular beds expressing E-selectin. 2 These and several other independent lines of evidence 3,4,[7][8][9] have highlighted a central role for E-selectin-dependent interactions in HSPC recruitment to marrow. However, our knowledge of the cognate E-selectin coreceptors expressed on human and mouse HSPCs remains incomplete.E-selectin, together with L-and P-selectin, comprise the selectin family of adhesion molecules, which are cell membrane calcium-dependent lectins that function as principal regulators ofStep 1 rolling interactions that are requisite for tissue-specific cell migration. 10 All 3 selectins bind to specialized carbohydrate determinants, comp...

This study was undertaken to investigate the molecular constituents mediating LS174T colon adenocarcinoma cell adhesion to 4-h TNF-alpha-stimulated human umbilical vein endothelial cells (HUVECs) under flow. At 1 dyn/cm(2), approximately 57% of cells rolled and then became firmly adherent, whereas others continuously rolled on endothelium. Initial cell binding was primarily mediated by endothelial E-selectin. By using neuraminidase, glycolipid biosynthesis inhibitor d,l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol. HCl, trypsin, and flow cytometry, LS174T cells were shown to express sialyl Lewis(x) (sLe(x))- and di-sLe(x)-decorated, but not sLe(a)-decorated, glycolipid and glycoprotein ligands for E-selectin. The cells preferentially employed sialylated glycoproteins over glycolipids in adhesion as measured by conversion of rolling to firm adhesion, resistance to detachment by increased shear stress, and rolling velocity. However, a nonsialylated E-selectin counterreceptor also exists. Furthermore, LS174T alpha(2), alpha(6), and beta(1) integrins support a minor pathway in adhesion to HUVECs. Finally, tumor cell attachment specifically increases HUVEC endocytosis of E-selectin. Altogether, the data indicate the complexity of carcinoma cell-endothelium adhesion via sialylated glycoconjugates, integrins, and their respective counterreceptors.

Selectins on activated vascular endothelium mediate inflammation by binding to complementary carbohydrates on circulating neutrophils. The human neutrophil receptor for E-selectin has not been established. We report here that sialylated glycosphingolipids with 5 Nacetyllactosamine (LacNAc, Gal␤1-4Glc-NAc␤1-3) repeats and 2 to 3 fucose residues are major functional E-selectin receptors on human neutrophils. Glycolipids were extracted from 10 10 normal peripheral blood human neutrophils. Individual glycolipid species were resolved by chromatography, adsorbed as model membrane monolayers and selectinmediated cell tethering and rolling under fluid shear was quantified as a function of glycolipid density. E-selectin-expressing cells tethered and rolled on selected glycolipids, whereas P-selectin-expressing cells failed to interact. Quantitatively minor terminally sialylated glycosphingolipids with 5 to 6 LacNAc repeats and 2 to 3 fucose residues were highly potent E-selectin receptors, constituting more than 60% of the E-selectin-binding activity in the extract. These glycolipids are expressed on human blood neutrophils at densities exceeding those required to support E-selectin-mediated tethering and rolling. Blocking glycosphingolipid biosynthesis in cultured human neutrophils diminished E-selectin, but not Pselectin, adhesion. The data support the conclusion that on human neutrophils the glycosphingolipid NeuAc␣2-3Gal IntroductionMultiple mechanisms ensure that granulocytes efficiently move into tissues when and where needed. 1 Activation of the blood vessel endothelium results in circulating granulocytes tethering and rolling, stopping, flattening, and squeezing into the surrounding tissue. A 3-step model describes leukocyte extravasation 2 : (1) Eand P-selectin on activated endothelia bind to glycans on leukocytes, initiating tethering and rolling under the shear stress of blood flow (L-selectin on some leukocytes contributes to this process).(2) Upon tethering, leukocytes are exposed to chemoattractants (eg, chemokines) generated by activated endothelium and tissueresident cells, resulting in integrin mobilization. (3) Strong cell adhesion is mediated by leukocyte integrin binding to immunoglobulin superfamily proteins on the endothelium. Each step is required for recruitment of leukocytes; blocking any one diminishes inflammation. Despite exceptions, 3 this scheme is broadly applicable.Each selectin (E-, L-, and P-) 4 has a carbohydrate recognition domain that mediates binding to specific glycans on apposing cells. They have remarkably similar protein folds and carbohydrate binding residues, 5 leading to overlap in the glycans to which they bind. Nevertheless, each selectin has distinct receptors to which it binds with high affinity.Selectins bind to the sialyl Lewis x (SLe x ) determinant "NeuAc␣2-3Gal␤1-4(Fuc␣1-3)GlcNAc." [6][7][8][9] However, SLe x , per se, does not constitute an effective selectin receptor. [10][11][12] Instead, SLe x and related sialylated, fucosylated glycans are components of more ext...

When confined to a primary site (early stage disease), the five-year survival rate from colon cancer is ϳ90%. However, when disseminated from primary sites (metastatic), survival drops precipitously to 8%. These dramatic survival statistics underscore the need to define the pathobiology of the metastatic process to devise novel interventions to prevent or inhibit colon cancer spread. Hematogenous metastasis occurs as a highly regulated cascade of events, initiated by the escape of tumor cells from the primary site into the blood stream and culminating in the formation of secondary colonies in distant organs. This process critically involves the binding of circulating colon cancer cells to the endothelium of target tissue(s) under fluid shear conditions, as well as the dynamic formation of leukocyte/cancer cell emboli, each of which are directed by selectin-selectin ligand interactions.The selectin family of adhesion molecules, E-, P-, and L-selectin, are Ca 2ϩ -dependent lectins that bind sialofucosylated carbohydrate structures, the prototypes of which are sialyl Lewis X (sLe x ) and sialyl Lewis A (sLe a ) (1, 2). E-and P-selectin are typically inducible endothelial molecules (P-selectin is also expressed on activated platelets), and L-selectin expression is restricted to leukocytes (3). There are multiple reports that tumor cell expression of E-selectin ligand(s) promotes the metastatic spread of numerous cancer types in vivo, including colon cancer (4 -10). Colon carcinoma cells also tether and roll under dynamic flow conditions on E-selectin purified and immobilized on plastic (11, 12), as well as E-selectin presented by cytokine-stimulated human umbilical vein endothelial cells (HUVEC) 2 (11-13). Independently, L-selectin has been shown to promote colon cancer metastasis in vivo (14), presumably mediated by the physical association of leukocytes with tumor cells resulting in leukocyte-colon cancer cell emboli (15, 16). Although expression of E-selectin ligand(s) on tumor cells itself promotes metastasis, one model holds that selectins work cooperatively to promote the spread of cancer; leukocyte L-selectin engagement of relevant tumor ligand(s) mediates the formation of leukocyte-tumor aggregates, which possess heightened binding capacity to endothelium in an E-selectindependent manner (16).Colon carcinoma cells express selectin ligands, and there is abundant evidence that selectin-dependent adhesive events are central to the metastatic process (4 -10,14). However, these selectin ligands have yet to be fully characterized or identified other than by general classifications (e.g. mucins). We recently identified the sialofucosylated hematopoietic cell E-and L-selectin ligand (HCELL) glycoform of CD44 on the LS174T colon carcinoma cell line and demonstrated its function as a high affinity E-and L-selectin ligand using the blot rolling assay (17, 18). However, these studies did not specifically address the relative contribution(s) of HCELL to the observed potent E-and L-selectin ligand activity of intact LS174...

Selectins and fibrin(ogen) play key roles in the hematogenous dissemination of tumor cells, and especially of colon carcinomas. However, the fibrin(ogen) receptor(s) on colon carcinoma cells has yet to be defined along with its relative capacity to bind fibrinogen versus fibrin under flow. Moreover, the functional P-selectin ligand has yet to be validated using intact platelets rather than purified selectin substrates. Using human CD44-knockdown and control LS174T cells, we demonstrate the pivotal involvement of CD44 in the P-selectin-mediated binding to platelets in shear flow. Quantitative comparisons of the binding kinetics of LS174T versus P-selectin glycoprotein ligand-1 (PSGL-1)-expressing THP-1 cells to activated platelets reveal that the relative avidity of P-selectin-CD44 binding is more than sevenfold lower than that of P-selectin-PSGL-1 interaction. Using CD44-knockdown LS174T cells and microspheres coated with CD44 immunoprecipitated from control LS174T cells, and purified fibrin(ogen) as substrate, we provide the first direct evidence that CD44 also acts as the major fibrin, but not fibrinogen, receptor on LS174T colon carcinoma cells. Interestingly, binding of plasma fibrin to CD44 on the colon carcinoma cell surface interferes with the P-selectin-CD44 molecular interaction and diminishes platelet-LS174T heteroaggregation in the high shear regime. Cumulatively, our data offer a novel perspective on the apparent metastatic potential associated with CD44 overexpression on colon carcinoma cells and the critical roles of P-selectin and fibrin(ogen) in metastatic spread and provide a rational basis for the design of new therapeutic strategies to impede metastasis.

This study was undertaken to investigate the kinetics and molecular requirements of platelet binding to tumor cells in bulk suspensions subjected to a uniform linear shear field, using a human colon adenocarcinoma cell line (LS174T) as a model. The effects of shear rate (20-1000 s(-1)), shear exposure time (30-300 s), shear stress (at constant shear rate by adjusting the viscosity of the medium from 1.3-2.6 cP), cell concentration, and platelet activation on platelet-LS174T heteroaggregation were assessed. The results indicate that hydrodynamic shear-induced collisions augment platelet-LS174T binding, which is further potentiated by thrombin/GPRP-NH(2). Peak adhesion efficiency occurs at low shear and decreases with increasing shear. Intercellular contact duration is the predominant factor limiting heteroaggregation at shear rates up to 200 s(-1), whereas these interactions become shear stress-sensitive at > or = 400 s(-1). Heteroaggregation increases with platelet concentration due to an elevation of the intercellular collision frequency, whereas adhesion efficiency remains nearly constant. Moreover, hydrodynamic shear affects the receptor specificity of activation-dependent platelet binding to LS174T cells, as evidenced by the transition from a P-selectin-independent/Arg-Gly-Asp (RGD)-dependent process at 100 s(-1) to a P-selectin/alpha(IIb)beta(3)-dependent interaction at 800 s(-1). This study demonstrates that platelet activation and a fluid-mechanical environment representative of the vasculature affect platelet-tumor cell adhesive interactions pertinent to the process of blood-borne metastasis.