Hematopoietic Cell E-/L-selectin Ligand Research Articles

CD44 fucosylation on bone marrow-derived mesenchymal stem cells enhances homing and promotes enteric nervous system remodeling in diabetic mice

BackgroundDiabetes can cause extensive enteric nervous system (ENS) injuries and gastrointestinal motility disorder. In developing possible treatments, researchers have engaged in tissue regeneration engineering with the very promising bone marrow-derived mesenchymal stem cells (BMSCs). However, BMSCs have poor homing ability to the targeted tissues after intravenous injection. Thus, we aimed to investigate whether enhancing the expression of E-selectin ligand on BMSCs could improve their homing ability and subsequently influence their role in ENS remodeling in diabetic mice.MethodsFirst, we constructed the fucosylation modification of CD44 on BMSCs through a fucosyltransferase VII (FTVII) system to generate a Hematopoietic Cell E-/L-selectin Ligand (HCELL) property, a fucosylated sialyllactosaminyl glycovariant of CD44 that potently binds E-selectin. Next, FTVII-modified and unmodified BMSCs labeled with green fluorescent protein (GFP) were injected into diabetic mice through the tail vein to compare their homing ability to the gastrointestinal tract and their effect on ENS remodeling, respectively. A bioluminescent imaging system was used to evaluate the homing ability of GFP-labeled BMSCs with and without FTVII modification, to the gastrointestinal tract. Gastrointestinal motility was assessed by gastrointestinal transient time, defecation frequency, stool water content and colon strips contractility. Immunofluorescence staining and western blotting were used to assess the expression levels of protein gene product 9.5 (PGP9.5), glial fibrillary acidic protein (GFAP) and glial cell line-derived neurotrophic factor (GDNF).ResultsThe FTVII-mediated α(1,3)-fucosylation modification of CD44 on BMSCs generated a HCELL property. Bioluminescent imaging assays showed that FTVII-modified BMSCs had enhanced homing ability to gastrointestinal tract, mainly to the colon, 24 h after injection through the tail vein. Compared with diabetic mice, FTVII-modified BMSCs significantly promoted the gastrointestinal motility and the ENS remodeling, including intestinal peristalsis (P < 0.05), increased feces excretion (P < 0.05) and the water content of the feces (P < 0.05), restored the spontaneous contraction of the colon (P < 0.05), and upregulated the protein expression levels of PGP9.5 (P < 0.01), GFAP (P < 0.001), and GDNF (P < 0.05), while unmodified BMSCs did not (P > 0.05).ConclusionsCD44 fucosylation modification on murine BMSCs promotes homing ability to the gastrointestinal tract and ENS remodeling in diabetic mice.

Production via good manufacturing practice of exofucosylated human mesenchymal stromal cells for clinical applications

BackgroundThe regenerative and immunomodulatory properties of human mesenchymal stromal cells (hMSCs) have raised great hope for their use in cell therapy. However, when intravenously infused, hMSCs fail to reach sites of tissue injury. Fucose addition in α(1,3)-linkage to terminal sialyllactosamines on CD44 creates the molecule known as hematopoietic cell E-/L-selectin ligand (HCELL), programming hMSC binding to E-selectin that is expressed on microvascular endothelial cells of bone marrow (BM), skin and at all sites of inflammation. Here we describe how this modification on BM-derived hMSCs (BM-hMSCs) can be adapted to good manufacturing practice (GMP) standards. MethodsBM-hMSCs were expanded using xenogenic-free media and exofucosylated using α(1,3)-fucosyltransferases VI (FTVI) or VII (FTVII). Enforced fucosylation converted CD44 into HCELL, and HCELL formation was assessed using Western blot, flow cytometry and cell-binding assays. Untreated (unfucosylated), buffer-treated and exofucosylated BM-hMSCs were each analyzed for cell viability, immunophenotype and differentiation potential, and E-selectin binding stability was assessed at room temperature, at 4°C, and after cryopreservation. Cell product safety was evaluated using microbiological testing, karyotype analysis, and c-Myc messenger RNA (mRNA) expression, and potential effects on genetic reprogramming and in cell signaling were analyzed using gene expression microarrays and receptor tyrosine kinase (RTK) phosphorylation arrays. ResultsOur protocol efficiently generates HCELL on clinical-scale batches of BM-hMSCs. Exofucosylation yields stable HCELL expression for 48 h at 4°C, with retained expression after cell cryopreservation. Cell viability and identity are unaffected by exofucosylation, without changes in gene expression or RTK phosphorylation. DiscussionThe described exofucosylation protocol using xenogenic-free reagents enforces HCELL expression on hMSCs endowing potent E-selectin binding without affecting cell viability or native phenotype. This described protocol is readily scalable for GMP-compliant clinical production.

A Glycovariant of Human CD44 is Characteristically Expressed on Human Mesenchymal Stem Cells.

The clinical effectiveness of systemically administered human mesenchymal stem cells (hMSCs) depends on their capacity to engage vascular endothelium. hMSCs derived from bone marrow (BM-hMSCs) natively lack endothelial binding capacity, but express a CD44 glycovariant containing N-linked sialyllactosamines that can be α(1,3)-fucosylated using fucosyltransferase-VI (FTVI) to enforce sLeX decorations, thereby creating hematopoietic cell E-/L-selectin ligand (HCELL). HCELL expression programs potent shear-resistant adhesion of circulating cells to endothelial beds expressing E-selectin. An alternative source of hMSCs is adipose tissue (A-hMSCs), and we assessed whether A-hMSCs bind E-selectin and/or possess sialyllactosamine-decorated CD44 accessible to α(1,3)-fucosylation. Similar to BM-hMSCs, we found that A-hMSCs natively lack E-selectin ligands, but FTVI-mediated cell surface α(1,3)-fucosylation induces sLeX expression and robust E-selectin binding secondary to conversion of CD44 into HCELL. Moreover, treatment with the α(1,3)-fucosyltransferase-FTVII also generated expression of HCELL on both BM-hMSCs and A-hMSCs, with sLeX decorations created on N-linked glycans of the "standard" CD44 (CD44s) isoform. The finding that hMSCs from both source tissues each lack native E-selectin ligand expression prompted examination of the expression of glycosyltransferases that direct lactosaminyl glycan synthesis. These studies reveal that both types of hMSCs conspicuously lack transcripts encoding α(1,3)-fucosyltransferases, but equally express glycosyltransferases critical to creation of sialyllactosamines. Collectively, these data indicate that assembly of a sialyllactosaminyl-decorated CD44s glycovariant is a conserved feature of hMSCs derived from adipose tissue and marrow, thus identifying a CD44 glycosignature of these cells and supporting the applicability of cell surface α(1,3)-fucosylation in programming migration of systemically administered A-hMSCs to sites of tissue injury/inflammation. Stem Cells 2017;35:1080-1092.

CD44 fucosylation on mesenchymal stem cell enhances homing and macrophage polarization in ischemic kidney injury

The lack of homing ability possibly reduces the healing potential of bone-marrow-derived mesenchymal stem cells (MSCs). Therefore, transforming native CD44 on MSCs into a hematopoietic cell E-/L-selectin ligand (HCELL) that possesses potent E-selectin affinity might enhance the homing and regenerative abilities of MSCs. Through fucosyltransferase VI (FTVI) transfection, MSCs were fucosylated on N-glycans of CD44 to become HCELL positive, thus interacting with E-selectin on injured endothelial cells. HCELL expression facilitated MSC homing in kidneys within 24h after injury and reduced lung stasis. An in vitro adhesion assay revealed that transfection enhanced the association between MSCs and hypoxic endothelial cells. In mice treated with HCELL-positive MSCs, the injured kidneys exhibited clusters of homing MSCs, whereas MSCs were rarely observed in mouse kidneys treated with HCELL-negative MSCs. Most MSCs were initially localized at the renal capsule, and some MSCs later migrated inward between tubules. Most homing MSCs were in close contact with inflammatory cells without tubular transdifferentiation. Furthermore, HCELL-positive MSCs substantially alleviated renal injury, partly by enhancing the polarization of infiltrating macrophages. In conclusion, engineering the glycan of CD44 on MSCs through FTVI transfection might enhance renotropism and the regenerating ability of MSCs in ischemic kidney injury.

Cell surface glycan engineering of neural stem cells augments neurotropism and improves recovery in a murine model of multiple sclerosis.

Neural stem cell (NSC)-based therapies offer potential for neural repair in central nervous system (CNS) inflammatory and degenerative disorders. Typically, these conditions present with multifocal CNS lesions making it impractical to inject NSCs locally, thus mandating optimization of vascular delivery of the cells to involved sites. Here, we analyzed NSCs for expression of molecular effectors of cell migration and found that these cells are natively devoid of E-selectin ligands. Using glycosyltransferase-programmed stereosubstitution (GPS), we glycan engineered the cell surface of NSCs ("GPS-NSCs") with resultant enforced expression of the potent E-selectin ligand HCELL (hematopoietic cell E-/L-selectin ligand) and of an E-selectin-binding glycoform of neural cell adhesion molecule ("NCAM-E"). Following intravenous (i.v.) injection, short-term homing studies demonstrated that, compared with buffer-treated (control) NSCs, GPS-NSCs showed greater neurotropism. Administration of GPS-NSC significantly attenuated the clinical course of experimental autoimmune encephalomyelitis (EAE), with markedly decreased inflammation and improved oligodendroglial and axonal integrity, but without evidence of long-term stem cell engraftment. Notably, this effect of NSC is not a universal property of adult stem cells, as administration of GPS-engineered mouse hematopoietic stem/progenitor cells did not improve EAE clinical course. These findings highlight the utility of cell surface glycan engineering to boost stem cell delivery in neuroinflammatory conditions and indicate that, despite the use of a neural tissue-specific progenitor cell population, neural repair in EAE results from endogenous repair and not from direct, NSC-derived cell replacement.

HCELL Expression on Murine MSC Licenses Pancreatotropism and Confers Durable Reversal of Autoimmune Diabetes in NOD Mice.

Type 1 diabetes (T1D) is an immune-mediated disease resulting in destruction of insulin-producing pancreatic beta cells. Mesenchymal stem cells (MSCs) possess potent immunomodulatory properties, garnering increasing attention as cellular therapy for T1D and other immunologic diseases. However, MSCs generally lack homing molecules, hindering their colonization at inflammatory sites following intravenous (IV) administration. Here, we analyzed whether enforced E-selectin ligand expression on murine MSCs could impact their effect in reversing hyperglycemia in nonobese diabetic (NOD) mice. Although murine MSCs natively do not express the E-selectin-binding determinant sialyl Lewis(x) (sLe(x) ), we found that fucosyltransferase-mediated α(1,3)-exofucosylation of murine MSCs resulted in sLe(x) display uniquely on cell surface CD44 thereby creating hematopoietic cell E-/L-selectin ligand (HCELL), the E-selectin-binding glycoform of CD44. Following IV infusion into diabetic NOD mice, allogeneic HCELL(+) MSCs showed threefold greater peri-islet infiltrates compared to buffer-treated (i.e., HCELL(-) ) MSCs, with distribution in proximity to E-selectin-expressing microvessels. Exofucosylation had no effect on MSC immunosuppressive capacity in in vitro assays; however, although engraftment was temporary for both HCELL(+) and HCELL(-) MSCs, administration of HCELL(+) MSCs resulted in durable reversal of hyperglycemia, whereas only transient reversal was observed following administration of HCELL(-) MSCs. Notably, exofucosylation of MSCs generated from CD44(-/-) mice induced prominent membrane expression of sLe(x) , but IV administration of these MSCs into hyperglycemic NOD mice showed no enhanced pancreatotropism or reversal of hyperglycemia. These findings provide evidence that glycan engineering to enforce HCELL expression boosts trafficking of infused MSCs to pancreatic islets of NOD mice and substantially improves their efficacy in reversing autoimmune diabetes. Stem Cells 2013;33:1523-1531.

Adhesion Of Acute Myeloid Leukemia Blasts To E-Selectin In The Vascular Niche Enhances Their Survival By Mechanisms Such As Wnt Activation

BackgroundAdhesion within the bone marrow microenvironment enhances leukemia survival and chemoresistance. Both normal hematopoietic stem cells and cancer cells are known to express E-selectin ligands, and adhesion of colon carcinoma cells to E selectin activates survival pathways such as NFκB (Porquet et al., BMC Cancer 11:285, 2011). E-selectin within the bone marrow vascular niche induces proliferation of normal hematopoietic stem cells (HSC), and a selectin inhibitor enhances HSC quiescence and self-renewal (Winkler et al., Nat Med 18:1651, 2012). We therefore initiated a study of E-selectin ligand expression and function by acute myeloid leukemia (AML) blasts to elucidate the potential role of E-selectin in AML biology and chemotherapy resistance. MethodsPrimary AML blasts and leukemia stem cells (LSCs) (CD34+CD38-CD123+) obtained with informed consent from 40 patients were analyzed for E-selectin ligands by flow cytometry for binding of E-selectin-Fc chimera or by labeling with the HECA452 antibody. Primary AML blasts were engrafted in NODscid IL2Rgc-/- mice for studies of E-selectin inhibitors in combination with chemotherapy. Human Stem Cell Signaling, Leukemia, Apoptosis, and NFκB PCR Arrays (SA Biosciences) were used to analyze gene expression by quantitative RT-PCR after adhesion of primary AML patient blasts to E-selectin coated plates, compared to bovine serum albumin (BSA) coated plates. The activation of the Wnt pathway was studied by luciferase reporter assay. ResultsWe find that the majority of primary patient acute myeloid leukemia blasts and leukemia stem cells express an E-selectin ligand, as demonstrated by flow cytometry by binding of E-selectin-Fc chimera and by staining with HECA-452 antibody [that recognizes hematopoietic cell E-/L-selectin ligand (HCELL) and cutaneous lymphocyte antigen (CLA)], as well as by binding to E-selectin coated plates. Flow cytometry analysis reveals that the mean percent binding of E-selectin-Fc chimera is 28% ± 24% (SD) by AML blasts, the mean % staining by the HECA-452 antibody 51% ± 35% (SD). De novo patients tended to have smaller mean fluorescence intensity (MFI) values than relapsed/refractory patients, as follows: for blasts, de novo mean 1441 ± 1127 (SD) vs. relapsed/refractory 4488 ± 4920 (SD) (Wilcoxon p=0.024), and for LSCs, de novo mean 1578 ± 1560 (SD) vs. relapsed/refractory 6601 ± 8498 (SD) (Wilcoxon p=0.061), suggesting upregulation of expression of E-selectin ligand for relapsed as compared to newly diagnosed patients. The specific E-selectin small molecule inhibitor GMI-1271 is able to overcome adhesion mediated chemotherapy resistance of AML in vitro and reduce the leukemia burden of primary AML engrafted NODscid IL2Rgc-/- mice in combination with chemotherapy agents daunorubicin and cytarabine. Addition of GMI1271 to chemotherapy in this xenograft model reduced the spleen burden at 2 weeks post treatment from 17.1 ± 10.4 X 106 hCD45+ cells/spleen to 7.6 ± 5.8 (p=0.04).To assess the molecular mechanism by which adhesion to E-selectin might protect AML blasts, we first screened with quantitative RT-PCR arrays. We found that adhesion to E-selectin caused upregulation of members of the Wnt and sonic hedgehog pathways for primary AML patient blasts grown on E-selectin vs. BSA coated plates, as well as members of other pathways critical to leukemia such as GM-CSF and IL-3 receptors and Fos. We then confirmed adhesion to E-selectin by AML blasts from 4 different patients enhanced activity of Wnt target genes by Wnt reporter assays. The Wnt reporter assay demonstrated 2-3 fold enhanced activity of Wnt target genes for AML blasts on E-selectin as compared to those on BSA, which increased to 3.3-4.5 fold with addition of Wnt3a. The inhibitor GMI-1271 reduced Wnt activity to 1.4-2.5 fold, similar to XAV939, an inhibitor of the Wnt/β catenin pathway that reduced activity to 1.1-1.8 fold. Similar reduction of Wnt pathway gene expression by GMI-1271 was also observed by the quantitative RT-PCR assay. ConclusionThese data support a critical role for E-selectin, likely in the vascular bone marrow niche, that promotes survival of AML, that can be targeted with therapeutic intent, and suggests that GMI-1271 should be explored as a treatment for AML in combination with chemotherapy. Disclosures:Chien:GlycoMimetics, Inc.: Research Funding. Haq:GlycoMimetics, Inc.: Research Funding. Magnani:GlycoMimetics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Becker:GlycoMimetics, Inc.: Research Funding.

A Novel Small Molecule E-Selectin Inhibitor GMI-1271 Blocks Adhesion of AML Blasts to E-Selectin and Mobilizes Blood Cells in Nodscid IL2Rgc−/− Mice Engrafted with Human AML

AbstractAbstract 4092The E-selectin ligand HCELL (hematopoietic cell E-/L-selectin ligand) is expressed by normal hematopoietic stem cells (Merzaban et al Blood 2011) as a functional glycoform of CD44. We observe high level CD44 expression (99%± 1.4%) by blasts from 55 consecutive patients with acute myeloid leukemia (AML) and by putative CD34+CD38−CD123+ leukemia stem cells (LSCs) (99.8% ± 0.6%). The mean fluorescence intensity (MFI) for CD44 expression by AML blasts is one to two logs higher than the MFI for 16 other adhesion receptors. We find that the majority of blasts from patients with AML also express an E-selectin ligand by flow cytometry: >75%% of 22 primary gated blast samples exhibit ≥10% binding of E selectin-IgG chimeric protein with a mean of 22.7% ± 0.17%SD, range 1.8 to 66.2%. We demonstrated that this ligand was HCELL by immunoprecipitation of CD44 from AML cell membranes, followed by staining with HECA 452 antibody that recognizes a functional trisaccharide domain shared by sialyl Lea and sialyl Lex and known to bind to E-selectin. HECA 452 not only detects the functional glycoform of CD44 known as HCELL, a major ligand for E-selectin, but also identifies the human lymphocyte homing receptor CLA (cutaneous lymphocyte antigen). HECA 452 labeled 5 of 6 patient leukemia blast populations, with mean expression 59.0% ± 24.8%. We also observed that HECA 452 antibody labeled CD34+CD38−CD123+ LSCs in addition to leukemic blasts, with a higher percent expression in most cases for the LSCs than the corresponding unfractionated blast population. Moreover, HECA 452 labeled 94% of human AML cells that had been serially engrafted in NODscid IL2Rgc−/− animals, fulfilling the functional definition of LSCs (scid repopulating cells), suggesting that HCELL may be enriched on LSCs. We observe a change in morphology with binding of AML blasts to E-selectin coated plastic, in that they elongate, become more cuboidal and less reflective, in contrast to the non-adherent cells, which remain round and refractile. AML blasts appear to bind to the elongated ends of spindle shaped endothelial cells. The E-selectin-specific inhibitor GMI-1271 (concentration 20 μM) inhibited adhesion of primary human AML cells to E-selectin by an average of 45.0%± 9.1%SD for all patients. For one patient, AML 035, for example, the percent inhibition with GMI-1271 compared to media control was 33.4%± 15.3%SD, p=0.000018. Adhesion to E-selectin did not confer adhesion mediated chemotherapy resistance to daunorubicin or cytarabine observed with adhesion to recombinant fibronectin peptide or immobilized VCAM-1 (Becker et al Blood 2009). We demonstrated that a dual inhibitor of E selectin and CXCR4 (GMI-1215) mobilized human AML engrafted in NODscid IL2Rgc−/− mice (Chien et al Abstract 579, ASH 2011), to a greater degree than we observed with CXCR4 inhibitor plerixafor (Chien et al Abstract 1432, ASH 2011) alone (3–4 fold vs. ∼2 fold). We now report that the E-selectin-specific inhibitor, GMI-1271 (40mg/kg), mobilizes both human and murine cells in immunodeficient xenograft mice engrafted with human AML: there is a 2 fold increase in WBC (p=0.00067) and 2 fold increase in human AML cells (p=0.14) at 3 hrs. An initial experiment with a combination of GMI-1271, daunorubicin, and cytarabine demonstrated greater depletion of human AML from the bone marrow (22% as many AML cells) and spleen (31% as many AML cells) than daunorubicin and cytarabine alone. Additional in vivo studies are in progress. We propose that residence in the bone marrow vascular niche may involve E-selectin and that migration of AML blasts involves critical interactions with the vascular endothelium through E-selectin. This interaction between HCELL expressed by AML and E-selectin might represent a new potential therapeutic target for AML. Disclosures:Patton:GlycoMimetics: Employment. Magnani:GlycoMimetics: Employment, Equity Ownership. Becker:GlycoMimetics, Inc: Research Funding.

Engineering cellular trafficking via glycosyltransferase‐programmed stereosubstitution

The proximate hurdle for cell trafficking to any anatomic site is the initial attachment of circulating cells to target tissue endothelium with sufficient strength to overcome prevailing forces of blood flow. E-selectin, an endothelial molecule that is inducibly expressed at all sites of inflammation, is a potent effector of this primary braking process. This molecule is a member of a family of C-type lectins known as selectins that bind sialofucosylated glycans displayed on either a protein (i.e., glycoprotein) or lipid (i.e., glycolipid) scaffold. On human cells, the predominant E-selectin ligand is a specialized glycoform of CD44 known as hematopoietic cell E-/L-selectin ligand (HCELL). This review focuses on the biology of HCELL/E-selectin interactions in cell migration, and discusses the utility and applicability of glycosyltransferase-programmed stereosubstitution (GPS) for glycoengineering HCELL expression. Without compromising cell viability or native phenotype, this exoglycosylation technology literally "sweetens" CD44, licensing E-selectin-dependent vascular delivery for all cell-based therapeutics.

The biology of CD44 and HCELL in hematopoiesis: the ‘step 2-bypass pathway’ and other emerging perspectives

The homing and egress of hematopoietic stem and progenitor cells (HSPCs) to and from marrow, respectively, and the proliferation and differentiation of HSPCs within marrow are complex processes critically regulated by the ordered expression and function of adhesion molecules that direct key cell-cell and cell-matrix interactions. The integral membrane molecule CD44, known primarily for its role in binding hyaluronic acid, is characteristically expressed on HSPCs. Conspicuously, human HSPCs uniquely display a specialized glycoform of CD44 known as hematopoietic cell E-/L-selectin ligand (HCELL), which is the most potent ligand for both E-selectin and L-selectin expressed on human cells. This review focuses on recent advances in our understanding of the biology of CD44 and HCELL in hematopoiesis. New data indicate that CD44-mediated events in hematopoiesis are more complex than previously imagined. Ex-vivo glycan engineering has established that HCELL serves as a 'bone marrow homing receptor'. Moreover, biochemical studies now show that CD44 forms bimolecular complexes with a variety of membrane proteins, one of which is VLA-4. Engagement of CD44 or of HCELL directly induces VLA-4 activation via G-protein-dependent signaling, triggering a 'step 2-bypass pathway' of cell migration, and extravascular lodgment, in absence of chemokine receptor engagement. Recent studies have further clarified the roles of CD44 and its glycoform HCELL in hematopoietic processes, providing key insights on how targeting these molecules may be beneficial in promoting hematopoiesis and in treating hematologic malignancies.

Glycosyltransferase‐programmed stereosubstitution (GPS) to create HCELL: engineering a roadmap for cell migration

During evolution of the vertebrate cardiovascular system, the vast endothelial surface area associated with branching vascular networks mandated the development of molecular processes to efficiently and specifically recruit circulating sentinel host defense cells and tissue repair cells at localized sites of inflammation/tissue injury. The forces engendered by high-velocity blood flow commensurately required the evolution of specialized cell surface molecules capable of mediating shear-resistant endothelial adhesive interactions, thus literally capturing relevant cells from the blood stream onto the target endothelial surface and permitting subsequent extravasation. The principal effectors of these shear-resistant binding interactions comprise a family of C-type lectins known as 'selectins' that bind discrete sialofucosylated glycans on their respective ligands. This review explains the 'intelligent design' of requisite reagents to convert native CD44 into the sialofucosylated glycoform known as hematopoietic cell E-/L-selectin ligand (HCELL), the most potent E-selectin counter-receptor expressed on human cells, and will describe how ex vivo glycan engineering of HCELL expression may open the 'avenues' for the efficient vascular delivery of cells for a variety of cell therapies.

HCELL Is the Major E- and L-Selectin Ligand Expressed on Human Hematopoietic Progenitor Cells and Colon Carcinoma Cells.

Selectins are a class of cell surface proteins specialized to mediate adhesive interactions under hydrodynamic shear flow conditions, and are critical in the trafficking of hematopoietic progenitor cells (HPC) into the bone marrow and dissemination of cancer cells in hematogenous metastasis, among other biological events. More specifically, E-selectin expressed by endothelial cells has been shown to mediate shear-resistant tethering/rolling of cells recruited from the fluid stream, while L-selectin expressed by distinct leukocyte subsets has been variously shown to mediate lymph node homing, as well as the formation of heterotypic cell aggregates in bulk flow. We have previously reported that the primitive CD34+ human hematopoietic progenitor cell (HPC) line KG1a exhibits stronger E-/L-selectin ligand activities than HL60, a more differentiated CD34− human hematopoietic cell line. These findings were attributed to the HPC/KG1a restricted expression of the sialofucosylated CD44 glycoform termed HCELL (Hematopoietic Cell E-/L-selectin Ligand), since both cell lines express approximately equivalent levels of CD44 and the well-characterized selectin ligand P-selectin glycoprotein ligand-1 (PSGL-1). To directly assess whether HCELL functions as the high affinity E-/L-selectin ligand on KG1a cells, HCELL expression was silenced using lentiviral siRNA targeting CD44, and the E-/L-selectin ligand activities were subsequently compared between untreated and transduced KG1a cells. CD44 siRNA transduction of KG1a cells decreased CD44 (i.e. HCELL) expression levels >90% mean fluorescence intensity (MFI) relative to untreated and vector alone-transduced cells as measured by flow cytometry. CD44 targeting was specific, as expression of PSGL-1 was not reduced upon CD44 siRNA transduction, nor were CD29, CD49d, or CD49e levels affected. Functionally, at venous shear stress levels (≤4 dyn/cm2), CD44 siRNA-transduced KG1a cells rolled markedly faster than untreated and vector alone-transduced cells on a monolayer of E-selectin transfected Chinese hamster ovary (CHO-E) cells. At arterial and pathological shear (>4 dyn/cm2), control cells maintained rolling interactions on CHO-E cells, whereas CD44 siRNA-transduced cells readily detached from the monolayer. Comparable results were obtained for L-selectin-dependent peripheral blood mononuclear cell rolling on CD44 siRNA-transduced versus untreated and vector alone-transduced KG1a cells. Furthermore, the HCELL-negative HL60 cells supported similarly weak binding of E- and L-selectin as CD44 siRNA-transduced KG1a cells. Collectively, the data directly show that HCELL is the predominant ligand that stabilizes shear-resistant HPC binding to E- and L-selectin. We have also previously reported that the human colon carcinoma cell line LS174T expresses HCELL. To examine whether HCELL on LS174T cells functions as a selectin ligand, we again suppressed expression through targeted gene silencing by lentiviral CD44 siRNA transduction. Similar to KG1a cells, specific CD44/HCELL silencing on LS174T cells was achieved (>90% reduction in MFI relative to untreated and vector controls), resulting in significant inhibition of E- and L-selectin binding capabilities at physiologically relevant shear stress levels. In summary, these findings demonstrate that HCELL is the most potent E-/L-selectin ligand mediating shear-resistant adhesive interactions on human cells.

HCELL Is the Major E- and L-selectin Ligand Expressed on LS174T Colon Carcinoma Cells

Engagement of vascular E-selectin and leukocyte L-selectin with relevant counter-receptors expressed on tumor cells contributes to the hematogenous spread of colon carcinoma. We recently demonstrated that the LS174T colon carcinoma cell line expresses the CD44 glycoform known as hematopoietic cell E-/L-selectin ligand (HCELL), which functions as a high affinity E- and L-selectin ligand on these cells. To define the contribution of HCELL to selectin-mediated adhesion on intact tumor cells, we measured the binding of LS174T cells transduced with CD44 short interfering RNA (siRNA) or with vector alone to 6-h interleukin-1beta-stimulated human umbilical vein endothelial cells (HUVEC) and to human peripheral blood mononuclear cells (PBMC) under physiological flow conditions. LS174T cell attachment to HUVEC was entirely E-selectin-dependent, and PBMC tethering to tumor cell monolayers was completely L-selectin-dependent. At physiological shear stress, CD44 siRNA transduction led to an approximately 50% decrease in the number of LS174T cells binding to stimulated HUVEC relative to vector alone-transduced cells. CD44 siRNA-transduced cells also rolled significantly faster than vector-transduced cells on HUVEC, indicating prominent HCELL participation in stabilizing tumor cell-endothelial adhesive interactions against fluid shear. Furthermore, HCELL was identified as the principal L-selectin ligand on LS174T cells, as PBMC binding to CD44 siRNA-transduced tumor cells was reduced approximately 80% relative to vector-transduced cells. These data indicate that expression of HCELL confers robust and predominant tumor cell binding to E- and L-selectin, highlighting a central role for HCELL in promoting shear-resistant adhesive interactions essential for hematogenous cancer dissemination.