Lewis X Research Articles

NKG2D: Binding Properties for Glycan Ligands, and Mutagenesis Analysis

Killer lectin-like receptor NKG2D, which is found on natural killer cells, recognizes MHC class 1-related ligands and also interacts with glycan ligands, heparin-conjugated bovine serum albumin (heparin-BSA) and sialyl Lewis X (sLeX) on multi-antennary N-glycans on transferrin secreted by HepG2 cells (HepTF). Using the glutathione-Stransferase- fused extracellular domain (AA 73-216) of NKG2D (rGST-NKG2D) and seven site-directed mutants, we explored in detail the binding of NKG2D to sulfate-containing glycan-BSA and HepTF. rGST-NKG2D binds to sulfatecontaining glycan-BSA with Kd values of 25 nM ±15 for λ-carrageenan-BSA, 66 ±23 nM for fucoidan-BSA, and 1.5±0.5 μM for heparan sulfate-BSA. Of the site-directed rGST-NKG2D mutants, Y152A, Q185A, K197A, Y199A, E201A, and N207A reduced binding to these glycans. These results indicate that NKG2D interacts with highly sulfated- and α2,3- NeuAc-containing glycans and suggest that the glycan-binding sites on NKG2D are shared between sulfate- and α2,3- NeuAc-containing glycans, and might overlap with protein ligand binding sites.

Abstract 8567: Combination of Active Targeting Erythropoietin-Liposome with Sialyl Lewis X and G-CSF, is Markedly Protective against Ischemia Reperfusion Injury via EPCs Mobilization and Upregulation of p-Akt and p-ERK

Purpose: We previously reported that erythropoietin(EPO)-containing nano-size liposome with Sialyl Lewis X (SLX) and G-CSF prevent cardiac remodeling and improve cardiac function, respectively. Then we hypothesized that combination of EPO-liposome with SLX and G-CSF might be more protective against ischemia reperfusion injury than EPO-liposome with SLX or G-CSF alone. Methods: Japanese white rabbits (n=40) were subjected to 30-minute coronary occlusion followed by 14 days of reperfusion. Rabbits were administered saline (control group, i.v., n=10), G-CSF (G group, 10μ g/kg/day, i.c., for 5days, n=10), EPO-liposome with SLX (LE group, i.v., 5000IU/ml of EPO, 1ml, n=10), G-CSF + EPO-liposome with SLX (LE+G group, n=10) after reperfusion. Myocardial infarct size was calculated as a percentage of the risk area of the left ventricle. Western blot-analysis examined phosphorylation of Akt and ERK, and expression of E- and P-selectin in the ischemic myocardium at 14days of reperfusion. We also performed fluorescence-activated cell sorter (FACS) analysis of endothelial progenitor cells (EPCs) to evaluate CD34 + /CXCR4 + cell mobilization at 7 days after reperfusion. Results: The infarct size was significantly smaller in the LE+G group (14.0±2.5%) than in the control group (29.0±5.4%), G group (20.3±5.6%) or LE group (18.7±3.7%). Western blot-analysis showed a higher expression of phosphorylated(p)-Akt and p-ERK and expression of E- and P-selectin in the infarct area of the LE+G group than the others. Serum SDF-1 level at 2 days after reperfusion was higher in the G group and the LE+G group than in the Control group or the LE group. The percentages of CD34 + /CXCR4 + cells among total peripheral blood mononuclear cells of G group and LE+G group were higher than the others. Conclusions: Combination of post-infarct treatment with G-CSF and EPO-liposome with SLX markedly reduced the infarct size via EPCs mobilization and upregulation of p-Akt and p-ERK in the ischemic myocardium. This combination therapy may provide a new strategy for the treatment of myocardial infarction.

DNA aptamer blockade of Sialyl LewisX inhibits growth and metastasis of HepG2 hepatocellular carcinoma cells in vitro

Objective To study the effect of the Sialyl LewisX (SLeX) binding DNA aptamer on adhesion and metastasis of hepatocellular carcinoma (HCC) HepG2 cells in vitro,and to select possible anti-adhesion drugs for HCC metastasis.Methods The adhesive potential of HepG2 cells to E-selectin and alterations of the adhesive potential after HepG2 cells were sealed by the SLeX binding DNA aptamer were studied by means of the adhesive test.The effect of DNA aptamer on the metastasis of HepG2 cells was evaluated by tumor cell migration invasion assays.Results Most of the interactions between E-selectin and the SLeX on the HepG2 cells are specific and could be inhibited by the SLeX binding DNA aptamer.Cell number on the down-side of transwell membrane was significantly less in cells treated with 5,10,20 nmol DNA aptamer than in the control cells ( 159.00 ±3.27,142.00±5.50 and 115.00 ±5.07,vs.178.00 ±4.64,P ＜0.01 ).Conclusion These results indicate that the SLeX binding DNA aptamer can effectively and significantly inhibit the in vitroadhesion,migration,and invasion of HepG2 cells,and also suggest that the SLeX binding DNA aptamer may have potential as an agent to inhibit cancer growth and metastasis. Key words: Carcinoma,hepatocellular; Sialyl LweisX; DNA aptamer; Invasion

Interacting Lewis-X Carbohydrates in Condensed Phase: A First-Principles Molecular Dynamics Study

We performed first-principles molecular dynamics calculations at finite temperature, to study the interacting conformations of Lewis-X (LeX) trisaccharides in the crystalline phase. The calculated cell parameters and detailed atomic structure of the LeX molecule compare well to the experimental data obtained by X-ray diffraction. We identify and characterize the hydrogen-bond network, responsible for the mutual interaction of the LeX pairs, whereas we find the intramolecular conformation and stability to be mainly assured by dispersion forces. The relative contributions to the crystallization energy of the hydrogen bonds and of the dispersion forces are defined and quantified. From this study, candidate configurations for the fully hydrated, in vivo structures of homotypic LeX-LeX interactions at cell surfaces can be proposed. We discuss how these configurations could also be relevant for the adhesion and self-assembly of nanostructures.

196 Injectable matrix activates myogenic differentiation in embryonic stem cells and regenerates muscle in ischemic tissue

Previously, we demonstrated that an injectable collagen-based matrix containing sialyl LewisX (sLeX) applied to ischemic tissue amplified the endogenous progenitor cell response and increased vascular regeneration and perfusion, with evidence of enhanced myogenesis. In this study, we further explored the myogenic effects of this material.

Development and validation of a HPAE-PAD method for the quantification of CGP69669A, a sialyl Lewisx mimetic, in skin permeation studies

A simple, rapid, precise and specific isocratic HPAE-PAD method for quantification of CGP69669A was developed and validated. CGP69669A is a glycomimetic of sialyl Lewis(x) and an antagonist of E-selectin with potential application in the treatment of inflammatory skin disease. Quantification was performed using a Dionex CarboPac(TM) PA-200 anion-exchange column (3 × 250 mm) with 100 mm NaOH solution as mobile phase, a flow rate of 0.50 mL/min and an injection volume of 10 μL. A quadruple potential waveform was used to detect the carbohydrate (+0.1 V from 0.00 to 0.40 s, -2.0 V from 0.41 to 0.42 s, +0.6 V at 0.43 s and -0.1 V from 0.44 to 0.50 s with current integrated between 0.20 and 0.40 s for detection) and rafinose was employed as an internal standard. The optimized conditions enabled rapid elution of CGP69669A (at 3.0 min) without interference from solvent peaks or substances present in the skin. The method showed good intra- and inter-day precision and accuracy and the response was linear from 1.0 to 25 µg/mL. This is the first validated direct method for the quantification of CGP69669A. It will now be employed in studies investigating the topical and transdermal delivery of CGP69669A in vitro and in vivo and it should also be of use for other applications of this molecule.

Sialyl Lewisx-dependent binding of human monocyte-derived dendritic cells to selectins

The limited efficacy of monocyte-derived dendritic cell (mo-DC)-based vaccines is primarily attributed to the reduced mo-DC migratory capacity. One undefined aspect is the initial binding of mo-DCs to endothelial cells and vascular selectins. In this study, we investigated the role and modulation of the selectin binding determinant sialyl Lewisx (sLex) in selectin-dependent mo-DC binding. Our data reveal that sLex is required for maximal binding of mo-DCs to tumor necrosis factor (TNF)-α-activated endothelial cells under static conditions, as evidenced by the use of sialidase. Sialidase treatment also abrogated mo-DC cell tethering to immobilized, purified P-, L-, or E-selectin under flow. The requirement of sLex-dependent binding of mo-DC to selectins was further substantiated by using sLex free sugar and anti-sLex antibody, which significantly suppressed mo-DC-selectin binding. P-selectin glycoprotein ligand-1 is required for mo-DC binding to both P- and L-selectin, but it is dispensable for E-selectin recognition. Interestingly, the extent of mo-DC tethering was maximal on P-selectin, followed by E- and L- selectin. Accordingly, L-selectin mediated faster mo-DC rolling than E- or P-selectin. Interferon (IFN)-γ induces a significant increase in mo-DC surface sLex expression, which is probably due to the enhanced synthesis of C2GnT-I. These findings may contribute to improving mo-DC-based vaccination protocols.

Glycophenotype of prostatic carcinomas.

The factors that affect the progression of prostatic carcinoma are poorly understood, but it is known that carbohydrate antigens on the tumour cell surface play a role in the transforming and metastatic processes. The present report aimed to perform a comparative, lectin-histochemical study of benign and carcinomatous prostates, using a battery of lectins, in combination with monoclonal antibodies against Lewis antigens, and a semi quantitative study, to investigate the changes in glycosylation patterns that occur in prostatic carcinoma. Blocks from 27 necropsy cases of prostatic carcinoma were sectioned and stained with H+E, fifteen biotinylated lectins chosen to probe for a wide range of oligosaccharide sequences within several categories of glycoprotein glycans, using a lectin-biotin avidin-peroxidase method, and monoclonal antibodies against Lewisa, sialyl Lewisa and sialyl Lewisx antigens. The glycophenotype of prostatic carcinoma differed from that of the noncancerous prostate in revealing more intense staining with the following lectins (AAA, UEA-1, DBA, WFA, VVA, HPA, BSA-1B4, MPA, ECA, AHA, and CTA), while the binding patterns of (GNA and NPA) were almost similar in both prostatic carcinoma and the noncancerous prostate. Lewis antigens are found to be expressed in prostatic carcinomas but not in the noncancerous prostate. The observations of this study suggest that the gylcophenotype of transformed prostatic cells was modified. It showed a moderate increase in, and changing patterns of, fucosylation and galactosylation, increased branching of side chains and sharp rise in 2 deoxy, 2 acetamido galactosylation and masking process by sialylation, especially by α2-3 and α2-6 linkages. All these changes in the glycosylation pattern of the transformed prostatic cells were observed on O-glycans, no changes were observed on N-glycans.

Invasive behavior of ulcerative colitis-associated carcinoma is related to reduced expression of CD44 extracellular domain: comparison with sporadic colon carcinoma

BackgroundTo elucidate relations of invasion of ulcerative colitis (UC)-associated carcinoma with its prognosis, the characteristics of invasive fronts were analyzed in comparison with sporadic colonic carcinomas.MethodsPrognoses of 15 cases of UC-associated colonic carcinoma were compared with those of sporadic colon carcinoma cases, after which 75 cases of sporadic invasive adenocarcinoma were collected. Tumor budding was examined histologically at invasive fronts using immunohistochemistry (IHC) of pancytokeratin. Expressions of beta-catenin with mutation analysis, CD44 extracellular domain, Zo-1, occludin, matrix matalloproteinase-7, laminin-5γ2, and sialyl Lewis X (LeX) were immunohistochemically evaluated.ResultsUC-associated carcinoma showed worse prognosis than sporadic colon carcinoma in all the cases, and exhibited a tendency to become more poorly differentiated when carcinoma invaded the submucosa or deeper layers than sporadic carcinoma. When the lesions were compared with sporadic carcinomas considering differentiation grade, reduced expression of CD44 extracellular domain in UC-associated carcinoma was apparent. Laminin-5γ2 and sialyl-LeX expression showed a lower tendency in UC-associated carcinomas than in their sporadic counterparts. There were no differences in the numbers of tumor budding foci between the two lesion types, with no apparent relation to nuclear beta-catenin levels in IHC.ConclusionsUC-associated carcinoma showed poorer differentiation when the carcinoma invaded submucosa or deeper parts, which may influence the poorer prognosis. The invasive behavior of UC-associated carcinoma is more associated with CD44 cleavage than with basement membrane disruption or sialyl-Lewis-antigen alteration.

Adhesive behavior of dual-targeted microbubbles carrying both Sialyl Lewisx and anti-ICAM-1 monoclonal antibody in vitro

Objective To assess the adhesive behavior of dual-targeted microbubbles carrying both Sialyl Lewisx and anti-ICAM-1 monoclonal antibodies in vitro. Methods Selectin-targeted (with Sialyl Lewisx) microbubbles (MB-S),ICAM-1-targeted (with anti-ICAM-1 monoclonal antibodies) microbubbles (MB-Ⅰ),and dual-targeted (with both ligands) microbubbles(MB-D) were prepared by attaching the ligands to the biotinylated lipid-microbubbles via multi-step avidin biotin bridging chemistry. A parallel plate flow chamber combined with a novel automated tracking algorithm,were used to analyze the transient velocities,rolling and firmly adherent numbers of microbubbles at various shear stress (0. 6,2.0 and 4.0 dyn/cm2)over 6 min. Microbubbles detachments were tested by ramping up the shear stress at 30 s intervals. Results At 0.6 dyn/cm2 shear stress, the rolling numbers of MB-S and MB-D were remarkably more than that of MB-I( P＜0.05), while at 2.0 and 4.0 dyn/cm2 MB-S performed higher rolling efficiency as compared with either MB-I and MB-D ( P＜ 0.05). In all flow conditions, the adhesive numbers of MB-D to the targets were obviously greater than those of MB-S and MB-I ( P＜ 0.05). Half-maximal detachment decreased gradually in MB-I, MB-D and MB-S by turns ( P＜ 0.05). Conclusions MB-I, MB-S and MB-D have different adhesive behaviors. MB-I exhibites primarily firm adhesion with low rolling efficiency, while MB-S reveales unstable or transient adhesion with high rolling efficiency,and MB-D exhibites firm adhesion with high rolling efficiency. MB-D may be suitable for molecular imaging in high-flow vessels. Key words: Microbubbles; Gene targeting; Parallel plate flow chamber; Adhesive behavior

In vivo targeted molecular imaging for activated platelets by mri using USPIO-fucoidan in rat abdominal aortic aneuryms model

Background P-selectin, expressed on the activated platelets and endothelial cells, is one of the key adhesion molecules that regulate leukocyte trafficking in atherosclerosis. The natural and canonical ligand of selectins is a specific tetrasaccharide structure known as 6-sulfo sialyl Lewis X (SLeX). Fucoidans refer to a type of polysaccharide that contains L-fucose and sulfate ester groups, mainly derived from brown seaweed, and binds strongly to P-selectin. We assessed the hypothesis that a new MRI contrast agent made of fucoidan conjugated with ultra small paramagnetic iron oxide (USPIO) can demonstrate the presence of activated platelets in thrombus of an expanding aneurysm.

Role of c-Abl in L-selectin shedding from the neutrophil surface

L-selectin is a key molecule that participates in neutrophil tethering and subsequent rolling. It is cleaved from the surface of neutrophils activated in the presence of lipopolysaccharides, N-formyl-methionine-leucine-phenylalanine (fMLP), or Interleukin-8 (IL-8). We previously showed that L-selectin is also shed from the neutrophil surface during rolling on sialyl Lewis-x coated surfaces in a force-, ADAM-17 sheddase-, and p38 MAP kinase-dependent manner under flow. c-Abl tyrosine kinase is phosphorylated when L-selectin on the surface of neutrophils is cross-linked with anti-L-selectin antibodies. Here, we study the effect of c-Abl inhibition on L-selectin shedding from primary human neutrophils in static conditions following exposure to fMLP, IL-8, and hypotonic buffer and under flow through sialyl Lewis-x coated microtubes. Results indicate that c-Abl inhibition by STI571 significantly affects neutrophil adhesion via L-selectin, by decreasing the average rolling velocity and increasing the flux of rolling cells. The change in surface receptor expression was verified by flow cytometry. Interestingly, other forms of L-selectin shedding induced by fMLP, IL-8 or osmotic swelling were unaffected by STI571 treatment. These findings implicate the c-Abl signaling molecule in regulating L-selectin mechanical shedding in response to shear stress, setting this type of signaling apart from those triggered by the presence of a hypotonic environment, fMLP, or IL-8. This study sheds light on the role of c-Abl in neutrophil adhesion not previously reported in the literature.

Cloning and characterization of a viral α2–3-sialyltransferase (vST3Gal-I) for the synthesis of sialyl Lewisx

Sialyl Lewis(x) (SLe(x), Siaα2-3Galβ1-4(Fucα1-3)GlcNAcβOR) is an important sialic acid-containing carbohydrate epitope involved in many biological processes such as inflammation and cancer metastasis. In the biosynthetic process of SLe(x), α2-3-sialyltransferase-catalyzed sialylation generally proceeds prior to α1-3-fucosyltransferase-catalyzed fucosylation. For the chemoenzymatic synthesis of SLe(x) containing different sialic acid forms, however, it would be more efficient if diverse sialic acid forms are transferred in the last step to the fucosylated substrate Lewis(x) (Le(x)). An α2-3-sialyltransferase obtained from myxoma virus-infected European rabbit kidney RK13 cells (viral α2-3-sialyltransferase (vST3Gal-I)) was reported to be able to tolerate fucosylated substrate Le(x). Nevertheless, the substrate specificity of the enzyme was only determined using partially purified protein from extracts of cells infected with myxoma virus. Herein we demonstrate that a previously reported multifunctional bacterial enzyme Pasteurella multocida sialyltransferase 1 (PmST1) can also use Le(x) as an acceptor substrate, although at a much lower efficiency compared to nonfucosylated acceptor. In addition, N-terminal 30-amino-acid truncated vST3Gal-I has been successfully cloned and expressed in Escherichia coli Origami™ B(DE3) cells as a fusion protein with an N-terminal maltose binding protein (MBP) and a C-terminal His(6)-tag (MBP-Δ30vST3Gal-I-His(6)). The viral protein has been purified to homogeneity and characterized biochemically. The enzyme is active in a broad pH range varying from 5.0 to 9.0. It does not require a divalent metal for its α2-3-sialyltransferase activity. It has been used in one-pot multienzyme sialylation of Le(x) for the synthesis of SLe(x) containing different sialic acid forms with good yields.

Development of an active targeting liposome encapsulated with high-density colloidal gold for transmission electron microscopy

Active targeting of the liposome is an attractive strategy for drug delivery and in vivo bio-imaging. We previously reported the specific accumulation of Sialyl Lewis X (SLX) liposome to inflamed tissue in arthritic model mice or tumor-bearing mice. SLX-liposome encapsulation with fluorescent substances allows for the visualization of these liposomes by the time-dependent transvascular accumulation of fluorescent signals in the histological sections. In the present study, we developed a new SLX-liposome encapsulated with colloidal gold for transmission electron microscopic observation. We herein describe the characterization of the colloidal gold-loaded SLX-liposomes and demonstrate its specific targeting to the endothelial cells of tumor blood vessels in tumor-bearing mice.

Biosynthesis and distribution of Lewis X- and Lewis Y-containing glycoproteins in the murine male reproductive system

Although Lewis X (LeX) and Lewis Y (LeY) antigens were thought to play important roles in fertility, fucosyltransferase (Fut)-deficient (Fut1, Fut2 and Fut4) mice which lack LeX or LeY antigen are still fertile. In the present study, the Fut-deficient and wild-type mice were used to measure the expression of Fut mRNA along the mouse male reproductive tract and determine the role of each Fut in the biosynthesis of LeX/LeY antigens, which are conjugated to glycoproteins in the male reproductive system. LeX/LeY-containing glycoproteins were detected in the epididymis, vas deferens, seminal vesicle and coagulating gland, but not in the testis. We demonstrate that the synthesis of LeY-containing glycoproteins in the epididymis and vas deferens is catalyzed by Fut1 and Fut4. In the seminal vesicle and the coagulating gland, they are mainly synthesized by Fut2 and an α-(1,3)-Fut, but not Fut4. The synthesis of LeX-containing glycoproteins in the middle caput epididymis is catalyzed by Fut4 and by Fut4 and Fut2 in the seminal vesicle. We provide evidence that LeX is synthesized in the coagulating gland by Fut9. We found that the lack of activity by one Fut does not completely inhibit LeX/LeY antigen expression in the male reproductive tract. This redundancy may help to explain why in vivo studies with Fut-deficient mice do not support the presumption that LeX/LeY antigens play important roles in male fertility. We provide details regarding the phenotypes of established Fut-deficient mice and lay the foundation for elucidating the functions of LeX/LeY antigens in other aspects of the male reproductive system.

Optimization of ultrasound contrast agents with computational models to improve selection of ligands and binding strength

Diagnosis of cardiovascular disease is currently limited by the testing modality. Serum tests for biomarkers can provide quantification of severity but lack the ability to localize the source of the cardiovascular disease, while imaging technology such as angiography and ultrasound can only determine areas of reduced flow but not the severity of tissue ischemia. Targeted imaging with ultrasound contrast agents offers the ability to locally image as well as determine the degree of ischemia by utilizing agents that will cause the contrast agent to home to the affected tissue. Ultrasound molecular imaging via targeted microbubbles (MB) is currently limited by its sensitivity to molecular markers of disease relative to other techniques (e.g., radiolabeling). We hypothesize that computational modeling may provide a useful first approach to maximize microbubble binding by defining key parameters governing adhesion. Adhesive dynamics (AD) was used to simulate the fluid dynamic and stochastic molecular binding of microbubbles to inflamed endothelial cells. Sialyl Lewis(X) (sLe(x)), P-selectin aptamer (PSA), and ICAM-1 antibody (abICAM) were modeled as the targeting receptors on the microbubble surface in both single- and dual-targeted arrangements. Microbubble properties (radius [R(c)], kinetics [k(f), k(r)], and densities of targeting receptors) and the physical environment (shear rate and target ligand densities) were modeled. The kinetics for sLe(x) and PSA were measured with surface plasmon resonance. R(c), shear rate, and densities of sLe(x), PSA, or abICAM were varied independently to assess model sensitivity. Firm adhesion was defined as MB velocity <2% of the free stream velocity. AD simulations revealed an optimal microbubble radius of 1-2 µm and thresholds for kf(in) ( >10(2) s(-1)) and kr(o) (<10(-3) s(-1)) for firm adhesion in a multi-targeted system. State diagrams for multi-targeted microbubbles suggest sLe(x) and abICAM microbubbles may require 10-fold more ligand to achieve firm adhesion at higher shear rates than sLe(x) and PSA microbubbles. The AD model gives useful insight into the key parameters for stable microbubble binding, and may allow flexible, prospective design, and optimization of microbubbles to enhance clinical translation of ultrasound molecular imaging.

Synthesis of Glycopolymers for Microarray Applications via Ligation of Reducing Sugars to a Poly(acryloyl hydrazide) Scaffold

In this paper, we report on a general synthetic strategy for the assembly of glycopolymers that capitalizes on the intrinsic reactivity of reducing glycans toward hydrazides to form stable cyclic N-glycosides. We developed a poly(acryloyl hydrazide) (PAH) scaffold to which we conjugated a variety of reducing glycans ranging in structure from simple mono- and disaccharides to considerably more complex human milk and blood oligosaccharides. The conjugation proceeds under mild conditions with excellent ligation efficiencies and in a stereoselective manner, providing glycopolymers with pendant glycans accommodated mostly in their cyclic β-glycosidic form. Utilizing a biotin-terminated PAH scaffold prepared via RAFT polymerization, we quickly assembled a panel of glycopolymers that we microarrayed on streptavidin-coated glass. We then demonstrated that in these microarrays, the glycopolymer ligands bind lectins according to the structures of their pendant glycans. Importantly, glycopolymers containing biologically relevant branched oligosaccharides, such as sialyl Lewisx, as well as sulfated glycosaminoglycan-like epitopes can be readily prepared using our methodology.

Epigenetic Silencing of the Sulfate Transporter Gene DTDST Induces Sialyl Lewisx Expression and Accelerates Proliferation of Colon Cancer Cells

Colon cancer cells express the carbohydrate determinant sialyl Lewis(x), while they exhibit markedly decreased the expression of its sulfated derivative, sialyl 6-sulfo Lewis(x). In contrast, normal colonic epithelial cells strongly express sialyl 6-sulfo Lewis(x), but they virtually do not express sialyl Lewis(x). Impaired sulfation was therefore suggested to occur during the course of malignant transformation of colonic epithelial cells and was assumed to be responsible for the increased sialyl Lewis(x) expression in cancers. To elucidate the molecular biological background of the impaired sulfation in cancers, we studied the expression levels of mRNA for 6-O-sulfotransferase isoenzymes, PAPS synthases and transporters, and a cell membrane sulfate transporter, DTDST, in cancer tissues. The most striking decrease in cancer cells compared with nonmalignant epithelial cells was noted in the transcription of the DTDST gene (P = 0.0000014; n = 20). Most cultured colon cancer cells had a diminished DTDST transcription, which was restored when cultured with histone deacetylase inhibitors. Suppression of DTDST transcription under the control of a tet-off inducible promoter resulted in increased sialyl Lewis(x) expression and reduced sialyl 6-sulfo Lewis(x) expression. Unexpectedly, the growth rate of the cancer cells was markedly enhanced when transcription of DTDST was suppressed. These results show that the decrease in the transcription of the sulfate transporter gene is the major cause of decreased expression of sialyl 6-sulfo Lewis(x) and increased expression of sialyl Lewis(x) in colon cancers. The results also suggest that the diminished DTDST expression is closely related to enhanced proliferation of cancer cells.

Abstract LB-216: CSLEX (sialyl LewisX) is an important and useful tumor marker in monitoring breast cancer patients

Abstract Background: CSLEX-1 is one of the monoclonal antibodies for which the structure of the antigen is known. This antigen is called CSLEX (Sialyl Le X). It has been reported that CSLEX may be more useful than CEA in combination with CA15-3 in monitoring breast cancer patients. Methods: We measured and evaluated the serum levels of some tumor markers, including CSLEX, in a total of 482 breast cancer patients with or without metastasis who consecutively visited the outpatient clinic of the Division of Breast and Endocrine Surgery, Shinshu University Hospital, between April 2007 and September 2007. Results: All serum levels of tumor markers in a single measurement correlated significantly with the status of metastasis (P &amp;lt; 0.01). All combinations of combined measurements, such as CEA and CA15-3, CEA and NCC-ST439, CEA and CSLEX, CA15-3 and NCC-ST439, and CA15-3 and CSLEX, also correlated significantly with the status of metastasis (P &amp;lt; 0.01). The sensitivity, specificity, positive-predictive value, negative-predictive value and accuracy were almost the same between CSLEX and NCC-ST439, which belongs to the type II carbohydrate antigens. The cut-off values of serum CSLEX and NCC-ST439 were 38.8 ± 52.7 U/ml and 22.1 ± 27.8 U/ml, respectively (P = 0.16). Conclusions: These data support there are no differences between CSLEX and NCC-ST439 as diagnostic parameters in single or combined measurements. The cut-off value of serum CSLEX tended to be higher compared with that of NCC-ST439.Patient characteristics Without MetastasisWith MetastasisP-valueNumber363119 Mean Age ± SD (years)56.5 ± 11.758.9 ± 12.60.06CEA2.5 ± 6.311.9 ±34.50.008CA15-311.9 ± 11.166.4 ± 162.50.001BCA22561.0 ±51.5139.4 ± 228.60.001NCCST-4392.0 ± 2.234.2 ± 108.20.003CSLEX3.4 ± 3.765.6 ± 223.10.005Diagnostic parameters for the detection of breast cancer metastasis using tumor markers (%) SensitivitySpecificityPPVNPVAccuracySingle measurement CEA3095688179CA15-33498828282BCA2251698707878NCC-ST4392497727979CSLEX2397697978Combined measurement CEAandCA15-34893698482CEA and NCC-ST4394392648380CEA and CSLEX4392658380CA15-3 and NCC-ST4394595758483CA15-3 and CSLEX4495738482 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-216.

Engineered mesenchymal stem cells with self-assembled vesicles for systemic cell targeting

Cell therapy has the potential to impact the quality of life of suffering patients. Systemic infusion is a convenient method of cell delivery; however, the efficiency of engraftment presents a major challenge. It has been shown that modification of the cell surface with adhesion ligands is a viable approach to improve cell homing, yet current methods including genetic modification suffer potential safety concerns, are practically complex and are unable to accommodate a wide variety of homing ligands or are not amendable to multiple cell types. We report herein a facile and generic approach to transiently engineer the cell surface using lipid vesicles to present biomolecular ligands that promote cell rolling, one of the first steps in the homing process. Specifically, we demonstrated that lipid vesicles rapidly fuse with the cell membrane to introduce biotin moieties on the cell surface that can subsequently conjugate streptavidin and potentially any biotinylated homing ligand. Given that cell rolling is a pre-requisite to firm adhesion for systemic cell homing, we examined the potential of immobilizing sialyl Lewis X (SLeX) on mesenchymal stem cells (MSCs) to induce cell rolling on a P-selectin surface, under dynamic flow conditions. MSCs modified with SLeX exhibit significantly improved rolling interactions with a velocity of 8 μm/s as compared to 61 μm/s for unmodified MSCs at a shear stress of 0.5 dyn/cm 2. The cell surface modification does not impact the phenotype of the MSCs including their viability and multi-lineage differentiation potential. These results show that the transitory modification of cell surfaces with lipid vesicles can be used to efficiently immobilize adhesion ligands and potentially target systemically administered cells to the site of inflammation.