lewis-x-antigen and Neoplasms

Tumor-associated neutrophils (TAN) have been reported in a variety of malignancies. We conducted an up-to-date meta-analysis to evaluate the prognostic role of TAN in cancer.. Pubmed, Embase and web of science databases were searched for studies published up to April 2013. Pooled hazard ratios (HRs) and their corresponding 95% confidence intervals (CIs) were calculated. The impact of neutrophils localization and primary antibody were also assessed.. A total of 3946 patients with various solid tumors from 20 studies were included. High density of intratumoral neutrophils were independently associated with unfavorable survival; the pooled HRs were 1.68 (95%CI: 1.36-2.07, I2 = 55.8%, p<0.001) for recurrence-free survival (RFS)/disease-free survival (DFS), 3.36 (95%CI: 2.08-5.42, I2 = 0%, p<0.001) for cancer-specific survival (CSS) and 1.66 (95%CI: 1.37-2.01, I2 = 70.5%, p<0.001) for overall survival (OS). Peritumoral and stromal neutrophils were not statistically significantly associated with survival. When grouped by primary antibody, the pooled HRs were 1.80 (95%CI: 1.47-2.22, I2 = 67.7%, p<0.001) for CD66b, and 1.44 (95%CI: 0.90-2.30, I2 = 45.9%, p = 0.125) for CD15, suggesting that CD66b positive TAN might have a better prognostic value than CD15.. High levels of intratumoral neutrophils are associated with unfavorable recurrence-free, cancer-specific and overall survival.

Topics: Antigens, CD; Biomarkers, Tumor; Cell Adhesion Molecules; GPI-Linked Proteins; Humans; Lewis X Antigen; Neoplasms; Neutrophils; Predictive Value of Tests; Prognosis

Topics: Antigens, Tumor-Associated, Carbohydrate; Fucosyltransferases; Humans; Lewis X Antigen; Neoplasms; Neoplastic Stem Cells; Stage-Specific Embryonic Antigens

Sialyl Lewis X (sLeX) antigen, Neu5Acα2,3Galβ1,4(Fucα1,3)GlcNAc-R, is expressed on the glycoproteins in sera or the surface of the cells and the expression of sLeX is enhanced in various conditions such as the inflammation and cancer. SLeX in the serum is utilized as a tumor marker. To clarify the roles of sLeX on secreted glycoproteins in vivo, we investigate the regulation of natural killer (NK) cell-dependent cytotoxicity through sLeX. NK cells express many receptors to kill the target cells such as cancerous cells and non-self, and their protein ligands have been elucidated. Of the killer lectin-like receptors (KLRs) on NK cells, several have been reported to recognize glycans. Using recombinant extracellular domains of KLRs (rKLRs: rNKG2A, C, D and rCD94), we evaluated their glycan ligand specificity and binding affinities using EIA methods. We clarified that all of these rKLRs can bind to high sLeX-expressing glycoprotein and heparin, heparan sulfate and highly sulfated polysaccharides and that glycan binding sites on NKG2D are mostly overlapped with those of protein ligands. In this review, we show the recent findings concerning the glycan ligands of these KLRs.

Topics: Animals; Biomarkers; Cytotoxicity, Immunologic; Glycoproteins; Heparin; Heparitin Sulfate; Humans; Inflammation; Killer Cells, Natural; Lewis X Antigen; Ligands; Mice; Neoplasms; Polysaccharides; Protein Binding; Receptors, NK Cell Lectin-Like; Sialyl Lewis X Antigen

Tumor cells exhibit striking changes in cell surface glycosylation as a consequence of dysregulated glycosyltransferases and glycosidases. In particular, an increase in the expression of certain sialylated glycans is a prominent feature of many transformed cells. Altered sialylation has long been associated with metastatic cell behaviors including invasion and enhanced cell survival; however, there is limited information regarding the molecular details of how distinct sialylated structures or sialylated carrier proteins regulate cell signaling to control responses such as adhesion/migration or resistance to specific apoptotic pathways. The goal of this review is to highlight selected examples of sialylated glycans for which there is some knowledge of molecular mechanisms linking aberrant sialylation to critical processes involved in metastasis.

Topics: Animals; Antigens, Tumor-Associated, Carbohydrate; Cell Movement; Glycosylation; Humans; Integrins; Lewis X Antigen; N-Acetylneuraminic Acid; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Phenotype; Polysaccharides; Sialyl Lewis X Antigen

Tumor-associated carbohydrate antigens (TACA) result from the aberrant glycosylation that is seen with transformation to a tumor cell. The carbohydrate antigens that have been found to be tumor-associated include the mucin related Tn, Sialyl Tn, and Thomsen-Friedenreich antigens, the blood group Lewis related Lewis(Y), Sialyl Lewis(X) and Sialyl Lewis(A), and Lewis(X) (also known as stage-specific embryonic antigen-1, SSEA-1), the glycosphingolipids Globo H and stage-specific embryonic antigen-3 (SSEA-3), the sialic acid containing glycosphingolipids, the gangliosides GD2, GD3, GM2, fucosyl GM1, and Neu5GcGM3, and polysialic acid. Recent developments have furthered our understanding of the T-independent type II response that is seen in response to carbohydrate antigens. The selection of a vaccine target antigen is based on not only the presence of the antigen in a variety of tumor tissues but also on the role this antigen plays in tumor growth and metastasis. These roles for TACAs are being elucidated. Newly acquired knowledge in understanding the T-independent immune response and in understanding the key roles that carbohydrates play in metastasis are being applied in attempts to develop an effective vaccine response to TACAs. The role of each of the above mentioned carbohydrate antigens in cancer growth and metastasis and vaccine attempts using these antigens will be described.

Topics: Antigens, Tumor-Associated, Carbohydrate; B-Lymphocytes; Cancer Vaccines; Dendritic Cells; Epitopes; Gangliosides; Humans; Lewis X Antigen; Mucins; Neoplasms; Stage-Specific Embryonic Antigens

The carbohydrate determinants, sialyl Lewis A and sialyl Lewis X, which are frequently expressed on human cancer cells, serve as ligands for a cell adhesion molecule of the selectin family, E-selectin, which is expressed on vascular endothelial cells. These carbohydrate determinants are involved in the adhesion of cancer cells to vascular endothelium and thus contribute to hematogenous metastasis of cancer. The initial adhesion mediated by these molecules triggers activation of integrin molecules through the action of several cytokines and leads to the extravasation of cancer cells. Cancer cells also produce humoral factors that facilitate E-selectin expression on endothelial cells. The degree of expression of the carbohydrate ligands at the surface of cancer cells is well correlated with the frequency of hematogenous metastasis and prognostic outcome of patients with cancers. The alteration of glycosyltransferase activities that leads to the enhanced expression of these carbohydrate ligands on cancer cell surface are currently being investigated.

Topics: Animals; Biomarkers, Tumor; Cell Adhesion; E-Selectin; Glycosyltransferases; Humans; Lewis X Antigen; Models, Biological; Neoplasm Metastasis; Neoplasms; Oligosaccharides; P-Selectin; Prognosis; Sialyl Lewis X Antigen

Some cell surface carbohydrate determinants such as sialyl Lewis A and sialyl Lewis X serve as ligands for the cell adhesion molecules called selectins. These carbohydrate determinants are involved in the adhesion of cancer cells to vascular endothelial cells during the course of hematogenous metastasis of cancer. This review considers primary adhesion of cancer cells to endothelial cells mediated by carbohydrate-selectin interaction followed by the secondary involvement of the classical cell adhesion molecules called integrins in extravasation of cancer cells. Alteration in the glycosyltransferase activities that induce the accelerated synthesis of abnormal carbohydrate determinants on cancer cells is also discussed.

Topics: Antigens, Tumor-Associated, Carbohydrate; Carbohydrate Sequence; Cell Adhesion Molecules; Endothelium; Glycosyltransferases; Humans; Lewis X Antigen; Molecular Sequence Data; Neoplasm Metastasis; Neoplasms

Since S. Hakomori's pioneer work, many has studied on carbohydrate chains on cancer. Many monoclonal antibodies are developed and some of them are already evaluated as tumor markers. They are used routinely in daily clinical practice for immunohistochemistry and immunodetection of circulating antigens in patients' sera. However, most of them are not specific to cancers, are negative in early cancers, they are usually elevated in sera of patients with advanced or recurrent cancers and used in monitoring patients. In the major organic sites, positive rates of some serum markers are shown in a table and discussed their usefulness and limitations. Problems of similarities and discrepancies in many commercial systems of sugar chain tumor markers are discussed. Recent topic in the function of sialylated carbohydrate antigens is their strong affinities with cell-adhesion molecules, for that reason, sialyl Lewis A and sialyl Lewis X are thought to be responsible for the distant metastasis of those cancers that produce them.

Topics: Antigens, Tumor-Associated, Carbohydrate; Carbohydrate Sequence; Humans; Immunohistochemistry; Lewis X Antigen; Molecular Sequence Data; Neoplasms

Tumour development is usually associated with changes in cell surface carbohydrates. These are often divided into changes related to terminal carbohydrate structures, which include incomplete synthesis and modification of normally existing carbohydrates, and changes in the carbohydrate core structure. The latter includes chain elongation of both glycolipids and proteins, increased branching of carbohydrates in N-linked glycoproteins, and blocked synthesis of carbohydrates in O-linked mucin-like glycoproteins. In mature organisms, expression of distinct carbohydrates is restricted to specific cell types; within a given tissue, variation in expression may be related to cell maturation. Tumour-associated carbohydrate structures often reflect a certain stage of cellular development; most of these moieties are structures normally found in other adult or embryonic tissues. There is no unique tumour carbohydrate structure, since certain structures which are tumour-related in one organ may be normal constituents of other tissues. Tumour-associated carbohydrate changes have been used in the diagnosis of human cancers. Recently, however, it has been demonstrated that the expression of some carbohydrate structures is associated with prognosis. Tn, sialyl-Tn, and T are cell membrane-bound mucin-like carbohydrate structures that may be expressed in tumours due to blocked synthesis of the core carbohydrate chain of mucin-like structures. Their expression is strongly associated with prognosis in certain tumours, but the biological relationship between their expression and tumour progression is at present unknown. The blood group-related carbohydrate structures Le(x), sialyl-Le(x), ABH, and Le(y) are examples of terminal carbohydrate structures which are related to tumour prognosis. These structures are of increasing interest since they may function as adhesion molecules; adhesion of tumour cells to endothelial cells of blood vessels may be mediated by an interaction between sialosyl-Le(x) and E-selectin and studies indicate that Le(y) is related to cell motility. These findings are now the basis for tumour therapeutic experiments.

Topics: ABO Blood-Group System; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Disease Progression; Humans; Lewis X Antigen; Mucins; Neoplasms; Prognosis; Survival Rate

Sialosyl-Le(a) and sialosyl-Le(x) are tumor-associated carbohydrate antigens present in different types of human tumors. They are commonly found on the cell surface of a variety of adenocarcinomas such as lung cancer, gastric cancer, pancreatic cancer and colorectal cancer, and in serum of cancer patients. Both antigens have been proposed as important diagnostic markers and they are used in detecting and monitoring of these diseases. Recently, it has been shown that sialosyl-Le(a) and sialosyl-Le(x) carbohydrate structures are ligands for selectins, newly described family of adhesion molecules. Selectins function as lymphocyte-homing and leukocyte enrollment receptors, or as activation dependent cell surface receptors of platelets and endothelial cells. Several lines of evidence suggest that sialosyl-Le(a) and sialosyl-Le(x) are responsible for adhesion of human cancer cells to endothelium. It has been shown that E-selectin and P-selectin present on endothelial cells mediate these interactions. The mentioned facts suggest that selectins and their carbohydrate ligands can play an important role in a selective homing of tumor cells during metastasis.

Topics: Animals; Biomarkers, Tumor; CA-19-9 Antigen; Cell Adhesion; Disease Progression; E-Selectin; Endothelium; Gangliosides; Humans; Lewis X Antigen; Neoplasms; P-Selectin

Topics: alpha-Fetoproteins; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Carcinoembryonic Antigen; Genes, DCC; Genes, p53; Genes, ras; Humans; Lewis X Antigen; Neoplasms

Expression of some tumor-associated carbohydrate antigens may define the stage, rate and phenotype of tumor progression and may have prognostic value. Some of these antigens are now recognized as adhesion molecules that define the site of metastasis. Monoclonal antibodies to tumor-associated carbohydrate antigens, or the antigens themselves, may serve not only as classic immunological reagents but also as anti-adhesion reagents for the prevention of tumor progression.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Carbohydrate Sequence; Carbohydrates; Cell Adhesion; Cell Adhesion Molecules; Gene Expression Regulation, Neoplastic; Glycoproteins; Humans; Lewis Blood Group Antigens; Lewis X Antigen; Melanoma, Experimental; Mice; Molecular Sequence Data; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Proteins; Neoplasms; Peptides; Receptors, Immunologic; Receptors, Laminin; Vaccination

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Carbohydrate Sequence; Disaccharides; Glycolipids; Humans; Lewis X Antigen; Molecular Sequence Data; Neoplasms

The high-affinity receptor for IgG, Fc gamma RI, expressed on monocytes and interferon-gamma (IFN-gamma)-stimulated neutrophils, is a trigger molecule for cell-mediated cytotoxicity. We have prepared murine monoclonal antibodies (MoAb 22 and MoAb 32) that bind to Fc gamma RI outside the ligand binding site and thus bind to and trigger cytotoxicity that is not competed by other immunoglobulins. Because of these properties, it seemed that these MoAbs would be very useful for the development of bispecific antibodies (BsAb) for targeting normal cellular immune defense mechanisms as a new form of immunotherapy for treatment of cancer. BsAbs incorporate into a single molecule the binding specifities of two different antibodies, and, thus, can be used to target myeloid cells to tumors, ensure activation of cellular cytotoxic mechanisms, and target cell lysis and/or phagocytosis. BsAbs were prepared using anti-Fc gamma RI MoAb and an anti-myeloid cell MoAb, PM81, reactive with the CD15 antigen, for studies of antibody-dependent cellular cytotoxicity. Conjugates were made by cross-linking sulfhydryl groups of Fab fragments of MoAb 32 or 22 (both IgG1) and sulfhydryl groups added to intact PM81 (an IgM) using N-succinimdyl-acetyl-S-thioacetate (SATA). The resulting product was purified by high-performance size-exclusion chromatography. The ability of the BsAbs to mediate attachment of human monocytes to tumor target cells was confirmed in a microtiter well assay of binding of MTT-labeled U937 cells (a human Fc gamma RI-bearing cell line) to SKBR-3 (PM81-reactive breast carcinoma) target cells. The ability of the BsAbs to mediate killing of HL-60 promyelocytic leukemia cells was studied using a 6-hour Chromium-51 release assay. Effector cells were monocytes obtained by cytopheresis and cultured for 18 hours with IFN-gamma. Monocytes alone caused minimal killing (5-20%), monocytes plus BsAb caused moderate killing (20-50%), and monocytes plus BsAb plus human serum resulted in maximal killing (50-80%). Experiments were performed to test the ability of the BsAb to purge bone marrow of small numbers of leukemia cells using bone marrow mononuclear phagocytes treated for 18 hours with IFN-gamma prior to adding target cells. Without the addition of human serum as a source of complement, a 90% depletion of clonogenic HL-60 cells could be demonstrated. With human complement, up to 95% depletion was seen. Thus, this BsAb possessed the ability to lyse tumor cell targets by two

Topics: Antibodies, Monoclonal; Antibody Specificity; Cytotoxicity Tests, Immunologic; Humans; Immunotherapy; Leukemia, Myeloid, Acute; Lewis X Antigen; Neoplasms; Receptors, IgG; Risk Factors

The glycan moiety Lewis X (LeX) has been implicated in defining progenitor cells as well as playing a role in the progression of solid tumors, including breast cancer. Here, we used the original stage-specific embryonic antigen-1 (SSEA-1) antibody, MC-480, targeting the LeX motif to examine the expression pattern of this marker within the context of a differentiation hierarchy as well as functional properties of breast cancer cells. Immunohistochemical staining revealed the presence of SSEA-1 in a progenitor zone in the normal breast gland. In breast cancer, 81 of 220 carcinomas (37%) were positive for SSEA-1 and a distinct pattern could be correlated to major subtypes. Specifically, estrogen receptor alpha (ERα)-negative tumors showed a higher frequency of SSEA-1 expression compared to ERα-positive tumors, which are generally considered more differentiated (56% vs 29%,

Topics: Estrogen Receptor alpha; Humans; Lewis X Antigen; Neoplasms; Neoplastic Stem Cells; Phenotype

Immunotherapy has been successful in treating many tumour types. The development of additional tumour-antigen binding monoclonal antibodies (mAbs) will help expand the range of immunotherapeutic targets. Lewis histo-blood group and related glycans are overexpressed on many carcinomas, including those of the colon, lung, breast, prostate and ovary, and can therefore be selectively targeted by mAbs. Here we examine the molecular and structural basis for recognition of extended Lea and Lex containing glycans by a chimeric mAb. Both the murine (FG88.2) IgG3 and a chimeric (ch88.2) IgG1 mAb variants showed reactivity to colorectal cancer cells leading to significantly reduced cell viability. We determined the X-ray structure of the unliganded ch88.2 fragment antigen-binding (Fab) containing two Fabs in the unit cell. A combination of molecular docking, glycan grafting and molecular dynamics simulations predicts two distinct subsites for recognition of Lea and Lex trisaccharides. While light chain residues were exclusively used for Lea binding, recognition of Lex involved both light and heavy chain residues. An extended groove is predicted to accommodate the Lea-Lex hexasaccharide with adjoining subsites for each trisaccharide. The molecular and structural details of the ch88.2 mAb presented here provide insight into its cross-reactivity for various Lea and Lex containing glycans. Furthermore, the predicted interactions with extended epitopes likely explains the selectivity of this antibody for targeting Lewis-positive tumours.

Topics: Animals; Antibodies, Monoclonal, Murine-Derived; Antineoplastic Agents, Immunological; Cell Line, Tumor; Humans; Immunoglobulin Fab Fragments; Lewis Blood Group Antigens; Lewis X Antigen; Mice; Molecular Docking Simulation; Neoplasms; Oligosaccharides

Topics: E-Selectin; Glycomics; Glycoproteins; Humans; Immunotherapy, Adoptive; Lewis X Antigen; Ligands; Neoplasms; Receptors, Chimeric Antigen; T-Lymphocytes; Translational Research, Biomedical

Topics: Antineoplastic Agents; Apoptosis; Biosimilar Pharmaceuticals; Cell Line, Tumor; Decorin; Gangliosides; Glycolipids; Glycosyltransferases; Humans; Inhibitory Concentration 50; Lewis X Antigen; MCF-7 Cells; Microscopy, Fluorescence; Neoplasms; Sialyl Lewis X Antigen; Triterpenes

Immunohistochemical detection of CD15 is important in the diagnosis of Hodgkin lymphoma and may play a role in the classification of renal cell tumors (RCTs). In the NordiQC external quality assessment scheme, 4 CD15 tests, each with 71 to 121 participating laboratories, showed that 24% to 50% of the stains were insufficient. This was mainly because of very low primary antibody (Ab) concentration and insufficient heat-induced epitope retrieval, whereas the Ab clone performance seemed of little importance. The purpose of this study was to evaluate the performance of the most commonly used CD15 Abs on the basis of vendor-recommended and in-house optimized protocols. Multitissue blocks with 199 specimens including various malignant lymphomas, RCTs, and normal tissues were stained with 3 different concentrated (conc) CD15 Ab clones Carb-3, MMA, and BY87 according to predetermined in-house optimized protocols on 2 automated immunostaining platforms. Carb-3 and MMA were also applied in ready-to-use (RTU) formats utilized according to vendor protocols. Extension and intensity of stains was determined using the H-score method. Clone Carb-3-conc gave with an in-house optimized protocol the highest H-scores in Hodgkin lymphoma, RCTs, and normal kidney tissue. Clones Carb-3-RTU and MMA-conc gave slightly lower scores, whereas clones MMA-RTU and BY87-conc gave the lowest scores and a large proportion of false-negative reactions. For all concentrated Abs, in-house optimized protocols resulted in increased sensitivity and improved overall staining results compared with vendor-recommended protocols. The importance of Ab selection and protocol optimization in immunohistochemical laboratories is emphasized.

Topics: Antibodies, Monoclonal, Murine-Derived; Antibodies, Neoplasm; Female; Fucosyltransferases; Humans; Immunohistochemistry; Lewis X Antigen; Male; Neoplasms

As we age, the composition of our peripheral leukocytes changes dramatically. Many of these alterations contribute to the general immune dysfunction that burdens the elderly, which in turn, contributes to increased susceptibility to disease. MDSCs represent a heterogeneous population of immunosuppressive leukocytes that are elevated in the peripheral blood of cancer patients. Given the relation between cancer incidence and age, this study examined the frequency of peripheral blood CD33(+)HLA-DR(-) MDSCs across three cohorts: healthy adults (19-59 years old), community-dwelling seniors (61-76 years old), and frail elderly (67-99 years old). This analysis is the first to demonstrate that MDSCs and specifically the CD11b(+)CD15(+) MDSC subset are increased with age. Proinflammatory cytokines that are required for the differentiation of MDSCs (e.g., TNF-α, IL-6, and IL-1β) were similarly found to be increased in the serum of the frail elderly. Furthermore, the proportion of MDSCs and the CD11b(+)CD15(+) subset were found to be elevated significantly in elderly donors with a history of cancer. This age-related elevation in the frequency of MDSCs may contribute to the increased cancer incidence that occurs with age. Further investigation into the functional consequences of elevated MDSCs will provide valuable insight into the progression of age-related pathologies.

Topics: Adult; Aged; Aged, 80 and over; Aging; CD11b Antigen; Cell Differentiation; Female; Fucosyltransferases; Gene Expression; HLA-DR Antigens; Humans; Immunophenotyping; Interleukin-1beta; Interleukin-6; Lewis X Antigen; Male; Middle Aged; Myeloid Cells; Neoplasms; Sialic Acid Binding Ig-like Lectin 3; Tumor Necrosis Factor-alpha

Myeloid-derived suppressor cells (MDSCs) are a subpopulation of myeloid cells with immunosuppressive function whose numbers are increased in conditions such as chronic infection, trauma, and cancer. Unlike murine MDSCs defined as CD11b(+)/Gr-1(+), there are no specific markers for human MDSCs. The goal of this study was to delineate a specific human MDSCs subpopulation in granulocytes from terminal cancer patients and investigate its clinical implications. Here, we show that the CD15(+)/CD16(low) subset was increased in terminal cancer patients compared with healthy donors (P = 0.009). Phorbol 12-myristate 13-acetate-activated granulocytes (CD16(low)/CD66b(++)/CD15(+)) that have a phenotype similar to MDSCs from cancer patients, effectively suppressed both proliferation and cytotoxicity of normal T cells. Among cancer patients, T-cell proliferation was highly suppressed by granulocytes isolated from terminal cancer patients with a high proportion of CD15(+)/CD16(low) cells. Patients with low peripheral blood levels of CD15(+)/CD16(low) cells had significantly longer survival than those with high levels (P = 0.0011). Patients with higher levels of CD15(+)/CD16(low) also tended to have poor performance status (P = 0.05). These data suggest that CD15(+)/CD16(low) granulocytes found in terminal cancer patients may play a role in the progression of cancer by inhibiting tumor immunity.

Topics: Adult; Aged; Female; Granulocytes; Humans; Immune Tolerance; Leukocyte Count; Lewis X Antigen; Male; Middle Aged; Myeloid Cells; Neoplasms; Receptors, IgG; T-Lymphocytes; Young Adult

Myeloid-derived suppressor cells (MDSC) present in the human peripheral blood, represent a heterogeneous population of cells with monocytic and granulocytic features. To provide guidelines for reliable assessments of the frequency and function of MDSC, we compared fresh vs. cryopreserved peripheral blood mononuclear cell (PBMC) samples obtained from normal controls and patients with cancer. PBMC were obtained from 4 healthy donors and 21 patients with cancer. They were stained with labeled antibodies, and the frequency of DR⁻/LIN⁻/CD11b+, DR⁻/LIN⁻/CD15+, DR⁻/LIN⁻/CD33+ and DR(-/low)/CD14+ cells was determined by flow cytometry before and after cryopreservation. CFSE-based suppressor assays were used to test inhibitory functions of MDSC. Arginase I expression and reactive oxygen species (ROS) upregulation in MDSC subsets were evaluated by flow cytometry. The DR(-/low)/CD14+ and DR⁻/LIN⁻/CD11b+ subsets of MDSC were found to be more resistant to the cryopreservation/thawing procedure compared to the DR⁻/LIN⁻/CD15+ and DR⁻/LIN⁻/CD33+ subsets. The frequency of the latter two MDSC subsets was significantly reduced after cryopreservation. All but DR⁻/LIN⁻/CD15+ cells inhibited proliferation of autologous CSFE-labeled CD4+ cells but lost suppressor activity after cryopreservation. Only DR⁻/LIN-/CD15+ cells were positive for Arginase I, but lost its expression after cryopreservation. Only fresh DR⁻/LIN⁻/CD11b+ and DR⁻/LIN⁻/CD15+ cells produced ROS after in vitro stimulation. Studies of human MDSC should be performed in fresh blood samples. If samples have to be cryopreserved, monitoring of CD11b+ and CD14+ MDSC subsets provides the most reliable results. Arginase I expression or stimulated ROS production assessed by flow cytometry are useful markers for MDSC subsets only in fresh samples.

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; Arginase; Cryopreservation; Flow Cytometry; Granulocytes; HLA-DR Antigens; Humans; Leukocytes, Mononuclear; Lewis X Antigen; Lipopolysaccharide Receptors; Monocytes; Myeloid Cells; Neoplasms; Reactive Oxygen Species; Sialic Acid Binding Ig-like Lectin 3

The purpose of the study was to develop a flow cytometric assay for quantitative determination of adhesive interactions of human endothelial cells (ECs) with tumor cells. EC lines established from human lymph node, appendix, lung, skin and intestine microvessels, labeled with PKH26-GL fluorescent dye, were grown to confluency in 24-well TC plates. Human colon adenocarcinoma cell suspension was overlaid onto labeled ECs, and allowed to adhere for 20 min at 4 degrees C under static conditions. Non-adhering cells were collected first, and adhering tumor cells together with ECs were detached from the culture plate. Collected cell fractions were evaluated by flow cytometry. Results were re-calculated as a ratio (R) of adhering colon carcinoma cells per one EC. We demonstrated that immortalized human microvascular ECs preserved their organ specificity. Colon carcinoma cells adhere preferentially to ECs of intestine origin. The immunofluorescent staining of adhering and non-adhering cancer cell subpopulations has revealed an augmented level of Lewis(x) antigen on adhering cancer cells. The organ specificity of endothelial cell interactions with colon carcinoma cells demonstrated in static conditions was verified and confirmed with flow adhesion assay. The method elaborated is suitable for quantifying of tumor cells adhering to ECs, with simultaneous evaluation of cell surface phenotypic markers of both partner cells participating in adhesive interactions. Validated by comparison to dynamic shear stress adhesion assay in blood flow reconstituted conditions this assay greatly facilitates evaluation of tumor cell-endothelial cell mutual interactions taking place during metastatic process.

Topics: Cell Adhesion; Cell Communication; Cell Line, Tumor; Cells, Cultured; Coculture Techniques; Colonic Neoplasms; Endothelial Cells; Flow Cytometry; Fluorescent Antibody Technique; Humans; Intestine, Small; Lewis X Antigen; Lung; Lymph Nodes; Neoplasms; Oligosaccharides; Sialyl Lewis X Antigen; Skin

Lewis Y (LeY) antigen is highly expressed in a variety of human carcinomas of epithelial cell origin. Recent studies suggest functional blockade of LeY may provide a novel therapeutic approach for the treatment of cancers. However, suppressing LeY expression by genetic manipulation and its impact on neoplastic cell proliferation has not been investigated. We report here that different fucosyltransferases (FUTs) were expressed with the greatest expression of fucosyltransferase I or IV (FUT1/4), the two key enzymes for the synthesis of LeY in human epidermoid carcinoma A431 cells. Knocking down FUT1/4 expression by short interfering RNA technique dramatically reduced the expression of FUT1/4 and LeY and inhibited cell proliferation through decreasing epidermal growth factor receptor (EGFR) signaling pathway. Treatment of A431 cells that were inoculated into the nude mice with FUT1 siRNA or FUT4 siRNA greatly impeded tumor growth. Suppressing FUT1/4 expression also blocked EGF-induced tyrosine phosphorylation of EGFR and mitogen-activated protein kinases. In conclusion, suppressing the expression of FUT1/4 by RNAi technology reduces the synthesis of LeY and inhibits cancer growth. It may serve as a potential methodology for the treatment of cancers that express LeY glycoconjugates.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Epidermal Growth Factor; ErbB Receptors; Fucosyltransferases; Galactoside 2-alpha-L-fucosyltransferase; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Lewis Blood Group Antigens; Lewis X Antigen; MAP Kinase Signaling System; Mice; Mice, Nude; Mitogen-Activated Protein Kinases; Neoplasms; Phosphotyrosine; RNA, Small Interfering; Transfection; Xenograft Model Antitumor Assays

There are many molecular tumor markers for diagnosing and monitoring cancer patients. Especially, quantitative assay for serum levels of tumor markers; such as AFP, CEA, PSA, hCG, CA 19-9 and CA 125, are frequently used in daily practice because of their relative specificities and usefulness to the common cancers. Though not suitable for early diagnosis, but they are used in monitoring patients with advanced caner, especially after treatments. Two of them, AFP and PSA, are also used in the screening and monitoring of high-risk groups, namely patients with chronic viral hepatitis and old male, who are the high risk for hepatoma and proste cancer respectively. Problems in using serum markers are; relatively low specificity and low sensitivity to cancer, confusing naming for similar markers that recognize almost the same molecule of cancer. Users must understand that CA 19-9, CA 50, KM-O 1 and SPAN-1 are in the same sialylated Lewis A group, and CA 125, CA 130 and CA 602; in the mucin antigen group, and STN, CA 54/61 and CA 72-4; in the sialyl Tn antigen group. Combination of two or more markers may inform us the biological characteristics of the cancer. For example, a germ-cell tumors may produce hCG and placental marker. That is of the choriocarcinoma type. Those with hCG and fetal antigens are the ordinal type of germ cell tumors, and those with AFP, CEA and cytokeratin are teratoma, and those with LDH and ALP only but negative for hCG and AFP must be seminoma. For the bronchial and alveolar carcinomas, CEA, SCC, NSE and cytokeratin 19 fragments are useful. Combination may be difficult for beginners but once understood, it will be an art in clinical oncology.

Topics: alpha-Fetoproteins; Antigens, Neoplasm; Biomarkers; Biomarkers, Tumor; Carcinoembryonic Antigen; Diagnosis, Differential; Female; Hepatitis, Viral, Human; Humans; Keratin-19; Keratins; Lewis X Antigen; Male; Neoplasms; Prostate-Specific Antigen; Prostatic Neoplasms; Protein Precursors; Prothrombin

Unique specificities of the cloned alpha 1,3-L-fucosyltransferases (FTs), FT III (Lewis type), FT IV (myeloid type), and FT V (plasma type), and the alpha 1,3-FTs of Colo 205 (colon carcinoma), HL 60 (myeloid), B142 (lymphoid), EKVX (lung carcinoma), and calf mesenteric lymph nodes (CMLN) were discerned with sulfated, sialylated, and/or fucosylated Gal beta 1,3/4GlcNAc beta-based acceptor moieties. (a) FT V was 1.0-, 20.8-, and 4.6-fold active in forming Lewis x, Lewis y, and 3'-alpha-galactosyl Lewis x, respectively. (b) FT III and FT V formed approximately 4-fold 3'-sulfo Lewis x, as compared to 3'-sialyl Lewis x. (c) FT IV showed great efficiency in forming 3'-sulfo Lewis x (249%) and Lewis x (345%) in mucin-type branched chains. (d) FT III, FT IV, and FT V formed 19%, 62%, and 47% 6-sulfo Lewis x as compared to Lewis x. (e) 6'-Sulfo Lewis x and 3'-sialyl-6'-sulfo Lewis x (GLYCAM ligand) were not synthesized from their immediate precursors by FT III, FT IV, or FT V. (f) FT III, FT IV, and FT V were 311%, 9%, and 188% active, respectively, with 2'-fucosyl lactose but were not active with 2'- fucosyl-6'-sulfo lactose. (g) FT III and FT V were 7.0- and 0.5-fold active in forming Lewis a as compared to Lewis x, whereas, FT IV was inactive. (h) FT III was -2.0-fold more active in forming 3'-alpha-galactosyl Lewis a than Lewis b. (i) FT III synthesized 6-sialyl Lewis a (40% efficiency as compared to Lewis a) from 6-sialyl type 1. (j) FT III did not act on 6'-sulfo or 6'-sialyl type 1 but was 106% and 22% active with 3'-sulfo and 6-sulfo type 1, respectively. (k) The Colo 205 FT activities with type 1 compounds almost paralleled that of FT III except for the low activity (9%) with Gal beta 1,3(NeuAc alpha 2, 6)GlcNAc beta-O-Bn, but with type 2 considerable differences between Colo 205 FT and FT III were noticed. (l) The alpha 1,3-FTs of CMLN, HL60, B142, and EKVX were 1.2-1.7 times active with Fuc alpha 1,2Gal beta 1,4GlcNAc beta- O-pNP and Gal alpha 1,3Gal beta 1,4 GlcNAc beta-O-Bn with respect to Gal beta 1,4GlcNAc beta-O-Al. (m) Both CMLN and HL60 FTs were 2-fold active with 3-sulfoGal beta 1,4GlcNAc in a mucin-type branch structure such as 3-sulfoGal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn. (n) The 3'-sulfoLacNAc/acrylamide copolymer, either as an acceptor or as a competitive inhibitor, had the potential to distinguish myeloid type alpha 1,3-FT from the plasma type.

Topics: Animals; Bone Marrow; Carbohydrate Sequence; Cattle; Cell Line; Fucosyltransferases; Humans; Lewis X Antigen; Lymph Nodes; Molecular Sequence Data; Neoplasms; Oligosaccharides; Recombinant Proteins; Sialyl Lewis X Antigen; Substrate Specificity

CD15 is expressed on a wide variety of tumor cells including myeloid leukemia, breast, colorectal, lung cancer cells. It is probable that the core proteins and lipids differ on the different cell types since there is marked variability in the binding of the mAb within and among the different cell types. Previously, there had only been a single mAb of the IgG class directed to CD15. The present study demonstrates the existence of one more IgG mAb (an IgG3). Unfortunately, a third mAb submitted as an IgG1, MA14, did not appear to be an IgG in our studies and a fourth mAb, MA9, submitted as an IgG1 did not have significant binding activity and also could not be confirmed as anti-CD15. An interesting finding was that all of the mAb could mediate complement-dependent cytotoxicity with rabbit serum. However, the two IgG3 mAb, MA63 (MCS-1) and MA88 (7C3), could not mediate lysis with human complement, while all of the IgM mAb could do so. It is not clear why this difference exists. CD15 is known to be expressed on mature myeloid cells in the hematopoietic lineage. The expression of CD15 on progenitor cells has been mapped by the indirect method of C'-dependent lysis and measurement of colony-forming cells. These studies have estimated that about 50% of normal CFU-GM express CD15. In the present workshop, we examined the expression of CD15 on a quantifiable cell population, the CD34 positive population.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Antigen-Antibody Reactions; Antigens, Neoplasm; Hematopoietic Stem Cells; Humans; Immunoglobulin G; Immunoglobulin M; Leukemia, Myeloid; Lewis X Antigen; Macromolecular Substances; Mice; Neoplasms; Neuraminidase; Rabbits; Tumor Cells, Cultured

The reactivity of monoclonal antibodies from the 5th workshop Selectins/Selectin Ligands panel, directed to the sialylated Lewis(x) and sialylated Lewis(a) determinants, with the human breast carcinoma (BT-20, ZR-75-1 and MDA-MB-468) cell lines, human ovarian carcinoma cell line A2780, fibrosarcoma (HT-1080, B-6FS) and hematopoietic neoplastic (U-937, HL-60, K-562) cell lines were determined with the aid of flow immunocytofluorometry. The examined monoclonal antibodies to sialylated Lewis determinants reacted with examined breast carcinoma, but not with the examined ovarian carcinoma and fibrosarcoma cell lines.

Topics: Antibodies, Monoclonal; Breast Neoplasms; Epitopes; Female; Fibrosarcoma; Flow Cytometry; Fluorescent Antibody Technique; Humans; Leukemia, Promyelocytic, Acute; Lewis Blood Group Antigens; Lewis X Antigen; Melanoma; Neoplasms; Ovarian Neoplasms; Tumor Cells, Cultured

The mouse monoclonal antibody anti-Leu-M1 (CD15) recognizes the carbohydrate determinant lacto-N-fucopentaose III, an oligosaccharide believed to be involved in cell-cell interactions. Anti-Leu-M1 is used in surgical pathology as an aid in the diagnosis of Hodgkin's disease. Additionally, adenocarcinomas derived from various organs stained positively with anti-Leu-M1 at the light microscopic level. Since mesotheliomas do not display positive reactivity to this antibody, Leu-M1 is clinically useful as part of a panel of antibodies in distinguishing adenocarcinomas from mesotheliomas. Previous work was carried out using post-embedding protein A-gold immunocytochemistry on thin sections embedded in Lowicryl K4M from a patient with Hodgkin's disease of the nodular sclerosing type; intense and precise labeling by gold particles was revealed in cytoplasmic granules, which were often clustered in a perinuclear location, in the Golgi apparatus, and focally along the plasma membrane of Reed-Sternberg cells. Moreover, polymorphonuclear leukocytes demonstrated similar labelling along the plasma membrane and over cytoplasmic granules. To define precisely the intracellular localization of Leu-M1 in human adenocarcinomas, we have performed post-embedding immunoelectron microscopy with the protein A-gold technique on sections embedded in Lowicryl K4M from neoplasms of the lung, stomach, colon, and breast. The pattern of labeling by gold particles indicative of Leu-M1 binding varied in adenocarcinomas of the different organs.

Topics: Adenocarcinoma; Antigens, Neoplasm; Hodgkin Disease; Humans; Immunohistochemistry; Lewis X Antigen; Microscopy, Immunoelectron; Neoplasms; Neutrophils

ELAM-1 (endothelial-leukocyte adhesion molecule-1, E-selectin) is a cell adhesion molecule which is specifically expressed on cytokine-activated endothelial cells. It is known to bind a carbohydrate antigen sialyl Le(x) (sialyl SSEA-1) present on leukocytes, and the sialyl Le(x)/ELAM-1 adhesion system is suggested to play a physiologically important role in leukocyte recruitment in the process of inflammation. Some leukemia cells also express the sialyl Le(x) antigen, and in such a case, the sialyl Le(x)/ELAM-1 adhesion system will be involved in the organ infiltration of leukemia cells. On the other hand, in the adhesion of human cancer cells to endothelial cells, another carbohydrate antigen, sialyl Le(a), serves as the ligand for ELAM-1, as well as sialyl Le(x). These two carbohydrate determinants, sialyl Le(a) and sialyl Le(a), on cancer cells will be involved in the hematogenous metastasis of cancer cells. The physiological function of these two carbohydrate determinants at the surface of normal epithelial cells is most probably to mediate stage-specific cell-to-cell recognition and adhesion during the course of organogenesis in developing embryos, and the abnormal cell-adhesion behaviors of cancer cells are the results of aberrant expression of cell adhesion molecules which would play physiologically important roles under normal condition.

Topics: Cell Adhesion; Cell Adhesion Molecules; E-Selectin; Endothelium; Epitopes; Humans; Leukemia; Leukocytes; Lewis X Antigen; Neoplasm Metastasis; Neoplasms

C203 and C242 are mouse monoclonal antibodies (MAbs) generated using a human colon carcinoma cell line. They recognize novel tumour-associated epitopes present in elevated levels in sera from patients with colon and pancreatic cancer. These epitopes were found to be co-expressed with sialylated Lewisa on the CanAg molecule. To study the association and distribution of the epitopes of CanAg in sera, these new antibodies, together with C50, were used in different combinations in time-resolved fluoroimmunoassays. Relative serum concentrations were examined in patients with various types of carcinoma and in patients with ulcerative colitis and benign pancreatic, and hepatobiliary diseases. A double-determinant assay using C50 and C242 was shown to distinguish carcinoma from benign biliary and hepatocellular diseases better than a single-determinant assay based on C50 as both catching and tracing antibody. The number of sera with elevated CanAg levels from patients with benign obstructive biliary disease was 17 out of 29 using the single-determinant CA50 assay. This was reduced to 4 out of 29 in the double-determinant assay. When sera from patients with liver cirrhosis were analyzed, 16 of 23 patients showed elevated CanAg levels with the C50-C50 combination, but only 4 of 23 patients had elevated antigen values using the C50-C242 assay. The increased specificity was obtained without loss of sensitivity. MAb C203 was evaluated both in a double-determinant combination with C50 and in an homologous assay, but did not contribute either increased sensitivity or specificity as compared with C50-C50.

Topics: Animals; Antibodies, Monoclonal; Antigens, Tumor-Associated, Carbohydrate; Cell Line; Cholestasis; Colitis, Ulcerative; Epitopes; Female; Humans; Immunoenzyme Techniques; Immunoglobulin G; Immunoglobulin M; Lewis X Antigen; Liver Cirrhosis; Mice; Mice, Inbred BALB C; Neoplasms; Pancreatitis; Reference Values

Endothelial leukocyte adhesion molecule-1 (ELAM-1) is an endothelial cell adhesion molecule that allows myeloid cells to attach to the walls of blood vessels adjacent to sites of inflammation. ELAM-1 recognizes the sialyl-Lewis X (sialyl-Lex) determinant, NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc-, a granulocyte carbohydrate also found on the surface of some tumor cell lines. Binding of myeloid cells to soluble ELAM-1 is inhibited by a monoclonal antibody recognizing sialyl-Lex or by proteins bearing sialyl-Lex, some of which may participate in humoral regulation of myeloid cell adhesion. Stimulated granulocytes also release an inhibitor of ELAM-1 binding that can be selectively adsorbed by monoclonal antibody to sialyl-Lex.

Topics: Amniotic Fluid; Antibodies, Monoclonal; Carbohydrate Sequence; Cell Adhesion; Cell Adhesion Molecules; Cell Membrane; E-Selectin; Endothelium, Vascular; Fucose; Fucosyltransferases; Granulocytes; Immunosorbent Techniques; Interleukin-1; Interleukin-8; Lewis X Antigen; Molecular Sequence Data; Neoplasms; Neuraminidase; Oligosaccharides; Orosomucoid; Sialyl Lewis X Antigen; Tumor Cells, Cultured

Topics: ABO Blood-Group System; Animals; Antigens, Tumor-Associated, Carbohydrate; Biomarkers, Tumor; Glycolipids; Humans; Lewis Blood Group Antigens; Lewis X Antigen; Neoplasms

Topics: Antibodies, Monoclonal; Antigens, Surface; Brain; Cell Differentiation; Cell Line; Epitopes; Female; Granulocytes; Humans; Lewis X Antigen; Milk, Human; Neoplasms; Oligosaccharides; Pregnancy; Tissue Distribution

Monoclonal antibodies were produced by immunizing rats with human small cell lung carcinoma (SCLC) cell lines. Monoclonal antibodies 600D11 and 624A12 were found to be directed against the ceramide pentasaccharide that contains the lacto-N-fucopentaose III (LNFP III) sequence of sugars, an isomer of the Lewis A blood group antigen. LNFP III is an immunodominant antigen whose reactivity is maintained in formalin-fixed paraffin-embedded sections (PS). LNFP III has been recognized in a number of human tumors including: SCLC; adenocarcinomas of the breast, gastrointestinal tract, genitourinary tract, and lung; renal cell carcinoma; neuroblastoma; and myelogenous leukemia. We now report the normal adult and fetal tissue distribution of the LNFP III antigen by immunoperoxidase staining on PS utilizing 600D11 and 624A12. Binding was demonstrated in bronchial epithelium and bronchial glands; squamous epithelium of the esophagus; gastric crypts, duodenal enterocytes and Brunners glands; argentaffin cells; jejunal and colonic goblet cells; pancreatic acinar cells; salivary glands; endocervical and exocervical cells; skin epidermis; myelinated motor fibers; cells of the adrenal medulla and anterior pituitary gland; polymorphonuclear leukocytes (PMNs); tissue macrophages and renal proximal tubules and loops of Henle. Staining was localized to cell membranes and within the cytoplasm, with greatest intensity at the apical and basal portions of the cells. These staining patterns were noted in adult and neonatal tissues, and initial expression could be traced to approximately the second trimester of fetal development. Knowledge of the normal tissue distribution of this immunodominant antigenic determinant may offer insight into its structural and functional role in benign and malignant tissues.

Topics: Adolescent; Animals; Antibodies, Monoclonal; Antigens, Surface; Cell Line; Female; Fetus; Genes, Dominant; Humans; Immunoenzyme Techniques; Lewis X Antigen; Neoplasms; Oligosaccharides; Pregnancy; Rats; Rats, Inbred Strains; Tissue Distribution

Normal human tissues and various human tumors were surveyed by immunohistochemical techniques for expression of the stage-specific embryonic antigen 1 (SSEA-1). The antibody reacted with many normal and neoplastic human tissues. In most instances, equivalent human and mouse tissues expressed SSEA-1; however, different tissue localization patterns were sometimes seen between these two species. Most SSEA-1-positive tumors originate from tissues that normally expressed this antigen; however, some breast and ovarian tumors are SSEA-1 positive, and these organs are SSEA-1 negative. SSEA-1-positive tumors were composed of both immunoreactive and nonreactive tumor cells. These data show that SSEA-1, initially defined as a mouse embryonic antigen, represents a heterogenetic antigen present in many normal human tissues. It is retained on many but not all neoplastic cells originating in these normal tissues and also appears on the surface of some tumor cells developing in SSEA-1-negative tissues.

Topics: Antigens, Neoplasm; Cell Line; Female; Glycolipids; Humans; Immunoenzyme Techniques; Lewis X Antigen; Male; Neoplasms; Tissue Distribution

Two monoclonal antibodies (MC631 and MC813-70) raised against 4- to 8-cell stage mouse embryos and a human teratocarcinoma cell line, respectively, detect the stage-specific embryonic antigens, the previously defined SSEA-3 and SSEA-4, described herein. These antibodies were both reactive with a unique globo-series ganglioside with the structure shown below: (formula; see text) The antibodies were found to recognize sequential regions of this ganglioside, i.e., MC813-70 recognizes the terminal 'a' structure whereas antibody MC631 recognizes the internal 'b' structure. Thus, a set of two antibodies defines this unique embryonic antigen. During differentiation of human teratocarcinoma 2102Ep cells, the globo-series glycolipids defined by these antibodies decrease and the lacto-series glycolipids, reacting with the SSEA-1 antibody appear. This antigenic conversion suggests that a shift of glycolipid synthesis from globo-series to lacto-series glycolipids occurs during differentiation of human teratocarcinoma and perhaps of pre-implantation mouse embryos.

Topics: Animals; Antibodies, Monoclonal; Antigen-Antibody Complex; Antigens, Neoplasm; Antigens, Surface; Blastocyst; Carbohydrate Conformation; Carbohydrate Sequence; Erythrocytes; Female; Glycolipids; Granulocytes; Humans; Lewis X Antigen; Lymphocytes; Mice; Neoplasms; Oocytes; Species Specificity; Teratoma

Topics: Animals; Blastocyst; Blood Group Antigens; Cell Line; Embryo, Mammalian; Epitopes; Erythrocytes; Glycolipids; Granulocytes; Humans; I Blood-Group System; Lewis X Antigen; Mice; Neoplasms