Lymphocyte Homing Receptor CD44 Research Articles

Multifunctionalized iron oxide nanoparticles for selective drug delivery to CD44-positive cancer cells

Nanomedicine nowadays offers novel solutions in cancer therapy and diagnosis by introducing multimodal treatments and imaging tools in one single formulation. Nanoparticles acting as nanocarriers change the solubility, biodistribution and efficiency of therapeutic molecules, reducing their side effects. In order to successfully apply these novel therapeutic approaches, efforts are focused on the biological functionalization of the nanoparticles to improve the selectivity towards cancer cells. In this work, we present the synthesis and characterization of novel multifunctionalized iron oxide magnetic nanoparticles (MNPs) with antiCD44 antibody and gemcitabine derivatives, and their application for the selective treatment of CD44-positive cancer cells. The lymphocyte homing receptor CD44 is overexpressed in a large variety of cancer cells, but also in cancer stem cells (CSCs) and circulating tumor cells (CTCs). Therefore, targeting CD44-overexpressing cells is a challenging and promising anticancer strategy. Firstly, we demonstrate the targeting of antiCD44 functionalized MNPs to different CD44-positive cancer cell lines using a CD44-negative non-tumorigenic cell line as a control, and verify the specificity by ultrastructural characterization and downregulation of CD44 expression. Finally, we show the selective drug delivery potential of the MNPs by the killing of CD44-positive cancer cells using a CD44-negative non-tumorigenic cell line as a control. In conclusion, the proposed multifunctionalized MNPs represent an excellent biocompatible nanoplatform for selective CD44-positive cancer therapy in vitro.

Hyaluronan-CD44v3 Interaction with Oct4-Sox2-Nanog Promotes miR-302 Expression Leading to Self-renewal, Clonal Formation, and Cisplatin Resistance in Cancer Stem Cells from Head and Neck Squamous Cell Carcinoma

Human head and neck squamous cell carcinoma (HNSCC) is a highly malignant cancer associated with major morbidity and mortality. In this study, we determined that human HNSCC-derived HSC-3 cells contain a subpopulation of cancer stem cells (CSCs) characterized by high levels of CD44v3 and aldehyde dehydrogenase-1 (ALDH1) expression. These tumor cells also express several stem cell markers (the transcription factors Oct4, Sox2, and Nanog) and display the hallmark CSC properties of self-renewal/clonal formation and the ability to generate heterogeneous cell populations. Importantly, hyaluronan (HA) stimulates the CD44v3 (an HA receptor) interaction with Oct4-Sox2-Nanog leading to both a complex formation and the nuclear translocation of three CSC transcription factors. Further analysis reveals that microRNA-302 (miR-302) is controlled by an upstream promoter containing Oct4-Sox2-Nanog-binding sites, whereas chromatin immunoprecipitation (ChIP) assays demonstrate that stimulation of miR-302 expression by HA-CD44 is Oct4-Sox2-Nanog-dependent in HNSCC-specific CSCs. This process results in suppression of several epigenetic regulators (AOF1/AOF2 and DNMT1) and the up-regulation of several survival proteins (cIAP-1, cIAP-2, and XIAP) leading to self-renewal, clonal formation, and cisplatin resistance. These CSCs were transfected with a specific anti-miR-302 inhibitor to silence miR-302 expression and block its target functions. Our results demonstrate that the anti-miR-302 inhibitor not only enhances the expression of AOF1/AOF2 and DNMT1 but also abrogates the production of cIAP-1, cIAP-2, and XIAP and HA-CD44v3-mediated cancer stem cell functions. Taken together, these findings strongly support the contention that the HA-induced CD44v3 interaction with Oct4-Sox2-Nanog signaling plays a pivotal role in miR-302 production leading to AOF1/AOF2/DNMT1 down-regulation and survival of protein activation. All of these events are critically important for the acquisition of cancer stem cell properties, including self-renewal, clonal formation, and chemotherapy resistance in HA-CD44v3-activated head and neck cancer.

Expression of CD44 and CD44v6 in primary cutaneous CD30 positive T-cell lymphoproliferative disorders.

Expression of the lymphocyte homing receptor CD44 and its variant form, CD44v6, have been linked to unfavorable prognosis and tumor dissemination in non-Hodgkin's lymphoma. The role of CD44 and CD44v6 in primary cutaneous lymphomas may not necessarily correlate with that observed in nodal lymphomas. Our study attempts to evaluate the expression pattern of CD44 and CD44v6 in primary cutaneous CD30 positive T-cell lymphoproliferative disorders including primary cutaneous anaplastic large cell lymphoma (cALCL) and lymphomatoid papulosis (LyP) and compared the expression between these two subgroups. Immunohistochemical staining of CD44 and CD44v6 was performed on 10 cALCL and 18 LyP cases. CD44 consistently expressed in all cases of primary cutaneous CD30 positive T-cell lymphoproliferative disorders. In contrast with previous studies, our results showed that CD44v6 expressed in 46% of all cases, including 50% cALCL and 44% LyP. There was no statistic difference in expression of CD44 or CD44v6 between cALCL and LyP subgroups. These results provide a new evidence that CD44v6 is expressed in a subset cases of primary cutaneous CD30 positive T-cell lymphoproliferative disorders and suggest that other factors or molecules rather than CD44 or CD44v6 are responsible for the difference between cALCL and LyP.

Cell Adhesion Proteins As Tumor Suppressors

We summarize recent progress on the role of cell adhesion molecules in biology and discuss the potential application of cell adhesion molecules for managing urological cancer. We comprehensively reviewed the literature from 1982 to 2001, including peer reviewed publications and recent abstracts from national meetings, relevant to cell adhesion molecules in urological cancer. A growing body of evidence suggests that alterations in the adhesion properties of neoplastic cells have a pivotal role in the development and progression of cancer. Loss of intercellular adhesion and desquamation of cells from the underlying lamina propria allows malignant cells to escape from their site of origin, degrade the extracellular matrix, acquire a more motile and invasion phenotype, and invade and metastasize. In addition to participating in tumor invasiveness and metastasis, adhesion molecules regulate or significantly contribute to various functions, including signal transduction, cell growth, differentiation, site specific gene expression, morphogenesis, immunological function, cell motility, wound healing and inflammation. To date a diverse system of transmembrane glycoproteins has been identified that mediates cell-cell and cell-extracellular matrix adhesion. The main families of adhesion molecules include members of the Ig superfamily, cadherins, integrins and selectins. Multiple and diverse cell adhesion molecules participate in intercellular and cell-extracellular matrix interactions of cancer. Cancer progression is a multistep process, in which some adhesion molecules have a pivotal role in the development of recurrent, invasive and distant metastasis. Recent data implicate some of these molecules in cell signaling and tumor suppression, which has important consequences for tumor growth.

Proteolytic Cleavage of the CD44 Adhesion Molecule in Multiple Human Tumors

Cell surface adhesion molecules are crucial for the development and/or pathogenesis of various diseases including cancer. CD44 has received much interest as a major adhesion molecule that is involved in tumor progression. We have previously demonstrated that the ectodomain of CD44 undergoes proteolytic cleavage by membrane-associated metalloproteases in various tumor cell lines. The remaining membrane-bound CD44 cleavage product can be detected using antibodies against the cytoplasmic domain of CD44 (anti-CD44cyto antibody). However, the cleavage of CD44 in primary human tumors has not been investigated. Using Western blots with anti-CD44cyto antibody to assay human tumor tissues, we show that the CD44 cleavage product can be detected in 58% (42 of 72) of gliomas but not in normal brain. Enhanced CD44 cleavage was also found in 67% (28 of 42) of breast carcinomas, 45% (5 of 11) of non-small cell lung carcinomas, 90% (9 of 10) of colon carcinomas, and 25% (3 of 12) of ovarian carcinomas. Tumors expressing a CD44 splice variant showed a significantly higher incidence of enhanced CD44 cleavage. The wide prevalence of CD44 cleavage suggests that it plays an important role in the pathogenesis of human tumors.

CD44 expression predicts disease outcome in localized large B cell lymphoma

Diffuse large B cell non-Hodgkin's lymphomas (DLCL) form a heterogeneous group of tumors with diverse morphology, clinical features, treatment response and prognosis. The biological variables underlying this heterogeneity are unknown. In the present study, we explored the value of the lymphocyte homing receptor CD44, a putative determinant of lymphoma dissemination, in predicting prognosis in DLCL. Expression of the standard form of CD44 (CD44s) and of CD44 isoforms containing exon v6 (CD44v6) on tumor cells was assessed by immunohistochemistry in a cohort of 276 DLCL patients from a population based lymphoma registry. We observed that CD44s as well as CD44v6 expression correlated with tumor dissemination in patients with primary nodal DLCL. Importantly, in patients with localized nodal disease, CD44s was a strong prognosticator predicting tumor related death independent of the other parameters of the International Prognostic Index (IPI). Incorporation of CD44s in the IPI parameter 'stage', increased the prognostic value of this parameter in nodal DLCL. Our data identify CD44 as a biological prognosticator, which can be used to 'fine-tune' the IPI for nodal DLCL.

Species‐specific detection of porcine xenografts with an antibody against a novel epitope of the lymphocyte homing receptor CD44

Abstract: We have isolated and characterized a murine monoclonal antibody, 10–14, that is reactive with an 85 kDa glycoprotein found on porcine peripheral blood lymphocytes, cultured aortic endothelial cells, fetal cardiomyocytes, and mixed neural/glial cell cultures. In contrast to the anti‐CD44 antibody Z062, which reacted with both porcine and murine CD44, the 10–14 antibody did not react with monkey, rabbit, rat, or murine cells, suggesting that 10–14 recognized a novel epitope found exclusively on the porcine CD44 molecule; 10–14 is thus a novel reagent for identification of xenotransplanted porcine cells. The reagent has been used in this study as an immunohistochemical probe to detect porcine neural cell grafts in the central nervous system of the rat and to label porcine fetal cardiomyocytes. Moreover, the antibody provides information on the expression of the CD44 molecule that is critical for lymphocyte adhesion and trafficking.

CD44 variant expression in cutaneous T-cell lymphoma.

Expression of the lymphocyte homing receptor CD44 and its splice variants have been linked to tumour dissemination and poor prognosis in non-Hodgkin's lymphoma. Specifically, the in vitro expression of variant exon V6 confers metastatic potential in rat pancreatic carcinoma cell lines. In this study, we investigated the expression of CD44 splice variants in cutaneous T-cell lymphomas, including patients with mycosis fungoides (MF), Sezary syndrome (SS), large-cell anaplastic lymphoma (LCAL) and HTLV1-associated cutaneous lymphoma. In addition, 4 involved lymph nodes from 2 patients with MF and 1 patient with SS were examined. Inflammatory dermatoses, lichen planus and psoriasis, and normal skin were also studied. Immunohistochemistry was performed using a panel of monoclonal antibodies, including those with specificity for CD44H (standard isoform) and variant exons V3, V6 and V8-9. Normal epidermal keratinocytes were consistently CD44H and CD44 V3, V6 and V8-9 positive. In all the different clinicopathological subtypes and stages of cutaneous T-cell lymphomas, including involved lymph nodes, tumour cells consistently expressed CD44H, but were CD44 V3 and V6 negative. CD44 V8-9 was expressed on a majority of tumour cells in 2/5 LCAL and on occasional tumour cells in 2/5 LCAL. Occasional V8-9 positive tumour cells were also identified in 6/13 MF, 1/4 SS and 3/4 HTLV1. In 2/3 lymph node samples from 2 patients with tumour-stage MF, CD44 V8-9 expression was found on a small percentage of atypical mononuclear cells. Scattered V8-9 positive dermal mononuclear cells were present in sections of lichen planus and psoriasis. We have found no evidence to suggest that the metastasis-associated CD44 variant exon (V6) is expressed in cutaneous T-cell lymphoma, or that CD44H expression is associated with an adverse prognostic group. It is not clear whether the strong expression of CD44 V8-9 in 2 patients with CD30 positive LCAL reflects activation status or metastatic potential.

Proteoglycan forms of the lymphocyte homing receptor CD44 are alternatively spliced variants containing the v3 exon.

The CD44 cell surface glycoprotein is expressed on a broad range of different tissues as multiple isoforms containing from one to ten alternatively spliced exons v1-v10 inserted within the extracellular domain. Differential glycosylation generates still further variability, yielding both N- and O-glycan-modified forms of CD44 in addition to proteoglycan-like variants containing chondroitin sulphate and heparan sulphate. These high molecular mass proteoglycan-like variants, previously identified in lymphocytes, melanomas, and keratinocytes have been implicated in cell-matrix adhesion, cell motility, and invasiveness. More recently, monocyte CD44 molecules presumed to carry glycosaminoglycan chains were shown to bind the chemokine MIP-1 beta (Tanaka, Y.,D. H. Adams, S. Hubscher, H. Hirano, U. Siebenlist, and S. Shaw. 1993. Nature (Lond). 361:79-82.) raising the intriguing possibility that proteoglycan-like CD44 variants might play a role in regulating inflammatory responses. Here we have investigated the molecular identity of these proteoglycan-like CD44 variants by generating a panel of recombinant CD44 isoforms using a novel cassette cloning strategy. We show that both chondroitin and heparan sulphate modifications are associated specifically with isoforms (CD44v3-10 and CD44v3,8-10) containing the v3 alternative exon which encodes a consensus motif SGXG for GAG addition. Other isoforms (CD44v10, CD44v8-10, CD44v7-10, and CD44v6-10) are shown to lack these GAG chains but to carry extensive O-glycan modifications, most likely within the mucin-like alternative exon inserts. We also demonstrate that the majority of endogenous GAG-modified CD44 isoforms present in epithelial cells constitute v3 isoforms thus establishing that in these cells the majority of proteoglycan-like CD44 variants are generated by alternative splicing. Finally we present evidence using transfected B lymphoma cells that the GAG-modified CD44 isoforms CD44v3-10 and CD44v3,8-10, unlike CD44H, bind only weakly to hyaluronan. Together with the demonstration in the accompanying paper (Bennett, K., D. G. Jackson, J.C. Simon, E. Tanczos, R. Peach, B. Modrell, I. Stamenkovic, G. Plowman, and A. Aruffo. 1995. J. Cell Biol. 128:687-698.), that CD44 molecules containing the v3 exon bind growth factors, these results highlight a new and potentially important role for CD44 alternative splicing in the control of cell-surface proteoglycan expression.

A monoclonal antibody that blocks poliovirus attachment recognizes the lymphocyte homing receptor CD44.

A monoclonal antibody, AF3, was previously shown to specifically inhibit poliovirus binding to HeLa cells and to detect a 100-kDa glycoprotein only in cell lines and tissues permissive for poliovirus infection. These results suggested that the 100-kDa protein may be involved in the pathogenesis of poliomyelitis and the cellular function of the poliovirus receptor site. To study further the role of the 100-kDa protein in poliovirus attachment, immunoaffinity purification, amino acid sequencing, and cDNA cloning were undertaken. The results demonstrate that antibody AF3 reacts with the lymphocyte homing receptor CD44, a multifunctional cell surface glycoprotein involved in the homing of circulating lymphocytes to lymph nodes and the modulation of lymphocyte adhesion and activation. Antibody AF3 reacts with a subset of CD44 molecules (AF3CD44H), which appears to be a small fraction of the heterogeneously glycosylated CD44 molecules expressed on hematopoietic and nonhematopoietic cells. Anti-CD44 monoclonal antibodies, previously reported to induce CD44-mediated modulation of lymphocyte activation and adhesion, compete with 125I-AF3 in binding assays, demonstrating functional overlap among the epitopes. The anti-CD44 monoclonal antibody A3D8, which binds to a greater molecular weight range of CD44 than does AF3, inhibits poliovirus binding to a similar degree. CD44 does not act as a poliovirus receptor, since CD44-expressing mouse L-cell transformants did not bind poliovirus. The poliovirus receptor and AF3CD44H may be noncovalently associated, or they may interact through the cytoskeleton or signal transduction pathways.

Adhesion molecule expression on B-cells from acute and chronic lymphoid leukemias.

Adhesion molecule expression on acute and chronic lymphoid leukemia cells of B lineage (B-ALL and B-CLL) may subserve several functions. Adhesion of leukemic cells to endothelial cells and to extracellular matrix components is relevant to homing, trafficking and spread of the malignant cells, and thus to clinical presentation, course and disease prognosis. Adhesive interactions between malignant cells and accessory cells, particularly stromal cells in the bone marrow environment, may support growth of the malignant cells via cytokine-delivered messages. They may also deliver signals that prevent or trigger programmed cell death of tumor cells. Here we review data on the adhesive phenotype of leukemic blasts from pro-B (CALLA +) ALL and of cells from B-CLL cases. We show that expression of certain adhesion molecules may help define disease subsets with distinctive clinical and prognostic features. One adhesion molecule, the lymphocyte homing receptor CD44, allows definition of two groups of B-CLL patients with significantly different survival.

Assembly of pericellular matrices by COS-7 cells transfected with CD44 lymphocyte-homing receptor genes.

The capacity to assemble and retain a pericellular matrix is correlated with the expression of the cell surface binding sites specific for the extracellular matrix macromolecule hyaluronan. These binding proteins have been termed hyaluronan receptors. The lymphocyte-homing receptor CD44 may have identity with these hyaluronan receptors. To determine whether hyaluronan receptors function independently in this capacity for matrix assembly, mammalian cells were transfected with cDNA encoding the putative hyaluronan receptor CD44. After transfection with CD44 cDNA, COS cells gained the capacity to assemble hyaluronan-dependent pericellular matrices in the presence of exogenously added hyaluronan and proteoglycan. Thus, CD44 receptors do function as matrix-organizing, matrix-anchoring hyaluronan-binding proteins. In addition, the expression of CD44/hyaluronan receptors alone is sufficient to direct this matrix assembly. If matrix assembly is a function of cells in vivo that express hyaluronan receptors, this raises interesting possibilities for the role of the receptors in cell migration, when new extracellular matrix environments are encountered.

The involvement of adhesion molecules in the biology of multiple myeloma.

Multiple myeloma represents a B cell malignancy characterized by a monoclonal proliferation of plasma cells. A striking feature of the disease is the tendency of the malignant plasma cells to affect mainly the bone marrow environment and to invade the peripheral blood only in the terminal stage. The growth of myeloma plasma cells is believed to be regulated by a functional interplay between the tumor cells and the bone marrow stroma, involving the action of various cytokines. This growth control is most probably mediated by close cellular contact of the myeloma cells and marrow stromal components. Therefore it can be assumed that myeloma plasma cells possess the ability to interact with the bone marrow stroma. Until now the adhesive mechanisms that may underlie this interaction, remain undetermined. We investigated the expression of several adhesion molecules on bone marrow plasma cells in myeloma patients and normal controls. Normal as well as malignant plasma cells were found to be strongly positive for the intercellular adhesion molecule ICAM-1, the fibronectin receptor VLA-4 and the lymphocyte homing receptor CD44. In addition, a much weaker expression of the second fibronectin receptor VLA-5, the laminin receptor VLA-6 and the vitronectin receptor CD51 was demonstrated. In contrast to normal plasma cells, myeloma cells can also express the neural cell adhesion molecule N-CAM. In this report we discuss the possible role of adhesion molecules in the pathogenesis and clinical evolution of multiple myeloma.

Ligation of the lymphocyte homing receptor CD44 triggers T-helper and cytolytic functions of human T cells

We show that antibodies to the CD44 molecule trigger proliferation of human CD3+/CD4+ T-cell clones. Such effect is IL2-dependent, as shown by IL2 production induced by anti-CD44 mAb and by inhibition of cell proliferation in the presence of anti-IL2 antibodies or cyclosporin A (CsA). Moreover, anti-CD44 mAb triggered human cytolytic CD4+ and CD8+ TCR α/β+ clones, and Vδ1 or Vδ2 TCR Y/δ+ clones to lyse Fc-gamma-R+ P815 cells and to release granule trypsin-like esterase enzymes. Anti-CD44 mAb-triggered proliferation and cytotoxicity were blocked by the PTK-inhibitor, genestein. In addition, ligation of the CD44 molecule induced tyrosine phosphorylation of proteins identical, by molecular weight, to those phosphorylated following anti-CD3 mAb-stimulation. Notably, anti-CD44 mAb does not induce tyrosine phosphorylation of a 21 kD protein (the phosphorylated zeta chain of the TcR molecular complex) typically observed upon anti-CD3 mAb stimulation.

Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons.

The CD44 molecule is known to display extensive size heterogeneity, which has been attributed both to alternative splicing and to differential glycosylation within the extracellular domain. Although the presence of several alternative exons has been partly inferred from cDNA sequencing, the precise intron-exon organization of the CD44 gene has not been described to date to our knowledge. In the present study we describe the structure of the human CD44 gene, which contains at least 19 exons spanning some 50 kilobases of DNA. We have identified 10 alternatively spliced exons within the extracellular domain, including 1 exon that has not been previously reported. In addition to the inclusion or exclusion of whole exons, more diversity is generated through the utilization of internal splice donor and acceptor sites within 2 of the individual exons. The variation previously reported for the cytoplasmic domain is shown to result from the alternative splicing of 2 exons. The genomic structure of CD44 reveals a remarkable degree of complexity, and we confirm the role of alternative splicing as the basis of the structural and functional diversity seen in the CD44 molecule.

Multiple variants of the human lymphocyte homing receptor CD44 generated by insertions at a single site in the extracellular domain.

The human CD44 cell-surface glycoprotein participates in a wide variety of cell-cell interactions including lymphocyte homing and tumor metastasis. The CD44 antigen is known to display extensive size heterogeneity when compared between different tissue sources although the structural basis for this variation is not yet clear. Recently, two further isotypes in addition to the basic hemopoietic form of the CD44 antigen have been cloned and sequenced and these have been found to contain all or part of a 200-400-base pair insert within the extracellular domain, suggesting that the characteristic heterogeneity in the molecule may be generated by a mechanism of alternative splicing. We have obtained further evidence for alternative splicing, and we report here the cloning and sequencing of six different CD44 sequence variants from a variety of cell lines using a combination of expression cloning and the polymerase chain reaction. Comparison of these variants indicates that each is probably assembled by the insertion of five different exon units in tandem into a discrete site within the membrane proximal region of the extracellular domain. One of the variants contains an exon that shares extensive amino acid sequence homology with a recently described rat CD44 variant that mediates tumor metastasis. Another variant contains a new exon that encodes a tandem repeat of the consensus sequence SG for covalent modification with chondroitin sulfate and is expressed predominantly on mammary tumors. We suggest that a mechanism of alternative exon splicing generates much of the observed structural heterogeneity of CD44 and that the particular set of CD44 variants expressed in a single cell may represent a precise postal code directing the final destination of migrating cells and metastatic tumors.

CD27 expression by a distinct subpopulation of human B lymphocytes

CD27 is present on the surface of a major subset of peripheral blood T lymphocytes. In this report we show that CD27 is also expressed on a subpopulation of the normal human B cell lineage which is absent from cord blood but present in tonsils and in the peripheral blood of adult individuals. CD27+ B lymphocytes are characterized by the following criteria: (a) in terms of physical properties, the CD27+ B cells form a population with an increased cell size combined with a decreased cell density; (b) the CD27 expression of tonsillar B lymphocytes is postively correlated with mIgA but negatively correlated with membrane IgM/membrane IgD positivity; (c) CD27 on B cells can be induced selectively by the combination of Staphylococcus aureus plus interleukin 2, but not by either treatment alone, and (d) CD27+ B lymphocytes express high levels of the adhesion structures LFA-1 (CD11a), ICAM-1 (CD54), LFA-3 (CD58) and of the lymphocyte homing receptor CD44. These latter findings suggest that CD27+ B cells are predispose to form cell-cell interactions. Accordingly, within 3 h of cell culture CD27+, but not CD27-, B lymphocytes were found to form LFA-1-mediated homotypic B cell clusters.