Mariana Silva

Overexpression of tumour-associated carbohydrate antigen sialyl-Tn in advanced bladder tumours

Little is known on the expression of the tumour‐associated carbohydrate antigen sialyl‐Tn (STn), in bladder cancer. We report here that 75% of the high‐grade bladder tumours, presenting elevated proliferation rates and high risk of recurrence/progression expressed STn. However, it was mainly found in non‐proliferative areas of the tumour, namely in cells invading the basal and muscle layers. STn was also found in tumour‐adjacent mucosa, which suggests its dependence on a field effect of the tumour. Furthermore, it was not expressed by the normal urothelium, demonstrating the cancer‐specific nature of this antigen. STn expression correlated with that of sialyltransferase ST6GalNAc.I, its major biosynthetic enzyme. The stable expression of ST6GalNAc.I in the bladder cancer cell line MCR induced STn expression and a concomitant increase of cell motility and invasive capability. Altogether, these results indicate for the first time a link between STn expression and malignancy in bladder cancer. Hence, therapies targeting STn may constitute new treatment approaches for these tumours.

Sialyl Tn-expressing bladder cancer cells induce a tolerogenic phenotype in innate and adaptive immune cells

Despite the wide acceptance that glycans are centrally implicated in immunity, exactly how they contribute to the tilt immune response remains poorly defined. In this study, we sought to evaluate the impact of the malignant phenotype‐associated glycan, sialyl‐Tn (STn) in the function of the key orchestrators of the immune response, the dendritic cells (DCs). In high grade bladder cancer tissue, the STn antigen is significantly overexpressed and correlated with the increased expression of ST6GALNAC1 sialyltransferase. Bladder cancer tissue presenting elevated expression of ST6GALNAC1 showed a correlation with increased expression of CD1a, a marker for bladder immature DCs and showed concomitant low levels of Th1‐inducing cytokines IL‐12 and TNF‐α. In vitro, human DCs co‐incubated with STn+ bladder cancer cells, had an immature phenotype (MHC‐IIlow, CD80low and CD86low) and were unresponsive to further maturation stimuli. When contacting with STn+ cancer cells, DCs expressed significantly less IL‐12 and TNF‐α. Consistent with a tolerogenic DC profile, T cells that were primed by DCs pulsed with antigens derived from STn+ cancer cells were not activated and showed a FoxP3high IFN‐γlow phenotype. Blockade of STn antigens and of STn+ glycoprotein, CD44 and MUC1, in STn+ cancer cells was able to lower the induction of tolerance and DCs become more mature.

The phagocytic capacity and immunological potency of human dendritic cells is improved by α2,6-sialic acid deficiency.

Dendritic cells (DCs) play an essential role in immunity against bacteria by phagocytosis and by eliciting adaptive immune responses. Previously, we demonstrated that human monocyte‐derived DCs (MDDCs) express a high content of cell surface α2,6‐sialylated glycans. However, the relative role of these sialylated structures in phagocytosis of bacteria has not been reported. Here, we show that treatment with a sialidase significantly improved the capacity of both immature and mature MDDCs to phagocytose Escherichia coli. Desialylated MDDCs had a significantly more mature phenotype, with higher expression of MHC molecules and interleukin (IL)‐12, tumour necrosis factor‐α, IL‐6 and IL‐10 cytokines, and nuclear factor‐κB activation. T lymphocytes primed by desialylated MDDCs expressed more interferon‐γ when compared with priming by sialylated MDDCs. Improved phagocytosis required E. coli sialic acids, indicating a mechanism of host–pathogen interaction dependent on sialic acid moieties. The DCs harvested from mice deficient in the ST6Gal.1 sialyltransferase showed improved phagocytosis capacity, demonstrating that the observed sialidase effect was a result of the removal of α2,6‐sialic acid. The phagocytosis of different pathogenic E. coli isolates was also enhanced by sialidase, which suggests that modifications on MDDC sialic acids may be considered in the development of MDDC‐based antibacterial therapies. Physiologically, our findings shed new light on mechanisms that modulate the function of both immature and mature MDDCs, in the context of host–bacteria interaction. Hence, with particular relevance to DC‐based therapies, the engineering of α2,6‐sialic acid cell surface is a novel possibility to fine tune DC phagocytosis and immunological potency.

Inhibition of fucosylation in human invasive ductal carcinoma reduces E‐selectin ligand expression, cell proliferation, and ERK1/2 and p38 MAPK activation

Breast cancer tissue overexpresses fucosylated glycans, such as sialyl‐Lewis X/A (sLeX/A), and α‐1,3/4‐fucosyltransferases (FUTs) in relation to increased disease progression and metastasis. These glycans in tumor circulating cells mediate binding to vascular E‐selectin, initiating tumor extravasation. However, their role in breast carcinogenesis is still unknown. Here, we aimed to define the contribution of the fucosylated structures, including sLeX/A, to cell adhesion, cell signaling, and cell proliferation in invasive ductal carcinomas (IDC), the most frequent type of breast cancer. We first analyzed expression of E‐selectin ligands in IDC tissue and established primary cell cultures from the tissue. We observed strong reactivity with E‐selectin and anti‐sLeX/A antibodies in both IDC tissue and cell lines, and expression of α‐1,3/4 FUTs FUT4, FUT5, FUT6, FUT10, and FUT11. To further assess the role of fucosylation in IDC biology, we immortalized a primary IDC cell line with human telomerase reverse transcriptase to create the ‘CF1_T cell line’. Treatment with 2‐fluorofucose (2‐FF), a fucosylation inhibitor, completely abrogated its sLeX/A expression and dramatically reduced adherence of CF1_T cells to E‐selectin under hemodynamic flow conditions. In addition, 2‐FF‐treated CF1_T cells showed a reduced migratory ability, as well as decreased cell proliferation rate. Notably, 2‐FF treatment lowered the growth factor expression of CF1_T cells, prominently for FGF2, vascular endothelial growth factor, and transforming growth factor beta, and negatively affected activation of signal‐regulating protein kinases 1 and 2 and p38 mitogen‐activated protein kinase signaling pathways. These data indicate that fucosylation licenses several malignant features of IDC, such as cell adhesion, migration, proliferation, and growth factor expression, contributing to tumor progression.

Cell-Specific Variation in E-Selectin Ligand Expression among Human Peripheral Blood Mononuclear Cells: Implications for Immunosurveillance and Pathobiology

Both host defense and immunopathology are shaped by the ordered recruitment of circulating leukocytes to affected sites, a process initiated by binding of blood-borne cells to E-selectin displayed at target endothelial beds. Accordingly, knowledge of the expression and function of leukocyte E-selectin ligands is key to understanding the tempo and specificity of immunoreactivity. In this study, we performed E-selectin adherence assays under hemodynamic flow conditions coupled with flow cytometry and Western blot analysis to elucidate the function and structural biology of glycoprotein E-selectin ligands expressed on human PBMCs. Circulating monocytes uniformly express high levels of the canonical E-selectin binding determinant sialyl Lewis X (sLeX) and display markedly greater adhesive interactions with E-selectin than do circulating lymphocytes, which exhibit variable E-selectin binding among CD4+ and CD8+ T cells but no binding by B cells. Monocytes prominently present sLeX decorations on an array of protein scaffolds, including P-selectin glycoprotein ligand-1, CD43, and CD44 (rendering the E-selectin ligands cutaneous lymphocyte Ag, CD43E, and hematopoietic cell E-selectin/L-selectin ligand, respectively), and B cells altogether lack E-selectin ligands. Quantitative PCR gene expression studies of glycosyltransferases that regulate display of sLeX reveal high transcript levels among circulating monocytes and low levels among circulating B cells, and, commensurately, cell surface α(1,3)-fucosylation reveals that acceptor sialyllactosaminyl glycans convertible into sLeX are abundantly expressed on human monocytes yet are relatively deficient on B cells. Collectively, these findings unveil distinct cell-specific patterns of E-selectin ligand expression among human PBMCs, indicating that circulating monocytes are specialized to engage E-selectin and providing key insights into the molecular effectors mediating recruitment of these cells at inflammatory sites.

E-Selectin Ligands in the Human Mononuclear Phagocyte System: Implications for Infection, Inflammation, and Immunotherapy

The mononuclear phagocyte system comprises a network of circulating monocytes and dendritic cells (DCs), and “histiocytes” (tissue-resident macrophages and DCs) that are derived in part from blood-borne monocytes and DCs. The capacity of circulating monocytes and DCs to function as the body’s first-line defense against offending pathogens greatly depends on their ability to egress the bloodstream and infiltrate inflammatory sites. Extravasation involves a sequence of coordinated molecular events and is initiated by E-selectin-mediated deceleration of the circulating leukocytes onto microvascular endothelial cells of the target tissue. E-selectin is inducibly expressed by cytokines (tumor necrosis factor-α and IL-1β) on inflamed endothelium, and binds to sialofucosylated glycan determinants displayed on protein and lipid scaffolds of blood cells. Efficient extravasation of circulating monocytes and DCs to inflamed tissues is crucial in facilitating an effective immune response, but also fuels the immunopathology of several inflammatory disorders. Thus, insights into the structural and functional properties of the E-selectin ligands expressed by different monocyte and DC populations is key to understanding the biology of protective immunity and the pathobiology of several acute and chronic inflammatory diseases. This review will address the role of E-selectin in recruitment of human circulating monocytes and DCs to sites of tissue injury/inflammation, the structural biology of the E-selectin ligands expressed by these cells, and the molecular effectors that shape E-selectin ligand cell-specific display. In addition, therapeutic approaches targeting E-selectin receptor/ligand interactions, which can be used to boost host defense or, conversely, to dampen pathological inflammatory conditions, will also be discussed.

Oxidative damage and response to Bacillus Calmette-Guérin in bladder cancer cells expressing sialyltransferase ST3GAL1

BackgroundTreatment with Bacillus Calmette-Guérin (BCG) is the gold standard adjuvant immunotherapy of non-muscle invasive bladder cancer (NMIBC), although it fails in one third of the patients. NMIBC expresses two tumor-associated O-linked carbohydrates: the disaccharide (Galβ1,3GalNAc) Thomsen-Friedenreich (T) antigen, and its sialylated counterpart (Siaα2,3Galβ1,3GalNAc) sialyl-T (sT), synthesized by sialyltransferase ST3GAL1, whose roles in BCG response are unknown.MethodsThe human bladder cancer (BC) cell line HT1376 strongly expressing the T antigen, was retrovirally transduced with the ST3GAL1 cDNA or with an empty vector, yielding the cell lines HT1376sT and HT1376T, that express, respectively, either the sT or the T antigens. Cells were in vitro challenged with BCG. Whole gene expression was studied by microarray technology, cytokine secretion was measured by multiplex immune-beads assay. Human macrophages derived from blood monocytes were challenged with the secretome of BCG-challenged BC cells.ResultsThe secretome from BCG-challenged HT1376sT cells induced a stronger macrophage secretion of IL-6, IL-1β, TNFα and IL-10 than that of HT1376T cells. Transcriptomic analysis revealed that ST3GAL1 overexpression and T/sT replacement modulated hundreds of genes. Several genes preserving genomic stability were down-regulated in HT1376sT cells which, as a consequence, displayed increased sensitivity to oxidative damage. After BCG challenge, the transcriptome of HT1376sT cells showed higher susceptibility to BCG modulation than that of HT1376T cells.ConclusionsHigh ST3GAL1 expression and T/sT replacement in BCG challenged-BC cancer cells induce a stronger macrophage response and alter the gene expression towards genomic instability, indicating a potential impact on BC biology and patient’s response to BCG.

Expression of sialyl-Tn sugar antigen in bladder cancer cells affects response to Bacillus Calmette Guérin (BCG) and to oxidative damage

The sialyl-Tn (sTn) antigen is an O-linked carbohydrate chain aberrantly expressed in bladder cancer (BC), whose biosynthesis is mainly controlled by the sialyltransferase ST6GALNAC1. Treatment with Bacillus Calmette-Guérin (BCG) is the most effective adjuvant immunotherapy for superficial BC but one third of the patients fail to respond. A poorly understood correlation between the expression of sTn and BC patients response to BCG was previously observed. By analyzing tumor tissues, we showed that patients with high ST6GALNAC1 and IL-6 mRNA expression were BCG responders. To investigate the role of sTn in BC cell biology and BCG response, we established the cell lines MCRsTn and MCRNc by retroviral transduction of the BC cell line MCR with the ST6GALNAC1 cDNA or with an empty vector, respectively. Compared with MCRNc, BCG-stimulated MCRsTn secreted higher levels of IL-6 and IL-8 and their secretome induced a stronger IL-6, IL-1β, and TNFα secretion by macrophages, suggesting the induction of a stronger inflammatory response. Transcriptomic analysis of MCRNc and MCRsTn revealed that ST6GALNAC1/sTn expression modulates hundreds of genes towards a putative more malignant phenotype and down-regulates several genes maintaining genomic stability. Consistently, MCRsTn cells displayed higher H2O2 sensitivity. In MCRsTn,, BCG challenge induced an increased expression of several regulatory non coding RNA genes. These results indicate that the expression of ST6GALNAC1/sTn improves the response to BCG therapy by inducing a stronger macrophage response and alters gene expression towards malignancy and genomic instability, increasing the sensitivity of BC cells to the oxidizing agents released by BCG.The sialyl-Tn (sTn) antigen is an O-linked carbohydrate chain aberrantly expressed in bladder cancer (BC), whose biosynthesis is mainly controlled by the sialyltransferase ST6GALNAC1. Treatment with Bacillus Calmette-Guérin (BCG) is the most effective adjuvant immunotherapy for superficial BC but one third of the patients fail to respond. A poorly understood correlation between the expression of sTn and BC patients response to BCG was previously observed. By analyzing tumor tissues, we showed that patients with high ST6GALNAC1 and IL-6 mRNA expression were BCG responders. To investigate the role of sTn in BC cell biology and BCG response, we established the cell lines MCRsTn and MCRNc by retroviral transduction of the BC cell line MCR with the ST6GALNAC1 cDNA or with an empty vector, respectively. Compared with MCRNc, BCG-stimulated MCRsTn secreted higher levels of IL-6 and IL-8 and their secretome induced a stronger IL-6, IL-1β, and TNFα secretion by macrophages, suggesting the induction of a stronger inflammatory response. Transcriptomic analysis of MCRNc and MCRsTn revealed that ST6GALNAC1/sTn expression modulates hundreds of genes towards a putative more malignant phenotype and down-regulates several genes maintaining genomic stability. Consistently, MCRsTn cells displayed higher H2O2 sensitivity. In MCRsTn,, BCG challenge induced an increased expression of several regulatory non coding RNA genes. These results indicate that the expression of ST6GALNAC1/sTn improves the response to BCG therapy by inducing a stronger macrophage response and alters gene expression towards malignancy and genomic instability, increasing the sensitivity of BC cells to the oxidizing agents released by BCG.

Could a plant derived protein potentiate the anticancer effects of a stem cell in brain cancer

Glioblastoma is the most aggressive brain tumor with poor overall survival bellow 2 years. The natural compounds with anti-cancer properties, are thus gaining attention for possible adjuvant GBM treatment. In various cancer models Enterolobium contortisiliquum Trypsin Inhibitor (EcTI) proved to have anti-cancer effects. Here, we investigated the EcTI effects on GBM U87 cells and on mesenchymal stem cells (MSC) compared to their direct coculture (MSC/U87). MSC are present in tumor stroma, modulating GBM cells phenotype, and also represent potential drug delivery vehicle due to their tumor tropism. We showed that in p53-wild type U87 cells, metabolic activity was less affected by EcTI as in MSC monocuture, but the metabolic rate of mixed coculture was significantly reduced at lower EcTI concentration. Under coculture condition, EcTI potentiated MSC induced cell cycle arrest, possible due to highly increased p53, p21 and lower D1 expression, but there was no effect on apoptosis. Accordingly, in the coculture EcTI also enhanced Ca2+ signalling mediated via bradykinin receptor 2, being associated with nitric oxide release that highly impaired proliferation and invasion. The mechanism did not seem to involve changes in cell adhesion but rather it down-regulated the β1 integrin signaling with associated p-FAK in U87 cells, both supporting inhibition of invasion. Finally, some cytokines were down-regulated, indicating that EcTI inhibition of signalling might be mediated by cytokines. In conclusion, these results indicate that in cocultured MSC/U87 cells EcTI impairs the metabolic activity, proliferation, and reduced invasion, possibly associated with observed cytokines secretion. In this context, we confirmed that the plant derived protein potentiated the anticancer effects, induced by MSC, as represented by GBM U87 cell line.

Ligation of the CD44 Glycoform HCELL on Culture-Expanded Human Monocyte-Derived Dendritic Cells Programs Transendothelial Migration

The success of dendritic cell (DC)–based immunotherapeutics critically hinges on the capacity of the vascularly administered cells to enter tissues. Transendothelial migration (TEM) is dictated by an ordered cascade of receptor/ligand interactions. In this study, we examined the key molecular effectors of TEM of human monocyte-derived DCs (mo-DCs) generated by clinically relevant methods: CD14 selection (CD14-S) and plastic adherence selection (PA-S). Without chemokine input, CD14-S cells undergo greater TEM than PA-S cells over TNF-α–stimulated HUVECs. TEM of CD14-S mo-DCs is E-selectin/very late Ag-4 (VLA-4) dependent, and engagement of E-selectin ligands activates VLA-4 on CD14-S mo-DCs but not on PA-S mo-DCs. E-selectin binding glycoforms of P-selectin glycoprotein ligand-1 (PSGL-1) (i.e., cutaneous lymphocyte Ag [CLA]) and CD44 (i.e., hematopoietic cell E-selectin/L-selectin ligand [HCELL]) are both expressed on CD14-S mo-DCs, but only CLA is expressed on PA-S mo-DCs. To elucidate the effect of CD44 or PSGL-1 engagement, mo-DCs were pretreated with their ligands. Ligation of CD44 on CD14-S mo-DCs triggers VLA-4 activation and TEM, whereas PSGL-1 ligation does not. HCELL expression on CD14-S mo-DC can be enforced by cell surface exofucosylation, yielding increased TEM in vitro and enhanced extravasation into bone marrow in vivo. These findings highlight structural and functional pleiotropism of CD44 in priming TEM of mo-DCs and suggest that strategies to enforce HCELL expression may boost TEM of systemically administered CD14-S mo-DCs.