Carcinoembryonic antigen is a sialyl Lewis x/a carrier and an E‑selectin ligand in non‑small cell lung cancer

LPCC/Pfizer 2011. Tagus TANK award 2018 (grant no. 1/2018). SFRH/BD/100970/2014 SFRH/BPD/108686/2015The formation of distant metastasis resulting from vascular dissemination is one of the leading causes of mortality in non-small cell lung cancer (NSCLC). This metastatic dissemination initiates with the adhesion of circulating cancer cells to the endothelium. The minimal requirement for the binding of leukocytes to endothelial E-selectins and subsequent transmigration is the epitope of the fucosylated glycan, sialyl Lewis x (sLex), attached to specific cell surface glycoproteins. sLex and its isomer sialyl Lewis a (sLea) have been described in NSCLC, but their functional role in cancer cell adhesion to endothelium is still poorly understood. In this study, it was hypothesised that, similarly to leukocytes, sLe glycans play a role in NSCLC cell adhesion to E-selectins. To assess this, paired tumour and normal lung tissue samples from 18 NSCLC patients were analyzed. Immunoblotting and immunohistochemistry assays demonstrated that tumour tissues exhibited significantly stronger reactivity with anti‑sLex/sLea antibody and E-selectin chimera than normal tissues (2.2- and 1.8-fold higher, respectively), as well as a higher immunoreactive score. High sLex/sLea expression was associated with bone metastasis. The overall α1,3-fucosyltransferase (FUT) activity was increased in tumour tissues, along with the mRNA levels of FUT3, FUT6 and FUT7, whereas FUT4 mRNA expression was decreased. The expression of E-selectin ligands exhibited a weak but significant correlation with the FUT3/FUT4 and FUT7/FUT4 ratios. Additionally, carcinoembryonic antigen (CEA) was identified in only 8 of the 18 tumour tissues; CEA-positive tissues exhibited significantly increased sLex/sLea expression. Tumour tissue areas expressing CEA also expressed sLex/sLea and showed reactivity to E-selectin. Blot rolling assays further demonstrated that CEA immunoprecipitates exhibited sustained adhesive interactions with E-selectin-expressing cells, suggesting CEA acts as a functional protein scaffold for E-selectin ligands in NSCLC. In conclusion, this work provides the first demonstration that sLex/sLea are increased in primary NSCLC due to increased α1,3-FUT activity. sLex/sLea is carried by CEA and confers the ability for NSCLC cells to bind E-selectins, and is potentially associated with bone metastasis. This study contributes to identifying potential future diagnostic/prognostic biomarkers and therapeutic targets for lung cancer.preprintpublishe

Sialyl LewisX/A and Cytokeratin Crosstalk in Triple Negative Breast Cancer

project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB. Publisher Copyright: © 2023 by the authors.Triple-negative breast cancer (TNBC) encompasses multiple entities and is generally highly aggressive and metastatic. We aimed to determine the clinical and biological relevance of Sialyl-Lewis X and A (sLeX/A)—a fucosylated glycan involved in metastasis—in TNBC. Here, we studied tissues from 50 TNBC patients, transcripts from a TNBC dataset from The Cancer Genome Atlas (TCGA) database, and a primary breast cancer cell line. All 50 TNBC tissue samples analysed expressed sLeX/A. Patients with high expression of sLeX/A had 3 years less disease-free survival than patients with lower expression. In tissue, sLeX/A negatively correlated with cytokeratins 5/6 (CK5/6, which was corroborated by the inverse correlation between fucosyltransferases and CK5/6 genes. Our observations were confirmed in vitro when inhibition of sLeX/A remarkably increased expression of CK5/6, followed by a decreased proliferation and invasion capacity. Among the reported glycoproteins bearing sLeX/A and based on the STRING tool, α6 integrin showed the highest interaction score with CK5/6. This is the first report on the sLeX/A expression in TNBC, highlighting its association with lower disease-free survival and its inverse crosstalk with CK5/6 with α6 integrin as a mediator. All in all, sLeX/A is critical for TNBC malignancy and a potential prognosis biomarker and therapeutic target.publishersversionpublishe

CuMV VLPs Containing the RBM from SARS-CoV-2 Spike Protein Drive Dendritic Cell Activation and Th1 Polarization.

Dendritic cells (DCs) are the most specialized and proficient antigen-presenting cells. They bridge innate and adaptive immunity and display a powerful capacity to prime antigen-specific T cells. The interaction of DCs with the receptor-binding domain of the spike (S) protein from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pivotal step to induce effective immunity against the S protein-based vaccination protocols, as well as the SARS-CoV-2 virus. Herein, we describe the cellular and molecular events triggered by virus-like particles (VLPs) containing the receptor-binding motif from the SARS-CoV-2 spike protein in human monocyte-derived dendritic cells, or, as controls, in the presence of the Toll-like receptors (TLR)3 and TLR7/8 agonists, comprehending the events of dendritic cell maturation and their crosstalk with T cells. The results demonstrated that VLPs boosted the expression of major histocompatibility complex molecules and co-stimulatory receptors of DCs, indicating their maturation. Furthermore, DCs' interaction with VLPs promoted the activation of the NF-kB pathway, a very important intracellular signalling pathway responsible for triggering the expression and secretion of proinflammatory cytokines. Additionally, co-culture of DCs with T cells triggered CD4+ (mainly CD4+Tbet+) and CD8+ T cell proliferation. Our results suggested that VLPs increase cellular immunity, involving DC maturation and T cell polarization towards a type 1 T cells profile. By providing deeper insight into the mechanisms of activation and regulation of the immune system by DCs, these findings will enable the design of effective vaccines against SARS-CoV-2

Epigenetically controlled tumor antigens derived from splice junctions between exons and transposable elements

Oncogenesis often implicates epigenetic alterations, including derepression of transposable elements (TEs) and defects in alternative splicing. Here, we explore the possibility that noncanonical splice junctions between exons and TEs represent a source of tumor-specific antigens. We show that mouse normal tissues and tumor cell lines express wide but distinct ranges of mRNA junctions between exons and TEs, some of which are tumor specific. Immunopeptidome analyses in tumor cell lines identified peptides derived from exon-TE splicing junctions associated to MHC-I molecules. Exon-TE junction-derived peptides were immunogenic in tumor-bearing mice. Both prophylactic and therapeutic vaccinations with junction-derived peptides delayed tumor growth in vivo. Inactivation of the TE-silencing histone 3-lysine 9 methyltransferase Setdb1 caused overexpression of new immunogenic junctions in tumor cells. Our results identify exon-TE splicing junctions as epigenetically controlled, immunogenic, and protective tumor antigens in mice, opening possibilities for tumor targeting and vaccination in patients with cancer

Biomaterial-based platforms for in situ dendritic cell programming and their use in antitumor immunotherapy

Dendritic cells (DCs) are central players in the immune system, with an exquisite capacity to initiate and modulate immune responses. These functional characteristics have led to intense research on the development of DC-based immunotherapies, particularly for oncologic diseases. During recent decades, DC-based vaccines have generated very promising results in animal studies, and more than 300 clinical assays have demonstrated the safety profile of this approach. However, clinical data are inconsistent, and clear evidence of meaningful efficacy is still lacking. One of the reasons for this lack of evidence is the limited functional abilities of the used ex vivo-differentiated DCs. Therefore, alternative approaches for targeting and modulating endogenous DC subpopulations have emerged as an attractive concept. Here, we sought to revise the evolution of several strategies for the in situ mobilization and modulation of DCs. The first approaches using chemokine-secreting irradiated tumor cells are addressed, and special attention is given to the cutting-edge injectable bioengineered platforms, programmed to release chemoattractants, tumor antigens and DC maturating agents. Finally, we discuss how our increasing knowledge of DC biology, the use of neoantigens and their combination with immune checkpoint inhibitors can leverage the refinement of these polymeric vaccines to boost their antitumor efficacy

Review Challenges in Antibody Development against Tn and Sialyl-Tn Antigens

www.mdpi.com/journal/biomolecules

Dendritic Cell Vaccines for Cancer Immunotherapy: The Role of Human Conventional Type 1 Dendritic Cells

Throughout the last decades, dendritic cell (DC)-based anti-tumor vaccines have proven to be a safe therapeutic approach, although with inconsistent clinical results. The functional limitations of ex vivo monocyte-derived dendritic cells (MoDCs) commonly used in these therapies are one of the pointed explanations for their lack of robustness. Therefore, a great effort has been made to identify DC subsets with superior features for the establishment of effective anti-tumor responses and to apply them in therapeutic approaches. Among characterized human DC subpopulations, conventional type 1 DCs (cDC1) have emerged as a highly desirable tool for empowering anti-tumor immunity. This DC subset excels in its capacity to prime antigen-specific cytotoxic T cells and to activate natural killer (NK) and natural killer T (NKT) cells, which are critical factors for an effective anti-tumor immune response. Here, we sought to revise the immunobiology of cDC1 from their ontogeny to their development, regulation and heterogeneity. We also address the role of this functionally thrilling DC subset in anti-tumor immune responses and the most recent efforts to apply it in cancer immunotherapy

Dendritic Cell Vaccines for Cancer Immunotherapy: The Role of Human Conventional Type 1 Dendritic Cells

Throughout the last decades, dendritic cell (DC)-based anti-tumor vaccines have proven to be a safe therapeutic approach, although with inconsistent clinical results. The functional limitations of ex vivo monocyte-derived dendritic cells (MoDCs) commonly used in these therapies are one of the pointed explanations for their lack of robustness. Therefore, a great effort has been made to identify DC subsets with superior features for the establishment of effective anti-tumor responses and to apply them in therapeutic approaches. Among characterized human DC subpopulations, conventional type 1 DCs (cDC1) have emerged as a highly desirable tool for empowering anti-tumor immunity. This DC subset excels in its capacity to prime antigen-specific cytotoxic T cells and to activate natural killer (NK) and natural killer T (NKT) cells, which are critical factors for an effective anti-tumor immune response. Here, we sought to revise the immunobiology of cDC1 from their ontogeny to their development, regulation and heterogeneity. We also address the role of this functionally thrilling DC subset in anti-tumor immune responses and the most recent efforts to apply it in cancer immunotherapy

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