Estudo da resposta imunológica desencadeada por células de cancro de bexiga que expressam antigénios sialil-Tn

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do Grau de Mestre em Genética Molecular e BiomedicinaAs células dendríticas (DCs) conseguem capturar antigénios tumorais e, se propriamente maturadas, activar linfócitos T específicos capazes de eliminar o tumor. No entanto, quando presentes nos tumores, apresentam um fenótipo pouco maduro, tornando-se frequentemente tolerogénicas. O antigénio sialil-Tn (sTn), expresso em diversos tipos de cancro, medeia interacções entre células, podendo influenciar o reconhecimento imune. O objectivo deste trabalho consistiu em avaliar se o sTn afecta a interacção das células tumorais com DCs, influenciando a função imunológica destas. Como modelo utilizaram-se, a linha celular, MCRpLenti.ST6GN1, sobreexpressando a sialiltransferase ST6GalNac.I e, consequentemente, sTn, e a linha controlo negativo MCRpLenti.transd. Verificou-se que as DCs tendem a aderir mais a células MCRpLenti.ST6GN1. A análise da expressão de moléculas apresentadoras de antigénios e co-estimulatórias, revelou que as DCs que aderiram a MCRpLenti.ST6GN1 apresentavam um perfil significativamente menos maduro, comparativamente a MCRpLenti.transd, sendo este fenómeno dependente da interacção célula-célula. Quando apoptóticas as células MCRpLenti.ST6GN1 são mais facilmente fagocitadas pelas DCs, potencialmente promovendo tolerância. Estes resultados sugerem que o antigénio sTn protege as células tumorais, suprimindo a imunogenicidade das DCs e favorecendo a tolerância imune. Pelo facto de promover a fagocitose, o sTn nos tumores poderá ser vantajoso no estabelecimento de uma terapia anti-cancro com DCs

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

Role of sialylated glycans in cancer progression : sialyl-Tn and sialyl-Lewisx

ABSTRACT: Glycosylation is a posttranslational modification of proteins and lipids, able to regulate a diversity of biological processes. Changes in glycosylation result in alterations in these processes and have been associated with cancer development and progression. One of the most commonly reported change in cancer glycosylation is an increased sialylation, which have been associated with malignant features and poor prognosis. Since sialic acid is a terminal sugar, it is normally involved in numerous biological processes, such as cellular recognition, cell adhesion and cell-to-cell signaling. In cancer cells, the increase of sialylated glycans seems to affect several cell-cell mechanisms, promoting cancer survival. However, the role of sialylated glycans in cancer progression is still poorly understood. In this thesis, we aimed to clarify the role of two specific sialylated glycans, sialyl-Tn (STn) and sialyl-Lewis X/A (sLeX/A), which are typically overexpressed in cancer. We aimed to understand whether they contribute to cancer progression, in particular in the recognition of tumor cells by immune cells and in the process of invasion and metastasis. In the immune recognition of cancer, dendritic cells (DCs) play a unique and decisive role in tumor immunity, being capable of activating antigen-specific cytotoxic T cells. However, to efficiently prime T cells, DCs have to undergo maturation, which is usually prevented by the tumor environment through many immunosuppressive strategies. STn glycan is expressed by more than 80% of human carcinomas and is associated with many malignant features, such as invasiveness and proliferation. Clinically, STn has been used as a target for cancer therapeutic strategies; however, STn-based vaccines tested in clinical trials have not been as efficient as expected, highlighting the low immunogenicity of STn. Here, we showed that STn expression in bladder cancer tissues is associated with an immature DCs phenotype. Using bladder cancer cells overexpressing ST6GalNAc1, which leads to STn expression, we demonstrated that DCs in contact with STn+ cancer cells have a more immature profile, expressing less MHC-II antigen presenting molecules and co-stimulatory molecules (e.g. CD80 and CD86), and producing less proinflammatory cytokines, in comparison with DCs with STn- cancer cells. Although DCs were able to uptake more STn+ apoptotic cancer cells, it led to a reduced ability to activate T cells. These findings showed that the expression of STn glycan in cancer cells hinder the immune response by inducing a more tolerogenic profile in DCs. In addition, we also verified that antibodies against STn, or its protein carriers (CD44 and MUC1), seem to revert this immature phenotype in DCs, suggesting that antibodies can be used to overcome the low immunogenicity of STn+ cancer cells. Regarding the sLeX/A antigens, we focused in their role as E-selectin ligand in breast cancer, as an important step for cell migration. Both sLeX and sLeA are the most common glycan structure in the glycoconjugates (proteins or lipids) able to bind E-selectin, which is expressed upon inflammation on vascular endothelium and is crucial during the process of extravasation of cancer cells from the bloodstream to form new metastasis. In order to better understand the role of these glycans in cancer progression, in particular the mechanisms mediated by E-selectin ligands (sLeX/A) in breast cancer, we established new primary breast cancer cell cultures from invasive ductal cancer tissues, which preliminary results had shown to have higher expression of E-selectin ligands. To understand these mechanisms, we inhibited sLeX/A expression by using a fucosylation inhibitor, named 2-fluorofucose (2-FF), in breast cancer cells. In the cell line showing higher expression of E-selecting ligands, namely the CF1_T cell line, we proved that 2-FF inhibitor abrogated the expression of functional E-selectin ligands, as well as reduced the proliferation index and migration capacity of the cells. We also showed that CF1_T cells treated with 2-FF inhibitor expressed lower levels of growth factors and consequently, ERK1/2 and p38 signaling pathways were less activated than in untreated cells, suggesting that the expression of E-selectin ligands, such as sLeX/A, has a major impact on malignant features, such as cell signaling/proliferation, adhesion and migration, contributing to tumor progression. E-selectin engagement is not only dependent on the sLeX/A glycan structures but also on its carrier molecules, whose nature is still poorly known in breast cancer. The identification of these molecules may reveal potential novel targets in therapy to circumvent cancer progression. Here, through the characterization of the glycoconjugates decorated with sLeX/A expressed by CF1_T breast cancer cell line, we revealed that they were mainly N-glycosylated proteins. Their nature was identified by mass spectrometry after immunoprecipitation with E-selectin chimera. From those, CD44 and CD13 glycoproteins were confirmed, by western blot, as carriers of sLeX/A glycans. CD44 had already been identified as an E-selectin ligand in cancer; however, CD13 glycoprotein, although being associated to cancer, has never before been described as being able to bind E-selectin. While further studies are still necessary to elucidate the exact contribution of CD13, this study suggest this glycoprotein as a potential interesting target for novel anticancer therapy. Within breast cancer subtypes, the triple negative breast cancer (TNBC) has a more aggressive course and increased probability of distant recurrence. So far the expression of sLeX/A glycans and general E-selectin ligands in TNBC is unknown. To address this, we develop a staining protocol using E-selectin chimera by immunohistochemistry. Our results showed that E-selectin ligands, such as sLeX/A, expression is negatively correlated with the expression of CK5/6, which is a basal-like biomarker associated with better survival rate in TNBC patients. The relevance of this association of E-selectin ligands and CK5/6 expression in TNBC are still under investigation. Overall, our findings have contributed to elucidate the role of two sialylated glycans, STn and sLeX/A, in cancer progression. Specifically, the data reported here shed light into the putative role of STn in the immunological escape of tumor cells and explore the role of E-selectin ligands, mainly sLeX/A, on cancer ability to growth and develop metastasis, pointing out to novel potential therapeutic targets in cancer.RESUMO: A glicosilação é uma modificação pós-traducional de proteínas e lípidos, capaz de regular uma diversidade de processos biológicos. As alterações na glicosilação resultam na alteração destes processos e têm sido associados com o desenvolvimento e progressão do cancro. Uma das alterações mais frequentemente relatadas na glicosilação em cancro é o aumento da sialilação, o qual tem sido associado a características malignas e a um mau prognóstico. Uma vez que o ácido siálico é um açúcar terminal, encontra-se normalmente envolvido em inúmeros processos biológicos, tais como o reconhecimento celular, a adesão celular e sinalização célula-a-célula. Em células cancerígenas, o aumento dos glicanos sialilados parece afetar vários mecanismos celulares, promovendo a sobrevivência do cancro. No entanto, o papel dos glicanos sialilados na progressão do cancro não é ainda totalmente compreendido. Nesta tese, pretendemos elucidar o papel de dois glicanos sialilados, o sialil-Tn (STn) e o sialil-Lewis X/A (sLeX/A), os quais estão tipicamente sobrexpressos em cancro. O nosso objetivo consistiu em compreender se eles contribuem para a progressão do cancro, em particular no reconhecimento de células tumorais por células imunológicas e no processo de invasão e metástases. No reconhecimento imunológico do cancro, as células dendríticas (DCs) desempenham um papel único e decisivo na imunidade tumoral, sendo capazes de ativar linfócitos T citotóxicos específicos. No entanto, de forma a ativar eficientemente os linfócitos T, as DCs têm de passar por um processo de maturação, o qual é geralmente comprometido pelo ambiente tumoral através de várias estratégicas imunossupressoras. O glicano STn é expresso em mais de 80% dos carcinomas humanos e está associado com muitas características malignas, tais como a capacidade de invasão e proliferação. Clinicamente, o STn tem sido utilizado como um alvo para estratégias terapêuticas em cancro; no entanto, vacinas com base no STn testadas em ensaios clínicos não foram tão eficientes quanto o esperado, destacando a baixa imunogenicidade do STn. Aqui mostramos que a expressão do STn em tecidos de cancro de bexiga está associada com um fenótipo de DCs imaturas. Usando células de cancro de bexiga que sobrexpressam ST6GalNAc1, o qual leva à expressão de STn, demonstrámos que as DCs em contacto com as células tumorais STn+ têm um perfil mais imaturo, expressando menos moléculas de apresentação de antigénio MHC-II e moléculas co-estimuladoras (por exemplo, CD80 e CD86), além de produzirem menos citocinas pró-inflamatórias em comparação com as DCs na presença de células tumorais STn-. Embora as DCs tenham sido capazes de internalizar mais células tumorais STn+ apoptóticas, isto levou a uma redução da sua capacidade de ativar os linfócitos T. Estes resultados demonstraram que a expressão do glicano STn em células tumorais compromete a resposta imunológica através da indução de um perfil mais tolerogénico nas DCs. Além disso, nós também verificámos que o uso de anticorpos contra STn, ou contra as proteínas decoradas por este glicano (CD44 e MUC1), parece reverter esse fenótipo mais imaturo das DCs, sugerindo que anticorpos podem ser usados de forma a ultrapassar a baixa imunegenicidade das células de cancro STn+. Em relação aos antigénios sLeX/A, nós focámo-nos no seu papel como ligando de Eselectina em cancro da mama, como um importante passo na migração celular. Ambos sLeX e sLeA são os glicanos mais comuns que decoram glicoconjugados (proteínas ou lípidos) com capacidade de ligação à E-selectina, que é expressa no endotélio vascular inflamado e é crucial no processo de extravasão de células de cancro da corrente sanguínea para formar novas metástases. A fim de melhor compreender o papel destes glicanos na progressão do cancro, em particular os mecanismos mediados por ligandos de E-selectina (sLeX/A) no cancro da mama, estabelecemos novas culturas celulares primárias de cancro de mama a partir de tecidos de cancro ductal invasivo, as quais resultados preliminares tinham demonstrado ter maior expressão de ligandos de Eselectina. Para melhor compreender estes mecanismos, nós inibimos a expressão de sLeX/A através da utilização de um inibidor de fucosilação, denominado 2-fluorofucose (2-FF), em células de cancro da mama. Na linha celular com maior expressão de ligandos de E-selectina, nomeadamente a linha celular CF1_T, provámos que o inibidor 2-FF impede a expressão de ligandos funcionais de E-selectina, bem como reduz o índice de proliferação e a capacidade de migração das células. Também mostrámos que as células CF1_T tratadas com o inibidor 2-FF expressam baixos níveis de fatores de crescimento e, consequentemente, as suas vias de sinalização ERK1/2 e p38 estão menos ativadas do que as das células não tratadas, sugerindo que a expressão de ligandos de E-selectina, tal como sLeX/A, tem um grande impacto nas características de malignidade, tais como sinalização/proliferação celular, migração e adesão, as quais contribuem para a progressão do tumor. A ligação a E-selectina não é só dependente do glicano sLeX/A, mas também das moléculas que estão decoradas com este glicano, cuja natureza é pouco conhecida no cancro da mama. A identificação destas moléculas pode revelar potenciais novos alvos em terapia para contornar a progressão do cancro. Aqui, através da caracterização dos glicoconjugados decorados com sLeX/A que são expressos pela linha celular de cancro da mama CF1_T, nós revelámos que são principalmente proteínas N-glicosiladas. A sua natureza foi identificada por espectrometria de massa depois de imunoprecipitação com uma molécula quimérica de E-selectina. Entre estas, as glicoproteínas CD44 e CD13 foram confirmadas, por Western blot, como estando decoradas com o glicano sLeX/A. A glicoproteina CD44 já foi antes identificada como ligando de E-selectina em cancro; no entanto, a glicoproteína CD13, apesar de ter sido associada a cancro, nunca foi antes descrita como sendo capaz de se ligar a E-selectina. Embora mais estudos sejam ainda necessários para elucidar a contribuição exata de CD13, este estudo sugere esta glicoproteína como um potencial interessante alvo para novas terapias anti-cancro. Dentro dos subtipos de cancro da mama, o cancro da mama triplo negativo (TNBC) tem um fenótipo mais agressivo e uma maior probabilidade de recorrência. Até agora, a expressão de glicanos sLeX/A e de todos os ligandos de E-selectina em TNBC é desconhecida. Para resolver isto, nós desenvolvemos um protocolo usando a molécula quimérica de E-selectina por imunohistoquímica. Os nossos resultados mostraram que a expressão de ligandos de E-selectina, como sLeX/A, está negativamente correlacionada com a expressão de CK5/6, o qual é um biomarcador do subtipo basal-like associado a uma melhor taxa de sobrevivência em doentes com TNBC. A relevância desta associação entre a expressão de ligandos de E-selectina e CK5/6 em TNBC está ainda sob investigação. No geral, os nossos resultados contribuíram para elucidar o papel de dois glicanos sialilados, STn e sLeX/A, na progressão do cancro. Especificamente, os dados aqui reportados explicam o papel putativo do STn no escape imunológico das células tumorais e exploram o papel dos ligandos de E-selectina, principalmente sLeX/A, na capacidade de crescimento do cancro e no desenvolvimento de metástases, apontando novos potenciais alvos terapêuticos em cancro

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