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Hepatocellular carcinoma (HCC) mortality rates continue to increase faster than those of other cancer types due to high heterogeneity, which limits diagnosis and treatment. Pathological and molecular subtyping have identified that HCC tumors with poor outcomes are characterized by intratumoral collagenous accumulation. However, the translational and post-translational regulation of tumor collagen, which is critical to the outcome, remains largely unknown. Here, we investigate the spatial extracellular proteome to understand the differences associated with HCC tumors defined by Hoshida transcriptomic subtypes of poor outcome (Subtype 1; S1; n = 12) and better outcome (Subtype 3; S3; n = 24) that show differential stroma-regulated pathways. Collagen-targeted mass spectrometry imaging (MSI) with the same-tissue reference libraries, built from untargeted and targeted LC-MS/MS was used to spatially define the extracellular microenvironment from clinically-characterized, formalin-fixed, paraffin-embedded tissue sections. Collagen α-1(I) chain domains for discoidin-domain receptor and integrin binding showed distinctive spatial distribution within the tumor microenvironment. Hydroxylated proline (HYP)-containing peptides from the triple helical regions of fibrillar collagens distinguished S1 from S3 tumors. Exploratory machine learning on multiple peptides extracted from the tumor regions could distinguish S1 and S3 tumors (with an area under the receiver operating curve of ≥0.98; 95% confidence intervals between 0.976 and 1.00; and accuracies above 94%). An overall finding was that the extracellular microenvironment has a high potential to predict clinically relevant outcomes in HCC.
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An overview of the role of glycosylation in prostate cancer (PCa) development and progression is presented, focusing on recent advancements in defining the N-glycome through glycomic profiling and glycoproteomic methodologies. Glycosylation is a common post-translational modification typified by oligosaccharides attached N-linked to asparagine or O-linked to serine or threonine on carrier proteins. These attached sugars have crucial roles in protein folding and cellular recognition processes, such that altered glycosylation is a hallmark of cancer pathogenesis and progression. In the past decade, advancements in N-glycan profiling workflows using Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) technology have been applied to define the spatial distribution of glycans in PCa tissues. Multiple studies applying N-glycan MALDI-MSI to pathology-defined PCa tissues have identified significant alterations in N-glycan profiles associated with PCa progression. N-glycan compositions progressively increase in number, and structural complexity due to increased fucosylation and sialylation. Additionally, significant progress has been made in defining the glycan and glycopeptide compositions of prostatic-derived glycoproteins like prostate-specific antigen in tissues and biofluids. The glycosyltransferases involved in these changes are potential drug targets for PCa, and new approaches in this area are summarized. These advancements will be discussed in the context of the further development of clinical diagnostics and therapeutics targeting glycans and glycoproteins associated with PCa progression. Integration of large scale spatial glycomic data for PCa with other spatial-omic methodologies is now feasible at the tissue and single-cell levels.
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Polisacáridos , Neoplasias de la Próstata , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Glicosilación , Humanos , Masculino , Polisacáridos/metabolismo , Glicómica/métodos , Glicoproteínas/metabolismo , Biomarcadores de Tumor/metabolismo , Líquidos Corporales/metabolismo , Líquidos Corporales/química , Procesamiento Proteico-Postraduccional , Animales , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Higher breast cancer mortality rates continue to disproportionally affect black women (BW) compared to white women (WW). This disparity is largely due to differences in tumor aggressiveness that can be related to distinct ancestry-associated breast tumor microenvironments (TMEs). Yet, characterization of the normal microenvironment (NME) in breast tissue and how they associate with breast cancer risk factors remains unknown. N-glycans, a glucose metabolism-linked post-translational modification, has not been characterized in normal breast tissue. We hypothesized that normal female breast tissue with distinct Breast Imaging and Reporting Data Systems (BI-RADS) categories have unique microenvironments based on N-glycan signatures that varies with genetic ancestries. Profiles of N-glycans were characterized in normal breast tissue from BW (n = 20) and WW (n = 20) at risk for breast cancer using matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). A total of 176 N-glycans (32 core-fucosylated and 144 noncore-fucosylated) were identified in the NME. We found that certain core-fucosylated, outer-arm fucosylated and high-mannose N-glycan structures had specific intensity patterns and histological distributions in the breast NME dependent on BI-RADS densities and ancestry. Normal breast tissue from BW, and not WW, with heterogeneously dense breast densities followed high-mannose patterns as seen in invasive ductal and lobular carcinomas. Lastly, lifestyles factors (e.g. age, menopausal status, Gail score, BMI, BI-RADS) differentially associated with fucosylated and high-mannose N-glycans based on ancestry. This study aims to decipher the molecular signatures in the breast NME from distinct ancestries towards improving the overall disparities in breast cancer burden.
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Manosa , Polisacáridos , Humanos , Femenino , Polisacáridos/metabolismo , Polisacáridos/química , Manosa/metabolismo , Manosa/química , Persona de Mediana Edad , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Glicómica , Mama/metabolismo , Mama/química , Mama/patología , Fucosa/metabolismo , Fucosa/química , Adulto , Microambiente TumoralRESUMEN
Ductal carcinoma in situ (DCIS) is a heterogeneous breast disease that remains challenging to treat due to its unpredictable progression to invasive breast cancer (IBC). Contemporary literature has become increasingly focused on extracellular matrix (ECM) alterations with breast cancer progression. However, the spatial regulation of the ECM proteome in DCIS has yet to be investigated in relation to IBC. We hypothesized that DCIS and IBC present distinct ECM proteomes that could discriminate between these pathologies. Tissue sections of pure DCIS, mixed DCIS-IBC, or pure IBC (n = 22) with detailed pathological annotations were investigated by multiplexed spatial proteomics. Across tissues, 1,005 ECM peptides were detected in pathologically annotated regions and their surrounding extracellular microenvironments. A comparison of DCIS to IBC pathologies demonstrated 43 significantly altered ECM peptides. Notably, eight fibrillar collagen peptides could distinguish with high specificity and sensitivity between DCIS and IBC. Lesion-targeted proteomic imaging revealed heterogeneity of the ECM proteome surrounding individual DCIS lesions. Multiplexed spatial proteomics reported an invasive cancer field effect, in which DCIS lesions in closer proximity to IBC shared a more similar ECM profile to IBC than distal counterparts. Defining the ECM proteomic microenvironment provides novel molecular insights relating to DCIS and IBC.
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Neoplasias de la Mama , Carcinoma Intraductal no Infiltrante , Matriz Extracelular , Proteómica , Microambiente Tumoral , Humanos , Femenino , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma Intraductal no Infiltrante/metabolismo , Carcinoma Intraductal no Infiltrante/patología , Proteómica/métodos , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Proteoma/metabolismo , Proteoma/análisis , Invasividad Neoplásica , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Persona de Mediana EdadRESUMEN
BACKGROUND: Bone metastasis is a common consequence of advanced prostate cancer. Bisphosphonates can be used to manage symptoms, but there are currently no curative treatments available. Altered tumour cell glycosylation is a hallmark of cancer and is an important driver of a malignant phenotype. In prostate cancer, the sialyltransferase ST6GAL1 is upregulated, and studies show ST6GAL1-mediated aberrant sialylation of N-glycans promotes prostate tumour growth and disease progression. METHODS: Here, we monitor ST6GAL1 in tumour and serum samples from men with aggressive prostate cancer and using in vitro and in vivo models we investigate the role of ST6GAL1 in prostate cancer bone metastasis. FINDINGS: ST6GAL1 is upregulated in patients with prostate cancer with tumours that have spread to the bone and can promote prostate cancer bone metastasis in vivo. The mechanisms involved are multi-faceted and involve modification of the pre-metastatic niche towards bone resorption to promote the vicious cycle, promoting the development of M2 like macrophages, and the regulation of immunosuppressive sialoglycans. Furthermore, using syngeneic mouse models, we show that inhibiting sialylation can block the spread of prostate tumours to bone. INTERPRETATION: Our study identifies an important role for ST6GAL1 and α2-6 sialylated N-glycans in prostate cancer bone metastasis, provides proof-of-concept data to show that inhibiting sialylation can suppress the spread of prostate tumours to bone, and highlights sialic acid blockade as an exciting new strategy to develop new therapies for patients with advanced prostate cancer. FUNDING: Prostate Cancer Research and the Mark Foundation For Cancer Research, the Medical Research Council and Prostate Cancer UK.
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Neoplasias Óseas , Ácido N-Acetilneuramínico , Neoplasias de la Próstata , Sialiltransferasas , Masculino , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Humanos , Sialiltransferasas/metabolismo , Sialiltransferasas/genética , Animales , Neoplasias Óseas/secundario , Neoplasias Óseas/metabolismo , Neoplasias Óseas/tratamiento farmacológico , Ratones , Ácido N-Acetilneuramínico/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Antígenos CD/metabolismo , Polisacáridos/farmacología , Glicosilación , beta-D-Galactósido alfa 2-6-SialiltransferasaRESUMEN
The extracellular matrix (ECM) proteome represents an important component of the tissue microenvironment that controls chemical flux and induces cell signaling through encoded structure. The analysis of the ECM represents an analytical challenge through high levels of post-translational modifications, protease-resistant structures, and crosslinked, insoluble proteins. This review provides a comprehensive overview of the analytical challenges involved in addressing the complexities of spatially profiling the extracellular matrix proteome. A synopsis of the process of synthesizing the ECM structure, detailing inherent chemical complexity, is included to present the scope of the analytical challenge. Current chromatographic and spatial techniques addressing these challenges are detailed. Capabilities for multimodal multiplexing with cellular populations are discussed with a perspective on developing a holistic view of disease processes that includes both the cellular and extracellular microenvironment.
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Proteínas de la Matriz Extracelular , Proteoma , Proteínas de la Matriz Extracelular/química , Proteoma/metabolismo , Proteómica/métodos , Matriz Extracelular/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
Circulating extracellular matrix (ECM) proteins are serological biomarkers of interest due to their association with pathologies involving disease processes such as fibrosis and cancers. In this study, we investigate the potential for serum biomarker research using differential protease specificity (DPS), leveraging alternate protease specificity as a targeting mechanism to selectively digest circulating ECM protein serum proteins. A proof-of-concept study is presented using serum from patients with cirrhotic liver or hepatocellular carcinoma. The approach uses collagenase DPS for digestion of deglycosylated serum and liquid-chromatography-trapped ion mobility-tandem mass spectrometry (LC-TIMS-MS/MS) to enhance the detection of ECM proteins in serum. It requires no sample enrichment and minimizes the albumin average precursor intensity readout to less than 1.2%. We further demonstrate the capabilities for using the method as a high-throughput matrix-assisted laser/desorption ionization mass spectrometry (MALDI-MS) assay coupled with reference library searching. A goal is to improve the depth and breadth of biofluid proteomics for noninvasive assays.
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Péptido Hidrolasas , Espectrometría de Masas en Tándem , Humanos , Proteómica/métodos , Cromatografía Liquida/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Colagenasas , BiomarcadoresRESUMEN
Metabolic dysfunction-associated steatotic liver disease (MASLD) is a progressive disease and comprises different stages of liver damage; it is significantly associated with obese and overweight patients. Untreated MASLD can progress to life-threatening end-stage conditions, such as cirrhosis and liver cancer. N-Linked glycosylation is one of the most common post-translational modifications in the cell surface and secreted proteins. N-Linked glycan alterations have been established to be signatures of liver diseases. However, the N-linked glycan changes during the progression of MASLD to liver cancer are still unknown. Here, we induced different stages of MASLD in mice and liver-cancer-related phenotypes and elucidated the N-glycome profile during the progression of MASLD by quantitative and qualitative profiling in situ using matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS). Importantly, we identified specific N-glycan structures including fucosylated and highly branched N-linked glycans at very early stages of liver injury (steatosis), which in humans are associated with cancer development, establishing the importance of these modifications with disease progression. Finally, we report that N-linked glycan alterations can be observed in our models by MALDI-IMS before liver injury is identified by histological analysis. Overall, we propose these findings as promising biomarkers for the early diagnosis of liver injury in MASLD.
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Dieta Occidental , Neoplasias Hepáticas , Humanos , Animales , Ratones , Polisacáridos/química , GlicosilaciónRESUMEN
N-glycosylation is an abundant post-translational modification of most cell-surface proteins. N-glycans play a crucial role in cellular functions like protein folding, protein localization, cell-cell signaling, and immune detection. As different tissue types display different N-glycan profiles, changes in N-glycan compositions occur in tissue-specific ways with development of disease, like cancer. However, no comparative atlas resource exists for documenting N-glycome alterations across various human tissue types, particularly comparing normal and cancerous tissues. In order to study a broad range of human tissue N-glycomes, N-glycan targeted MALDI imaging mass spectrometry was applied to custom formalin-fixed paraffin-embedded tissue microarrays. These encompassed fifteen human tissue types including bladder, breast, cervix, colon, esophagus, gastric, kidney, liver, lung, pancreas, prostate, sarcoma, skin, thyroid, and uterus. Each array contained both normal and tumor cores from the same pathology block, selected by a pathologist, allowing more in-depth comparisons of the N-glycome differences between tumor and normal and across tissue types. Using established MALDI-IMS workflows and existing N-glycan databases, the N-glycans present in each tissue core were spatially profiled and peak intensity data compiled for comparative analyses. Further structural information was determined for core fucosylation using endoglycosidase F3, and differentiation of sialic acid linkages through stabilization chemistry. Glycan structural differences across the tissue types were compared for oligomannose levels, branching complexity, presence of bisecting N-acetylglucosamine, fucosylation, and sialylation. Collectively, our research identified the N-glycans that were significantly increased and/or decreased in relative abundance in cancer for each tissue type. This study offers valuable information on a wide scale for both normal and cancerous tissues, serving as a reference for future studies and potential diagnostic applications of MALDI-IMS.
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Procesamiento Proteico-Postraduccional , Sarcoma , Masculino , Femenino , Humanos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Glicosilación , Polisacáridos/metabolismoRESUMEN
Parkinson's disease (PD) associated state of neuroinflammation due to the aggregation of aberrant proteins is widely reported. One type of post-translational modification involved in protein stability is glycosylation. Here, we aimed to characterize the human Parkinsonian nigro-striatal N-glycome, and related transcriptome/proteome, and its correlation with endoplasmic reticulum (ER) stress and unfolded protein response (UPR), providing a comprehensive characterization of the PD molecular signature. Significant changes were seen upon a PD: a 3% increase in sialylation and 5% increase in fucosylation in both regions, and a 2% increase in oligomannosylated N-glycans in the substantia nigra. In the latter, a decrease in the mRNA expression of sialidases and an upregulation in the UPR pathway were also seen. To show the correlation between these, we also describe a small in vitro study where changes in specific glycosylation trait enzymes (inhibition of sialyltransferases) led to impairments in cell mitochondrial activity, changes in glyco-profile, and upregulation in UPR pathways. This complete characterization of the human nigro-striatal N-glycome provides an insight into the glycomic profile of PD through a transversal approach while combining the other PD "omics" pieces, which can potentially assist in the development of glyco-focused therapeutics.
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Lupus nephritis (LN) is a serious complication for many patients who develop systemic lupus erythematosus, which primarily afflicts women. Our studies to identify biomarkers and the pathogenic mechanisms underlying LN will provide a better understanding of disease progression and sex bias, and lead to identification of additional potential therapeutic targets. The glycosphingolipid lactosylceramide (LacCer) and N-linked glycosylated proteins (N-glycans) were measured in urine and serum collected from LN and healthy control (HC) subjects (10 females and 10 males in each group). The sera from the LN and HC subjects were used to stimulate cytokine secretion and intracellular Ca2+ flux in female- and male-derived primary human renal mesangial cells (hRMCs). Significant differences were observed in the urine of LN patients compared to HCs. All major LacCers species were significantly elevated and differences between LN and HC were more pronounced in males. 72 individual N-glycans were altered in LN compared to HC and three N-glycans were significantly different between the sexes. In hRMCs, Ca2+ flux, but not cytokine secretion, was higher in response to LN sera compared to HC sera. Ca2+ flux, cytokine secretion, and glycosphingolipid levels were significantly higher in female-derived compared to male-derived hRMCs. Relative abundance of some LacCers and hexosylceramides were higher in female-derived compared to male-derived hRMCs. Urine LacCers and N-glycome could serve as definitive LN biomarkers and likely reflect renal disease activity. Despite higher sensitivity of female hRMCs, males may experience greater increases in LacCers, which may underscore worse disease in males. Elevated glycosphingolipid metabolism may poise renal cells to be more sensitive to external stimuli.
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Lupus Eritematoso Sistémico , Nefritis Lúpica , Humanos , Femenino , Masculino , Nefritis Lúpica/patología , Biomarcadores , Citocinas , Glicoesfingolípidos , PolisacáridosRESUMEN
N-linked glycans are complex biomolecules vital to cellular functions that have been linked to a wide range of pathological conditions. Mass spectrometry imaging (MSI) has been used to study the localization of N-linked glycans in cells and tissues. However, their structural diversity presents a challenge for MSI techniques, which stimulates the development of new approaches. In this study, we demonstrate for the first time spatial mapping of N-linked glycans in biological tissues using nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI). Nano-DESI MSI is an ambient ionization technique that has been previously used for imaging of metabolites, lipids, and proteins in biological tissue samples without special sample pretreatment. N-linked glycans are released from glycoproteins using an established enzymatic digestion with peptide N-glycosidase F, and their spatial localization is examined using nano-DESI MSI. We demonstrate imaging of N-linked glycans in formalin-fixed paraffin-embedded human hepatocellular carcinoma and human prostate tissues in both positive and negative ionization modes. We examine the localization of 38 N-linked glycans consisting of high mannose, hybrid fucosylated, and sialyated glycans. We demonstrate that negative mode nano-DESI MSI is well-suited for imaging of underivatized sialylated N-linked glycans. On-tissue MS/MS of different adducts of N-linked glycans proves advantageous for elucidation of the glycan sequence. This study demonstrates the applicability of liquid extraction techniques for spatial mapping of N-linked glycans in biological samples, providing an additional tool for glycobiology research.
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Neoplasias Hepáticas , Espectrometría de Masa por Ionización de Electrospray , Masculino , Humanos , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem , Imagen Molecular/métodos , Polisacáridos/análisisRESUMEN
The integration of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) with single cell spatial omics methods allows for a comprehensive investigation of single cell spatial information and matrisomal N-glycan and extracellular matrix protein imaging. Here, the performance of the antibody-directed single cell workflows coupled with MALDI-MSI are evaluated. Miralys™ photocleavable mass-tagged antibody probes (MALDI-IHC, AmberGen, Inc.), GeoMx DSP® (NanoString, Inc.), and Imaging Mass Cytometry (IMC, Standard BioTools Inc.) were used in series with MALDI-MSI of N-glycans and extracellular matrix peptides on formalin-fixed paraffin-embedded tissues. Single cell omics protocols were performed before and after MALDI-MSI. The data suggests that for each modality combination, there is an optimal order for performing both techniques on the same tissue section. An overall conclusion is that MALDI-MSI studies may be completed on the same tissue section as used for antibody-directed single cell modalities. This work increases access to combined cellular and extracellular information within the tissue microenvironment to enhance research on the pathological origins of disease.
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Anticuerpos , Polisacáridos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Polisacáridos/análisis , Péptidos/análisis , Colágeno , Rayos LáserRESUMEN
Although androgen deprivation treatment often effectively decreases prostate cancer, incurable metastatic castration-resistant prostate cancer (CRPC) eventually occurs. It is important to understand how CRPC metastasis progresses, which is not clearly defined. The loss of PTEN, a phosphatase to dephosphorylate phosphatidylinositol 3,4,5-trisphosphate in the PI3K pathway, occurs in up to 70% to 80% of CRPC. We generated a mouse androgen-independent prostate cancer cell line (PKO) from PTEN null and Hi-Myc transgenic mice in C57BL/6 background. We confirmed that this PKO cell line has an activated PI3K pathway and can metastasize into the femur and tibia of immunodeficient nude and immunocompetent C57BL/6 mice. In vitro, we found that androgen deprivation significantly enhanced PKO cell migration/invasion via the p110ß isoform-depended PAK1-MAPK activation. Inhibition of the p110ß-PAK1 axis significantly decreased prostate cancer cell migration/invasion. Of note, our analysis using clinical samples showed that PAK1 is more activated in CRPC than in advanced prostate cancer; high PAK1/phosphorylated-PAK1 levels are associated with decreased survival rates in patients with CRPC. All the information suggests that this cell line reflects the characteristics of CRPC cells and can be applied to dissect the mechanism of CRPC initiation and progression. This study also shows that PAK1 is a potential target for CRPC treatment. IMPLICATIONS: This study uses a newly generated PTEN null prostate cancer cell line to define a critical functional role of p110ß-PAK1 in CRPC migration/invasion. This study also shows that the p110ß-PAK1 axis can potentially be a therapeutic target in CRPC metastasis.
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Neoplasias de la Próstata Resistentes a la Castración , Animales , Humanos , Masculino , Ratones , Antagonistas de Andrógenos , Andrógenos/uso terapéutico , Línea Celular Tumoral , Ratones Endogámicos C57BL , Ratones Transgénicos , Quinasas p21 Activadas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Neoplasias de la Próstata Resistentes a la Castración/metabolismo , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Receptores Androgénicos/metabolismoRESUMEN
Aberrant glycosylation is a universal feature of cancer cells, and cancer-associated glycans have been detected in virtually every cancer type. A common change in tumour cell glycosylation is an increase in α2,6 sialylation of N-glycans, a modification driven by the sialyltransferase ST6GAL1. ST6GAL1 is overexpressed in numerous cancer types, and sialylated glycans are fundamental for tumour growth, metastasis, immune evasion, and drug resistance, but the role of ST6GAL1 in prostate cancer is poorly understood. Here, we analyse matched cancer and normal tissue samples from 200 patients and verify that ST6GAL1 is upregulated in prostate cancer tissue. Using MALDI imaging mass spectrometry (MALDI-IMS), we identify larger branched α2,6 sialylated N-glycans that show specificity to prostate tumour tissue. We also monitored ST6GAL1 in plasma samples from >400 patients and reveal ST6GAL1 levels are significantly increased in the blood of men with prostate cancer. Using both in vitro and in vivo studies, we demonstrate that ST6GAL1 promotes prostate tumour growth and invasion. Our findings show ST6GAL1 introduces α2,6 sialylated N-glycans on prostate cancer cells and raise the possibility that prostate cancer cells can secrete active ST6GAL1 enzyme capable of remodelling glycans on the surface of other cells. Furthermore, we find α2,6 sialylated N-glycans expressed by prostate cancer cells can be targeted using the sialyltransferase inhibitor P-3FAX -Neu5Ac. Our study identifies an important role for ST6GAL1 and α2,6 sialylated N-glycans in prostate cancer progression and highlights the opportunity to inhibit abnormal sialylation for the development of new prostate cancer therapeutics. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.
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Neoplasias de la Próstata , Sialiltransferasas , Masculino , Humanos , Glicosilación , Polisacáridos/química , Polisacáridos/metabolismo , Reino Unido , beta-D-Galactósido alfa 2-6-Sialiltransferasa , Antígenos CD/metabolismoRESUMEN
N-linked glycosylation plays an important role in both the innate and adaptive immune response through the modulation of cell surface receptors as well as general cell-to-cell interactions. The study of immune cell N-glycosylation is gaining interest but is hindered by the complexity of cell-type-specific N-glycan analysis. Analytical techniques such as chromatography, LC-MS/MS, and the use of lectins are all currently used to analyze cellular glycosylation. Issues with these analytical techniques include poor throughput, which is often limited to a single sample at a time, lack of structural information, the need for a large amount of starting materials, and the requirement for cell purification, thereby reducing their feasibility for N-glycan study. Here, we report the development of a rapid antibody array-based approach for the capture of specific nonadherent immune cells coupled with MALDI-IMS to analyze cellular N-glycosylation. This workflow is adaptable to multiple N-glycan imaging approaches such as the removal or stabilization and derivatization of terminal sialic acid residues providing unique avenues of analysis that have otherwise not been explored in immune cell populations. The reproducibility, sensitivity, and versatility of this assay provide an invaluable tool for researchers and clinical applications, significantly expanding the field of glycoimmunology.
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Anticuerpos , Espectrometría de Masas en Tándem , Glicosilación , Cromatografía Liquida , Reproducibilidad de los Resultados , Anticuerpos/metabolismo , Polisacáridos/químicaRESUMEN
Sialic acid isomers attached in either α2,3 or α2,6 linkage to glycan termini confer distinct chemical, biological, and pathological properties, but they cannot be distinguished by mass differences in traditional mass spectrometry experiments. Multiple derivatization strategies have been developed to stabilize and facilitate the analysis of sialic acid isomers and their glycoconjugate carriers by high-performance liquid chromatography, capillary electrophoresis, and mass spectrometry workflows. Herein, a set of novel derivatization schemes are described that result in the introduction of bioorthogonal click chemistry alkyne or azide groups into α2,3- and α2,8-linked sialic acids. These chemical modifications were validated and structurally characterized using model isomeric sialic acid conjugates and model protein carriers. Use of an alkyne-amine, propargylamine, as the second amidation reagent effectively introduces an alkyne functional group into α2,3-linked sialic acid glycoproteins. In tissues, serum, and cultured cells, this allows for the detection and visualization of N-linked glycan sialic acid isomers by imaging mass spectrometry approaches. Formalin-fixed paraffin-embedded prostate cancer tissues and pancreatic cancer cell lines were used to characterize the numbers and distribution of alkyne-modified α2,3-linked sialic acid N-glycans. An azide-amine compound with a poly(ethylene glycol) linker was evaluated for use in histochemical staining. Formalin-fixed pancreatic cancer tissues were amidated with the azide amine, reacted with biotin-alkyne and copper catalyst, and sialic acid isomers detected by streptavidin-peroxidase staining. The direct chemical introduction of bioorthogonal click chemistry reagents into sialic acid-containing glycans and glycoproteins provides a new glycomic tool set to expand approaches for their detection, labeling, visualization, and enrichment.
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Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Humanos , Ácidos Siálicos/química , Polisacáridos/química , Línea Celular TumoralRESUMEN
Glycosylation is an important posttranslational modifier of proteins and lipid conjugates critical for the stability and function of these macromolecules. Particularly important are N-linked glycans attached to asparagine residues in proteins. N-glycans have well-defined roles in protein folding, cellular trafficking and signal transduction, and alterations to them are implicated in a variety of diseases. However, the non-template driven biosynthesis of these N-glycans leads to significant structural diversity, making it challenging to identify the most biologically and clinically relevant species using conventional analyses. Advances in mass spectrometry instrumentation and data acquisition, as well as in enzymatic and chemical sample preparation strategies, have positioned mass spectrometry approaches as powerful analytical tools for the characterization of glycosylation in health and disease. Imaging mass spectrometry expands upon these strategies by capturing the spatial component of a glycan's distribution in-situ, lending additional insight into the organization and function of these molecules. Herein we review the ongoing evolution of glycan imaging mass spectrometry beginning with widely adopted tissue imaging approaches and expanding to other matrices and sample types with potential research and clinical implications. Adaptations of these techniques, along with their applications to various states of disease, are discussed. Collectively, glycan imaging mass spectrometry analyses broaden our understanding of the biological and clinical relevance of N-glycosylation to human disease.
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Polisacáridos , Humanos , Espectrometría de Masas/métodos , Glicosilación , Polisacáridos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodosRESUMEN
Colorectal cancer (CRC) stands as a leading cause of death worldwide, often arising from specific genetic mutations, progressing from pre-cancerous adenomas to adenocarcinomas. Early detection through regular screening can result in a 90% 5-year survival rate for patients. However, unfortunately, only a fraction of CRC cases are identified at pre-invasive stages, allowing progression to occur silently over 10-15 years. The intricate interplay between the immune system and tumor cells within the tumor microenvironment plays a pivotal role in the progression of CRC. Immune cell clusters can either inhibit or facilitate tumor initiation, growth, and metastasis. To gain a better understanding of this relationship, we conducted N-glycomic profiling using matrix-assisted laser desorption-ionization mass spectrometry imaging (MALDI-MSI). We detected nearly 100 N-glycan species across all samples, revealing a shift in N-glycome profiles from normal to cancerous tissues, marked by a decrease in high mannose N-glycans. Further analysis of precancerous to invasive carcinomas showed an increase in pauci-mannose biantennary, and tetraantennary N-glycans with disease progression. Moreover, a distinct stratification in the N-glycome profile was observed between non-mucinous and mucinous CRC tissues, driven by pauci-mannose, high mannose, and bisecting N-glycans. Notably, we identified immune clusters of CD20+ B cells and CD3/CD44+ T cells distinctive and predictive with signature profiles of bisecting and branched N-glycans. These spatial N-glycan profiles offer potential biomarkers and therapeutic targets throughout the progression of CRC.
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While mammograms are the standard tool for breast cancer screening, there remains challenges for mammography to effectively distinguish benign lesions from breast cancers, leading to many unnecessary biopsy procedures. A blood-based biomarker could provide a minimally invasive supplemental assay to increase the specificity of breast cancer screening. Serum N-glycosylation alterations have associations with many cancers and several of the clinical characteristics of breast cancer. The current study utilized a high-throughput mass spectrometry workflow to identify serum N-glycans with differences in intensities between patients that had a benign lesion from patients with breast cancer. The overall N-glycan profiles of the two patient groups had no differences, but there were several individual N-glycans with significant differences in intensities between patients with benign lesions and ductal carcinoma in situ (DCIS). Many N-glycans had strong associations with age and/or body mass index, but there were several of these associations that differed between the patients with benign lesions and breast cancer. Accordingly, the samples were stratified by the patient's age and body mass index, and N-glycans with significant differences between these subsets were identified. For women aged 50-74 with a body mass index of 18.5-24.9, a model including the intensities of two N-glycans, 1850.666 m/z and 2163.743 m/z, age, and BMI were able to clearly distinguish the breast cancer patients from the patients with benign lesions with an AUROC of 0.899 and an optimal cutoff with 82% sensitivity and 84% specificity. This study indicates that serum N-glycan profiling is a promising approach for providing clarity for breast cancer screening, especially within the subset of healthy weight women in the age group recommended for mammograms.