The HUSH complex controls brain architecture and protocadherin fidelity.

The HUSH (human silencing hub) complex contains the H3K9me3 binding protein M-phase phosphoprotein 8 (MPP8) and recruits the histone methyltransferase SETDB1 as well as Microrchidia CW-type zinc finger protein 2 (MORC2). Functional and mechanistic studies of the HUSH complex have hitherto been centered around SETDB1 while the in vivo functions of MPP8 and MORC2 remain elusive. Here, we show that genetic inactivation of Mphosph8 or Morc2a in the nervous system of mice leads to increased brain size, altered brain architecture, and behavioral changes. Mechanistically, in both mouse brains and human cerebral organoids, MPP8 and MORC2 suppress the repetitive-like protocadherin gene cluster in an H3K9me3-dependent manner. Our data identify MPP8 and MORC2, previously linked to silencing of repetitive elements via the HUSH complex, as key epigenetic regulators of protocadherin expression in the nervous system and thereby brain development and neuronal individuality in mice and humans.

Gruffi: an algorithm for computational removal of stressed cells from brain organoid transcriptomic datasets.

Organoids enable in vitro modeling of complex developmental processes and disease pathologies. Like most 3D cultures, organoids lack sufficient oxygen supply and therefore experience cellular stress. These negative effects are particularly prominent in complex models, such as brain organoids, and can affect lineage commitment. Here, we analyze brain organoid and fetal single-cell RNA sequencing (scRNAseq) data from published and new datasets, totaling about 190,000 cells. We identify a unique stress signature in the data from all organoid samples, but not in fetal samples. We demonstrate that cell stress is limited to a defined subpopulation of cells that is unique to organoids and does not affect neuronal specification or maturation. We have developed a computational algorithm, Gruffi, which uses granular functional filtering to identify and remove stressed cells from any organoid scRNAseq dataset in an unbiased manner. We validated our method using six additional datasets from different organoid protocols and early brains, and show its usefulness to other organoid systems including retinal organoids. Our data show that the adverse effects of cell stress can be corrected by bioinformatic analysis for improved delineation of developmental trajectories and resemblance to in vivo data.

Terminal α2,6-sialylation of epidermal growth factor receptor modulates antibody therapy response of colorectal cancer cells.

BACKGROUND: The epidermal growth factor receptor (EGFR) is a key protein involved in cancer development. Monoclonal antibodies targeting EGFR are approved for the treatment of metastatic colorectal cancer (CRC). Despite the beneficial clinical effects observed in subgroups of patients, the acquisition of resistance to treatment remains a major concern. Protein N-glycosylation of cellular receptors is known to regulate physiological processes leading to activation of downstream signaling pathways. In the present study, the role of EGFR-specific terminal ⍺2,6-sialylation was analyzed in modulation of the malignant phenotype of CRC cells and their resistance to monoclonal antibody Cetuximab-based therapy. METHODS: Glycoengineered CRC cell models with specific sialyltransferase ST6GAL1 expression levels were applied to evaluate EGFR activation, cell surface glycosylation and therapeutic response to Cetuximab. RESULTS: Glycoproteomic analysis revealed EGFR as a major target of ST6Gal1-mediated ⍺2,6-sialylation in a glycosite-specific manner. Mechanistically, CRC cells with increased ST6Gal1 expression and displaying terminal ⍺2,6-sialylation showed a marked resistance to Cetuximab-induced cytotoxicity. Moreover, we found that this resistance was accompanied by downregulation of EGFR expression and its activation. CONCLUSIONS: Our data indicate that EGFR ⍺2,6-sialylation is a key factor in modulating the susceptibility of CRC cells to antibody targeted therapy, thereby disclosing a potential novel biomarker and providing key molecular information for tailor made anti-cancer strategies.

Author Correction: iLoF: An intelligent Lab on Fiber Approach for Human Cancer Single-Cell Type Identification.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

iLoF: An intelligent Lab on Fiber Approach for Human Cancer Single-Cell Type Identification.

With the advent of personalized medicine, there is a movement to develop "smaller" and "smarter" microdevices that are able to distinguish similar cancer subtypes. Tumor cells display major differences when compared to their natural counterparts, due to alterations in fundamental cellular processes such as glycosylation. Glycans are involved in tumor cell biology and they have been considered to be suitable cancer biomarkers. Thus, more selective cancer screening assays can be developed through the detection of specific altered glycans on the surface of circulating cancer cells. Currently, this is only possible through time-consuming assays. In this work, we propose the "intelligent" Lab on Fiber (iLoF) device, that has a high-resolution, and which is a fast and portable method for tumor single-cell type identification and isolation. We apply an Artificial Intelligence approach to the back-scattered signal arising from a trapped cell by a micro-lensed optical fiber. As a proof of concept, we show that iLoF is able to discriminate two human cancer cell models sharing the same genetic background but displaying a different surface glycosylation profile with an accuracy above 90% and a speed rate of 2.3 seconds. We envision the incorporation of the iLoF in an easy-to-operate microchip for cancer identification, which would allow further biological characterization of the captured circulating live cells.

Analysis of the Effect of Increased α2,3-Sialylation on RTK Activation in MKN45 Gastric Cancer Spheroids Treated with Crizotinib.

In the scenario of personalized medicine, targeted therapies are currently the focus of cancer drug development. These drugs can block the growth and spread of tumor cells by interfering with key molecules involved in malignancy, such as receptor tyrosine kinases (RTKs). MET and Recepteur d'Origine Nantais (RON), which are RTKs frequently overactivated in gastric cancer, are glycoprotein receptors whose activation have been shown to be modulated by the cellular glycosylation. In this work, we address the role of sialylation in gastric cancer therapy using an innovative 3D high-throughput cell culture methodology that mimics better the in vivo tumor features. We evaluate the response to targeted treatment of glycoengineered gastric cancer cell models overexpressing the sialyltransferases ST3GAL4 or ST3GAL6 by subjecting 3D spheroids to the tyrosine kinase inhibitor crizotinib. We show here that 3D spheroids of ST3GAL4 or ST3GAL6 overexpressing MKN45 gastric cancer cells are less affected by the inhibitor. In addition, we disclose a potential compensatory pathway via activation of the Insulin Receptor upon crizotinib treatment. Our results suggest that cell sialylation, in addition of being involved in tumor progression, could play a critical role in the response to tyrosine kinase inhibitors in gastric cancer.

Glycosylation in the Era of Cancer-Targeted Therapy: Where Are We Heading?

This review provides insights on the impact of glycosylation in cancer biology and its influence in the current approaches of targeted cancer therapies in the clinical setting. The roles of glycosylation in cancer signaling, tumor progression, and metastasis are reviewed as well as glycans and glycan-binding proteins in tumor immunomodulation. Moreover, the latest reports on glycans influencing targeted therapeutic approaches in cancer are summarized. Finally, we discuss the future challenges of the field, outlining potential applications of glycan-based biomarkers for patient stratification and strategies for improving personalized cancer treatment.

Different isolation approaches lead to diverse glycosylated extracellular vesicle populations.

Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepTM density gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates.

O-glycan truncation enhances cancer-related functions of CD44 in gastric cancer.

CD44 isoforms are often upregulated in gastric cancer and have been associated with increased metastatic potential and poor survival. To evaluate the functional impact of O-glycan truncation on CD44 we have analysed glyco-engineered cancer cell models displaying shortened O-glycans. Here, we demonstrate that induction of aberrant O-glycan termination through various molecular mechanisms affects CD44 molecular features. We show that CD44 is a major carrier of truncated O-glycans and that this truncation is accompanied by an increased hyaluronan binding capacity and affects extracellular shedding. In addition, short O-glycans promoted the colocalization of CD44v6 with the receptor tyrosine kinase RON and concomitantly increased activation. Our in vitro findings were validated in gastric cancer clinical samples.

Multivariate data analysis for the detection of human alpha-acid glycoprotein aberrant glycosylation in pancreatic ductal adenocarcinoma.

Relative quantification of human alpha-acid glycoprotein (hAGP) glycan isomers using [12C6]/[13C6]-aniline in combination with multivariate data analysis is proposed as an efficient method for the identification of pancreatic ductal adenocarcinoma (PDAC) glycan biomarkers in serum samples. Intact and desialylated glycans from hAGP, purified from serum samples of patients with PDAC and chronic pancreatitis (ChrP), were labeled with aniline and analyzed by µZIC-HILIC-MS. Afterwards, partial least squares discriminant analysis (PLS-DA) was applied to the relative areas obtained for all glycan isomers in the different samples: pathological (ChrP or PDAC) versus healthy samples. Seven intact glycan isomers with α2-6 linked sialic acids, five of them also fucosylated, were the most meaningful to distinguish between PDAC and ChrP patients. The desialylated glycan isomers also identified by PLS-DA as potential biomarker candidates confirmed that antenna but also core fucosylation could be involved in PDAC. The analysis of intact and desialylated glycan isomers in combination with the multivariate data analysis revealed that the triantennary glycan with two fucoses of hAGP could have in the future a relevant role in the differentiation of patients with PDAC from those with ChrP. SIGNIFICANCE: Multivariate data analysis is currently being used in many omics fields for biomarker discovery. However, to date, no glycomics studies have applied chemometric tools combined with mass spectrometry in a preclinical research. In this work, this methodology has been used to identify altered glycosylation of human alpha-acid glycoprotein in pancreatic ductal adenocarcinoma (PDAC). The obtained results reveal that the triantennary glycan with two fucoses could have a great biomarker potential as it was relevant to differentiate PDAC and chronic pancreatitis (ChrP) patients.

Multicellular Human Gastric-Cancer Spheroids Mimic the Glycosylation Phenotype of Gastric Carcinomas.

Cellular glycosylation plays a pivotal role in several molecular mechanisms controlling cell⁻cell recognition, communication, and adhesion. Thus, aberrant glycosylation has a major impact on the acquisition of malignant features in the tumor progression of patients. To mimic these in vivo features, an innovative high-throughput 3D spheroid culture methodology has been developed for gastric cancer cells. The assessment of cancer cell spheroids' physical characteristics, such as size, morphology and solidity, as well as the impact of glycosylation inhibitors on spheroid formation was performed applying automated image analysis. A detailed evaluation of key glycans and glycoproteins displayed by the gastric cancer spheroids and their counterpart cells cultured under conventional 2D conditions was performed. Our results show that, by applying 3D cell culture approaches, the model cell lines represented the differentiation features observed in the original tumors and the cellular glycocalix underwent striking changes, displaying increased expression of cancer-associated glycan antigens and mucin MUC1, ultimately better simulating the glycosylation phenotype of the gastric tumor.

Hypoxia and serum deprivation induces glycan alterations in triple negative breast cancer cells.

Triple negative breast cancer (TNBC) is a major global public health problem. The lack of targeted therapy and the elevated mortality evidence the need for better knowledge of the tumor biology. Hypoxia and aberrant glycosylation are associated with advanced stages of malignancy, tumor progression and treatment resistance. Importantly, serum deprivation regulates the invasive phenotype and favors TNBC cell survival. However, in TNBC, the role of hypoxia and serum deprivation in the regulation of glycosylation remains largely unknown. The effects of hypoxia and serum deprivation on the expression of glycosyltransferases and glycan profile were evaluated in the MDA-MB-231 cell line. We showed that the overexpression of HIF-1α was accompanied by acquisition of epithelial-mesenchimal transition features. Significant upregulation of fucosyl- and sialyltransferases involved in the synthesis of tumor-associated carbohydrate antigens was observed together with changes in fucosylation and sialylation detected by Aleuria aurantia lectin and Sambucus nigra agglutinin lectin blots. Bioinformatic analysis further indicated a mechanism by which HIF-1α can regulate ST3GAL6 expression and the relationship within the intrinsic characteristics of TNBC tumors. In conclusion, our results showed the involvement of hypoxia and serum deprivation in glycosylation profile regulation of TNBC cells triggering breast cancer aggressive features and suggesting glycosylation as a potential diagnostic and therapeutic target.

Glycosylation in cancer: Selected roles in tumour progression, immune modulation and metastasis.

Tumour metastasis is the main cause of cancer related deaths. Metastasis is an intricate multi-step process that requires the acquisition of several cancer cell features, including the modulation of tumour cell migration, adhesion, invasion, and immune evasion. Changes in the cellular glycosylation are associated with malignant transformation of cancer cells, tumour progression and ultimately, metastasis formation. Glycans have major impact on cellular signalling and on the regulation of tumour cell-cell adhesion and cell-matrix interaction. Glycans drive the interplay between the cancer cells and the tumour microenvironment. In this review, we summarize the roles of glycan alterations in tumour progression, such as acquisition of oncogenic features due to modulation of receptor tyrosine kinases, proteoglycans, cadherins and integrins. We also highlight the importance of key glycan binding proteins such as selectins, siglecs and galectins, which are pivotal in the modulation of immune response. An overview on glycans as cancer biomarkers is also presented.

Analysis of sialyl-Lewis x on MUC5AC and MUC1 mucins in pancreatic cancer tissues.

Pancreatic adenocarcinoma (PDAC) lacks efficient biomarkers. Mucins are glycoproteins that can carry aberrant glycosylation in cancer. Our objective was to identify cancer-related glycan epitopes on MUC1 and MUC5AC mucins in PDAC as potential biomarkers. We have analysed the tumour-associated carbohydrate antigens sialyl-Lewis x (SLex) and sialyl-Tn (STn) on MUC1 and MUC5AC in PDAC tissues. The selected cohort for this study consisted of twenty-one PDAC tissues positive for SLex antigen and three normal pancreas specimens as controls. STn expression was shown in 76% of the PDAC tissues. MUC1 and MUC5AC were detected in 90% of PDAC tissues. We performed in situ proximity ligation assay combining antibodies against mucins and glycan epitopes to identify specific mucin glycoforms. MUC1-SLex and MUC5AC-SLex were found in 68% and 84% respectively, of the mucin expressing PDAC tissues, while STn hardly colocalized with any of the evaluated mucins. Further analysis by Western blot of MUC5AC and SLex in eight PDAC tissue lysates showed that six out of eight cases were positive for both markers. Moreover, immunoprecipitation of MUC5AC from positive PDAC tissues and subsequent SLex immunodetection confirmed the presence of SLex on MUC5AC. Altogether, MUC5AC-SLex glycoform is present in PDAC and can be regarded as potential biomarker.

Gastric Cancer Cell Glycosylation as a Modulator of the ErbB2 Oncogenic Receptor.

Aberrant expression and hyperactivation of the human epidermal growth factor receptor 2 (ErbB2) constitute crucial molecular events underpinning gastric neoplastic transformation. Despite ErbB2 extracellular domain being a well-known target for glycosylation, its glycosylation profile and the molecular mechanisms through which it actively tunes tumorigenesis in gastric cancer (GC) cells remain elusive. We aimed at disclosing relevant ErbB2 glycan signatures and their functional impact on receptor's biology in GC cells. The transcriptomic profile of cancer-relevant glycosylation enzymes, and the expression and activation of the ErbB receptors were characterized in four GC cell lines. Cellular- and receptor-specific glycan profiling of ErbB2-overexpressing NCI-N87 cells unveiled a heterogeneous glycosylation pattern harboring the tumor-associated sialyl Lewis a (SLea) antigen. The expression of SLea and key enzymes integrating its biosynthetic pathway were strongly upregulated in this GC cell line. An association between the expression of ERBB2 and FUT3, a central gene in SLea biosynthesis, was disclosed in GC patients, further highlighting the crosstalk between ErbB2 and SLea expression. Moreover, cellular deglycosylation and CA 19.9 antibody-mediated blocking of SLea drastically altered ErbB2 expression and activation in NCI-N87 cells. Altogether, NCI-N87 cell line constitutes an appealing in vitro model to address glycan-mediated regulation of ErbB2 in GC.

Glycomic Approaches for the Discovery of Targets in Gastrointestinal Cancer.

Gastrointestinal (GI) cancer is the most common group of malignancies and many of its types are among the most deadly. Various glycoconjugates have been used in clinical practice as serum biomarker for several GI tumors, however, with limited diagnose application. Despite the good accessibility by endoscopy of many GI organs, the lack of reliable serum biomarkers often leads to late diagnosis of malignancy and consequently low 5-year survival rates. Recent advances in analytical techniques have provided novel glycoproteomic and glycomic data and generated functional information and putative biomarker targets in oncology. Glycosylation alterations have been demonstrated in a series of glycoconjugates (glycoproteins, proteoglycans, and glycosphingolipids) that are involved in cancer cell adhesion, signaling, invasion, and metastasis formation. In this review, we present an overview on the major glycosylation alterations in GI cancer and the current serological biomarkers used in the clinical oncology setting. We further describe recent glycomic studies in GI cancer, namely gastric, colorectal, and pancreatic cancer. Moreover, we discuss the role of glycosylation as a modulator of the function of several key players in cancer cell biology. Finally, we address several state-of-the-art techniques currently applied in this field, such as glycomic and glycoproteomic analyses, the application of glycoengineered cell line models, microarray and proximity ligation assay, and imaging mass spectrometry, and provide an outlook to future perspectives and clinical applications.

Increased α1-3 fucosylation of α-1-acid glycoprotein (AGP) in pancreatic cancer.

Pancreatic cancer (PDAC) lacks reliable diagnostic biomarkers and the search for new biomarkers represents an important challenge. Previous results looking at a small cohort of patients showed an increase in α-1-acid glycoprotein (AGP) fucosylation in advanced PDAC using N-glycan sequencing. Here, we have analysed AGP glycoforms in a larger cohort using several analytical techniques including mass spectrometry (MS), capillary zone electrophoresis (CZE) and enzyme-linked lectin assays (ELLAs) for determining AGP glycoforms which could be PDAC associated. AGP from 31 serum samples, including healthy controls (HC), chronic pancreatitis (ChrP) and PDAC patients, was purified by immunoaffinity chromatography. Stable isotope labelling of AGP released N-glycans and their analysis by zwitterionic hydrophilic interaction capillary liquid chromatography electrospray MS (µZIC-HILIC-ESI-MS) showed an increase in AGP fucosylated glycoforms in PDAC compared to ChrP and HC. By CZE-UV analysis, relative concentrations of some of the AGP isoforms were found significantly different compared to those in PDAC and HC. Finally, ELLAs using Aleuria aurantia lectin displayed a significant increase in AGP fucosylation, before and after AGP neuraminidase treatment, in advanced PDAC compared to ChrP and HC, respectively. Altogether, these results indicate that α1-3 fucosylated glycoforms of AGP are increased in PDAC and could be potentially regarded as a PDAC biomarker.

Quantitative analysis of N-glycans from human alfa-acid-glycoprotein using stable isotope labeling and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry as tool for pancreatic disease diagnosis.

In this work we demonstrate the potential of glycan reductive isotope labeling (GRIL) using [(12)C]- and [(13)C]-coded aniline and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry (µZIC-HILIC-ESI-MS) for relative quantitation of glycosylation variants in selected glycoproteins present in samples from cancer patients. Human α1-acid-glycoprotein (hAGP) is an acute phase serum glycoprotein whose glycosylation has been described to be altered in cancer and chronic inflammation. However, it is not clear yet whether some particular glycans in hAGP can be used as biomarker for differentiating between these two pathologies. In this work, hAGP was isolated by immunoaffinity chromatography (IAC) from serum samples of healthy individuals and from those suffering chronic pancreatitis and different stages of pancreatic cancer, respectively. After de-N-glycosylation, relative quantitation of the hAGP glycans was carried out using stable isotope labeling and µZIC-HILIC-ESI-MS analysis. First, protein denaturing conditions prior to PNGase F digestion were optimized to achieve quantitative digestion yields, and the reproducibility of the established methodology was evaluated with standard hAGP. Then, the proposed method was applied to the analysis of the clinical samples (control vs. pathological). Pancreatic cancer samples clearly showed an increase in the abundance of fucosylated glycans as the stage of the disease increases and this was unlike to samples from chronic pancreatitis. The results gained here indicate the mentioned glycan in hAGP as a candidate structure worth to be corroborated by an extended study including more clinical cases; especially those with chronic pancreatitis and initial stages of pancreatic cancer. Importantly, the results demonstrate that the presented methodology combining an enrichment of a target protein by IAC with isotope coded relative quantitation of N-glycans can be successfully used for targeted glycomics studies. The methodology is assumed being suitable as well for other such studies aimed at finding novel cancer associated glycoprotein biomarkers.

Identification of potential pancreatic cancer serum markers: Increased sialyl-Lewis X on ceruloplasmin.

Pancreatic adenocarcinoma (PDAC) usually shows an enhanced expression of sialyl-Lewis X (sLe(x)) and related epitopes. PDAC may secrete some of the proteins carrying such increased sLe(x) determinant into serum, so they could be used as PDAC markers. Previously, we identified acute-phase proteins with increased sLe(x) in both PDAC and in chronic pancreatitis patients. In this study, depleted sera from the main acute-phase proteins has been analysed for the search of proteins with increased sLe(x) levels in PDAC. Sera from healthy controls, chronic pancreatitis and PDAC patients were depleted, electrophoresed and subjected to sLe(x) immunodetection. Proteins that differentially expressed sLe(x) in PDAC were trypsin digested and identified by LC-ESI-QTOF mass spectrometry. Five protein bands that differentially expressed sLe(x) in PDAC were identified and corresponded to seven different acute-phase proteins. Among them, ceruloplasmin (CP) was selected for further analysis. N-glycan sequencing of CP confirmed the increase of sLe(x) levels in CP in PDAC patients. Healthy controls, chronic pancreatitis and PDAC patients' sera were immunoprecipitated with anti-CP antibodies, and their sLe(x) and CP levels were analysed by western blot. The sLe(x)/CP ratio tended to be higher for the PDAC group, which altogether suggests that the sLe(x)/CP ratio could be a useful biomarker for PDAC.