Cancer remains a major cause of mortality, especially in advanced stages where current therapies are less effective. Antibody‐drug conjugates offer targeted treatment, but their efficacy is limited by on‐target/off‐tumor toxicity since most protein receptors are also present in healthy tissues. Abnormal glycosylation is a hallmark of cancer, with Thomsen‐nouveau (Tn) and Sialyl‐Tn (STn) antigens driving tumor progression. The human macrophage galactose‐type lectin (MGL) binds αGalNAc within these antigens through its carbohydrate recognition domain (CRD), distinguishing Tn/STn‐expressing cancer cells from healthy ones. This specificity positions the MGL‐CRD as a promising vehicle for safer drug delivery. Herein, an MGL‐based drug conjugate was engineered by introducing an exposed cysteine (mMGL) to allow chemical linkage to monomethyl auristatin E derivative (vcMMAE). The resulting conjugate (mMGL‐vcMMAE) is stable and preserves the thermal stability, the αGalNAc binding profile and, the affinity of the native MGL‐CRD, including its selectivity toward glycoengineered cancer models expressing Tn/STn antigens. Tissue validation confirmed selective binding to colorectal cancer (CRC) tissues. Metabolism and apoptosis analysis demonstrated that mMGL‐vcMMAE impacts glycoengineered cancer cells expressing Tn/STn O‐glycans compared to free vcMMAE, improving targeting precision. This strategy offers a novel glycan‐guided approach to potentially minimize off‐target effects, advancing in safer and more effective cancer therapies.

Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide, mainly due to resistance to targeted therapies such as Cetuximab, a chimeric monoclonal antibody targeting epidermal growth factor receptor (EGFR) used in RAS wild-type tumours. Aberrant glycosylation, specifically increased sialylation, has been implicated in tumour progression and therapy resistance. However, its role in the response to EGFR-targeted treatment is not fully understood. This study investigates the impact of inhibition of sialylation on CRC cells' malignant properties and on responsiveness to Cetuximab therapy. Three RAS wild-type CRC cell lines with distinct sialylation profiles, including an ST6GalI overexpressing model, were used as models to evaluate the effects of a pan-sialyltransferase inhibitor. Treatment effectively reduced both terminal α2,3- and α2,6-sialylation at the cell surface and induced a global remodelling of the N-glycome, with decreased sialylated and increased neutral glycans. Functionally, sialylation significantly impaired cell motility without affecting cell viability in any of the cell models. Total EGFR expression and basal activation were not altered, while EGFR glycosylation was directly modulated, particularly terminal α2,6-sialylation. Importantly, EGFR sialylation affected Cetuximab binding and receptor activation in a cell line-dependent manner, with cells bearing α2,6-sialylation showing modified antibody responsiveness. Overall, these findings demonstrate that EGFR sialylation modulates receptor behaviour and Cetuximab response in CRC, highlighting the inhibition of sialylation as a potential strategy to overcome glycosylation-mediated therapeutic resistance.

Aim: Tumor cells and extracellular vesicles (EVs) dynamically interact with adipocytes, contributing to tumor progression and systemic metabolic dysfunctions. While the role of glycosylation in this process remains unclear, glycans facilitate communication between tumors and their surrounding environment. Here, we aimed to elucidate the pivotal role of sialyl-Tn (STn), a tumor-associated glycan, in driving a bidirectional metabolic reprogramming between gastric cancer cells and adipocytes. Methods: EVs were isolated from gastric cancer cells with distinct glycosylation profiles by ultracentrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting. Flow cytometry was used to quantify EV uptake by adipocytes. EV-mediated adipocyte browning and lipolysis were measured by real-time quantitative polymerase chain reaction, glycerol and fatty acid release, perilipin immunofluorescence, and western blotting analysis of lipid synthesis and hydrolysis enzymes. The reciprocal effects of beige adipocytes on tumor cells were evaluated using fatty acid uptake assays, metabolomics, RNA sequencing, Seahorse metabolic flux, organelle number, proliferation, and migration assays. Results: STn-positive gastric cancer cells and EVs induced white-into-beige transdifferentiation. These cells exhibited an enhanced capacity to release glycerol and fatty acids, which were predominantly taken up by gastric cancer cells displaying STn. As a feedback mechanism, gastric cancer cells shift their metabolism towards fatty acid oxidation, concomitant with increased mitochondrial and endosomal biogenesis, and enhanced motility. Conclusion: Our findings reveal a key role for the tumor-associated glycan STn in metabolic reprogramming of adipocytes by tumor cells and EVs, opening new avenues for targeted therapies in cancer patients experiencing metabolic dysfunctions, such as cancer cachexia.

Accurate identification of tumor-specific markers is vital for developing chimeric antigen receptor (CAR)-based therapies. While cell surface antigens are seldom cancer-restricted, their post-translational modifications (PTMs), particularly aberrant carbohydrate structures, offer attractive alternatives. Among these, the sialyl-Tn (STn) antigen stands out for its prevalent presence in various epithelial tumors. Although monoclonal antibodies (mAbs) against STn have been developed, their clinical application has been hindered by concerns regarding specificity. Herein, we describe AM52.1, a mAb with unprecedented specificity for STn and lack of reactivity with healthy tissues. The single-chain variable fragment (scFv) of AM52.1 was assembled into a second-generation CAR scaffold. AM52.1CAR T cells efficiently targeted STn-expressing cancer cell lines and patient-derived organoids (PDOs), while sparing STn-negative cells. In further preclinical models, AM52.1CAR T cells robustly controlled gastric and tubo-ovarian tumors, as well as colorectal cancer mucinous peritoneal metastases, highlighting their strong therapeutic potential for targeting and managing complex solid tumors.

Plant-made PD1–Fc fusions engineered for optimized glycosylation and Fc-receptor engagement are highly efficient in blocking PD1/PDL1 interactions and can be cost-effective alternatives to antibody-based immune checkpoint inhibitors. Immune checkpoint inhibitors (ICIs) are antibodies to receptors that have pivotal roles during T-cell activation processes. The programmed cell death 1 (PD1) can be regarded as the primary immune checkpoint and antibodies targeting PD1 or its ligand PDL1 have revolutionized immunotherapy of cancer. However, the majority of patients fail to respond, and treatment resistance as well as immune-related adverse events are commonly associated with this therapy. Alternatives to antibody-based ICIs targeting the PD1 pathway may bear the potential to overcome some of these shortcomings. Here, we have used a plant expression platform based on the tobacco relative Nicotiana benthamiana to generate immunoglobulin fusion proteins harboring the wild type or an affinity-enhanced PD1 ectodomain. We have exploited the versatility of our system to generate variants that differed regarding their glycosylation profile as well as their capability to engage Fc-receptors. Unlike its wild-type counterpart, the affinity-enhanced versions showed strongly augmented capabilities to engage PDL1 in both protein- and cell-based assays. Moreover, in contrast with clinical antibodies, their binding is not affected by the glycosylation status of PDL1. Importantly, we could demonstrate that the plant-made PD1 fusion proteins are highly efficient in blocking inhibitory PD1 signaling in a T cell reporter assay. Taken together, our study highlights the utility of our plant-based protein expression platform to generate biologics with therapeutic potential. Targeting PDL1 with plant derived affinity-enhanced PD1 immunoglobulin fusion proteins may reduce overstimulation associated with antibody-based therapies while retaining favorable features of ICIs such as long serum half-life.

Glycosaminoglycans (GAGs) are major components of the cells glycocalyx and extracellular matrix (ECM), with important roles in both physiological and disease contexts. The biosynthesis of the long and structurally diverse GAG chains is orchestrated by a complex cellular glycosylation machinery and regulated in an organ-, and cell-specific way. Moreover, altered GAG expression levels and structural features have been described in different pathological conditions, including cancer. Noteworthy, GAG chains are endowed with important functional features during cancer progression, such as cancer cell growth, motility, and metastasis formation. Particularly in gastrointestinal (GI) tumors, GAGs have been frequently associated with tumorigenesis and disease progression. This review provides insights on the aberrant GAG expression profiles in GI cancers, highlighting illustrative examples of GAG structural features for each disease model. Relevance is given to the molecular mechanisms underlying altered GAG biosynthesis and post-synthesis editing in GI cancers. Finally, we address the potential of cancer-associated GAG expression signatures for improving GI clinical management.

The human macrophage galactose-type lectin (MGL) recognizes exposed GalNAc residues abundantly found in tumor O-glycans. Herein, we have used an integrative chemical, structural, and functional approach to unravel the intricate specificity and molecular determinants that underlie the recognition of Thomsen-nouveau (Tn), the sialylated variant (STn), and Thomsen-Friedenreich (TF) O-glycans by the carbohydrate recognition domain of the MGL (MGL-CRD) at the molecular and cellular levels. The MGL-CRD prefers binding to Tn > STn ≫ TF O-glycans. In this molecular context, NMR, isothermal titration calorimetry, and molecular dynamics simulations revealed quantitative key structural and dynamic differences in binding, depending on the O-glycan. Interestingly, the density of Tn epitopes was critical for engaging multiple MGL-CRDs to MUC1 Tn-glycopeptides; however, the enthalpy-entropy balance strongly influenced the affinity, and a higher Tn density did not improve the binding. Cell-based mucin arrays recapitulated the MGL-CRD binding preference (Tn > STn ≫TF), but no preference for a specific O-glycan pattern in mucins was observed. The MGL-CRD also selectively recognizes glycoengineered gastric cancer cells expressing Tn/STn. Conversely, in the cellular context, employing CHO cells expressing the full-length MGL (CHO+MGL) allowed analysis of the MGL binding properties in its native presentation toward tagged isolated mucin reporters. Specificity for short tumor-associated O-glycans without any preference for a specific mucin was confirmed. Stunningly, the CHO+MGL cells revealed that the MGL shows similar binding to the STn and TF mucin reporters, suggesting that its natural oligomeric state displays promiscuous binding to simple O-glycans. Conceptually, the key role of glycan and lectin presentations for binding is thus highlighted. Moreover, this suggests the compelling scenario that the MGL serves as a universal receptor for truncated cancer-associated O-glycans.

Aberrant cellular glycosylation is a key event that accompanies and actively sustains gastric neoplastic transformation. Patient-derived organoids (PDOs) have recently emerged as promising ex vivo models to study human gastric diseases; however, their glycosylation landscape remains unknown. To evaluate gastric PDOs as avatars of in vivo tissue glycosylation, a biobank of gastric PDOs (n = 56) was generated from gastric mucosa samples of non-tumoral obese patients (n = 11), adjacent tumor mucosa (n = 26), and gastric tumor tissue (n = 19). PDOs reproduce distinct stages of gastric carcinogenesis and recapitulate the gastric tissue-associated glycosylation profiles. PDOs capture glycan inter- and intra-tumoral heterogeneity, which is maintained over time and upon biobanking and xenografting. Furthermore, expression of type I/II Lewis antigens is dynamically controlled by the PDO's differentiation status, influencing Helicobacter pylori binding, mirroring the gastric epithelium-tissue interactions. This study establishes PDOs as robust ex vivo tools to study gastric glycan dynamics in both gastric physiological and pathological settings.

INTRODUCTION:Glycosylation is an essential enzymatic process of building glycan structures that occur mainly within the cell and gives rise to a diversity of cell surface and secreted glycoconjugates. These glycoconjugates play vital roles, for instance in cellcell adhesion, interaction and communication, activation of cell surface receptors, inflammatory response and immune recognition. This controlled and wellcoordinated enzymatic process is altered in cancer, leading to the biosynthesis of cancerassociated glycans, which impact glycandependent biological roles. AREAS COVERED:In this review, the authors discuss the importance of targeting cancerassociated glycans through potent glycan biosynthesis inhibitors. It focuses on the use of analogs, providing an overview of findings involving these in cancer. The highly explored fluorinated monosaccharide analogs targeting aberrant glycosylation are described, aiming to inspire advances in the field. EXPERT OPINION:Altered glycosylation, such as increased sialylation and fucosylation, is a feature in cancer and has been shown to play key roles in several malignant properties of cancer cells. Strategies aiming at remodeling cancer cells´ glycome are emerging and present a huge potential for cancer therapy. Fluorinated monosaccharides have been gathering promising preclinical results as novel cancer drugs. Nevertheless, cancer specific targeting strategies must be considered to avoid significant sideeffects.

BACKGROUND AND AIMS: Aberrant cellular glycosylation remains a key event that accompanies and actively sustains gastric neoplastic transformation. Patient-derived organoids (PDOs) have recently emerged as a promising ex vivo model to study human gastric disorders. Since the PDOs glycosylation landscape remains unknown, this study aims to evaluate PDOs as potential avatars of in vivo tissue glycosylation profiles in the gastric context. METHODS: Fresh gastric mucosa samples derived from non-tumoral obese patients (n=11), adjacent tumor mucosa samples (n=29), and tumor tissue samples derived from gastric cancer (GC) patients (n=30) were used to establish a biobank of gastric PDOs (n=56). The N- and O- glycophenotypes of normal, adjacent, and tumor PDOs and respective in vivo tissues were thoroughly characterized by immunostaining. Additionally, a comparative glycan analysis was performed over time, upon PDO biobanking and xenografting in mice. The binding of two Helicobacter pylori ( H. pylori ) isogenic strains with distinct glycan-binding affinities was assessed in parental gastric mucosa tissues and compared with the respective PDOs before and after modulation of their glycan landscape. RESULTS: Our results show that PDOs mimic different phenotypes of the carcinogenic cascade and recapitulate parental gastric tissues' glycosylation profile. Tumor PDOs recapitulate the inter- and intra-heterogeneity features observed in GC, which is maintained over time, upon biobanking and xenografting. We demonstrated that the expression of type I and type II Lewis antigens is dynamically controlled by PDOs differentiation status, which results in differential binding to H. pylori strains displaying distinct glycan-binding adhesins, mirroring the gastric epithelium tissue interactions. CONCLUSIONS: This study established PDOs as invaluable ex vivo tools to study the complex glycan dynamics in both gastric physiological and pathological settings. KEYWORDS: Glycosylation, Patient-derived organoids, Helicobacter pylori , Gastric carcinogenesis, Gastric cancer. ### Competing Interest Statement The authors have declared no competing interest.

Background: The World Health Organization has identified Helicobacter pylori, a Gram-negative bacterium responsible for several gastric disorders, as one of the pathogenic bacteria that requires newer non-antibiotic approaches for its management. We previously demonstrated that nanostructured lipid carriers (NLC) loaded with docosahexaenoic acid (DHA-NLC) have excellent in vitro performance against H. pylori.Materials and Methods: NLC were tested against different H. pylori strains and bacteria representative from human gut microbiota. For H. pylori, resistance development and membrane permeability assays were also performed. In vivo efficacy studies were done using an H. pylori-infected mouse model. Microbiome analysis (16S rRNA sequencing analysis) was performed on mice feces before and after DHA-NLC treatment.Results: NLC specifically killed different H. pylori strains by membrane disruption without inducing bacterial resistance. In vivo studies demonstrated that DHA-NLC (2 mg/mL containing 50 mu M of DHA) reduced 90%-95% of the H. pylori burden and eradicated infection in 50% of the animals when treatment was administrated ad libitum for 14 days. No significant differences were found between the administration procedure (ad libitum vs oral gavage). Also, increasing the DHA-NLC concentration to 4 and 8 mg/mL did not translate into an improvement in antibacterial performance. Notably, gut microbiome analysis showed no alterations, highlighting the safety to the gut microbiota. Finally, no histopathological changes were reported (stomach/liver sections).Conclusions: Overall, our results emphasize DHA-NLC as a promising approach for H. pylori infection management, since they can effectively reduce the H. pylori burden without affecting gut microbiota and, in opposition to antibiotics, without anticipating the development of resistance to this treatment.

Cancer cachexia is a complex metabolic syndrome characterized by unintentional loss of skeletal muscle and body fat. This syndrome is frequently associated with different types of cancer and negatively affects the prognosis and outcome of these patients. It involves a dynamic interplay between tumor cells and adipose tissue, where tumor-derived extracellular vesicles (EVs) play a crucial role in mediating intercellular communication. Tumor cells release EVs containing bioactive molecules such as hormones (adrenomedullin, PTHrP), pro-inflammatory cytokines (IL-6), and miRNAs (miR-1304-3p, miR-204-5p, miR-155, miR-425-3p, miR-146b-5p, miR-92a-3p), which can trigger lipolysis and induce the browning of white adipocytes contributing to a cancer cachexia phenotype. On the other hand, adipocyte-derived EVs can reprogram the metabolism of tumor cells by transporting fatty acids and enzymes involved in fatty acid oxidation, resulting in tumor growth and progression. These vesicles also carry leptin and key miRNAs (miR-155-5p, miR-10a-3p, miR-30a-3p, miR-32a/b, miR-21), thereby supporting tumor cell proliferation, metastasis formation, and therapy resistance. Understanding the intricate network underlying EV-mediated communication between tumor cells and adipocytes can provide critical insights into the mechanisms driving cancer cachexia. This review consolidates current knowledge on the crosstalk between tumor cells and adipose tissue mediated by EVs and offers valuable insights for future research. It also addresses controversial topics in the field and possible therapeutic approaches to manage cancer cachexia and ultimately improve patient outcomes and quality of life.

Pancreatic cancer (PC) is the sixth leading cause of cancer-related deaths worldwide, primarily due to late-stage diagnosis and limited treatment options. While novel biomarkers and immunotherapies are promising, further research into specific molecular targets is needed. Glycans, which are carbohydrate structures mainly found on cell surfaces, play crucial roles in health and disease. The Thomsen-Friedenreich-related carbohydrate antigen Sialyl-Tn (STn), a truncated O-glycan structure, is selectively expressed in epithelial tumors, including PC. In this study, we performed a comprehensive analysis of STn expression patterns in normal, premalignant, and malignant pancreatic lesions. Additionally, we analyzed the association between STn expression and various clinicopathological features. STn expression was statistically associated with pathological diagnosis; it was absent in normal pancreatic tissue but prevalent in pancreatic carcinoma lesions, including pancreatic ductal adenocarcinoma (PDAC), pancreatic acinar cell carcinoma, and pancreatic adenosquamous carcinoma. Moreover, we found a significant association between STn expression and tumor stage, with higher STn levels observed in stage II tumors compared to stage I. However, STn expression did not correlate with patient survival or outcomes. Furthermore, STn expression was assessed in PDAC patient-derived xenograft (PDX) models, revealing consistent STn levels throughout engraftment and tumor growth cycles. This finding supports the PDX model as a valuable tool for testing new anti-STn therapeutic strategies for PC in clinical setting.

Gastrointestinal cancers remain a global health burden, demanding more effective prevention and treatments. Phenethyl isothiocyanate (PEITC), a compound derived from cruciferous vegetables, stands out as a promising nutraceutical agent due to its chemopreventive and therapeutic properties. However, its therapeutic translation remains limited mainly due to its poor water solubility and rapid metabolism. Herein, we encapsulated PEITC into biocompatible chitosan-based microparticles with an extra virgin olive oil core to improve its bioavailability and stability. Pure PEITC's biocompatibility and microencapsulated PEITC's stability and antibacterial activity were evaluated. The antibacterial activity analysis showed microencapsulated PEITC as a promising antibacterial agent against gastrointestinal pathogenic bacteria (two Gram-positive and two Gram-negative). The impact of both pure and microencapsulated PEITC was assessed on gastrointestinal cancer cells (MKN45 gastric cancer and SW48 colon cancer cell lines). PEITC exhibited threshold or hormetic dose-dependent toxicity in colon fibroblasts and decreased gastric cancer cells' migration capacity, enhanced upon encapsulation into microparticles. In addition, microencapsulated PEITC induced downregulation of phosphorylated AKT, FAK, and ERK1/2 proteins, disrupting motility signaling pathways and tubulin expression. These findings suggest that the delivery of PEITC via chitosan-based microparticles holds promise as a nutraceutical delivery strategy against gastrointestinal disorders that predispose to cancer.

Colorectal (CRC) and gastric (GC) cancers remain the top lethal cancers and targeted therapies in this setting are still very limited. Sialyl Lewis X (SLeX), a cancer-associated glycan highly expressed in both CRC and GC, plays a crucial role in cancer cell dissemination and metastasis. Thus, presenting a promising but still underexplored therapeutic target. In this work, we performed a high-throughput screening (HTS) approach to identify potential inhibitors of SLeX expression on cancer cells. Two libraries including a total of 7836 compounds were screened and monensin emerged as a promising SLeX inhibitor. Monensin promoted structural alterations in the secretory pathway, particularly at the Golgi apparatus, impacting protein O-glycosylation and secretion. RNAseq transcriptomic analysis uncovered significant alterations in Gene Ontology (GO) terms associated with protein misfolding, target to the membrane, as well as, epithelial cell-cell adhesion protein. In vitro studies showed that, upon treatment with monensin, SLeX-positive cancer cells showed reduced viability, concomitant with decreased motility and invasive capacities. Using in vivo xenograft models of chick embryo chorioallantoic membrane (CAM) and nude mice, revealed that monensin reduced tumor formation and invasion. Pre-clinical validation using gastric cancer patient-derived organoids (PDOs) and organoid xenotransplants in mice further underscored the clinical potential of monensin in suppressing the growth of SLeX-positive tumors. Overall, our findings set the ground for further evaluation of monensin as a novel therapeutic agent in GC and CRC in the clinical setting### Competing Interest StatementThe authors have declared no competing interest.### Funding StatementThis study was funded by (i) Fundo Europeu de Desenvolvimento Regional (FEDER) funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalization (POCI), Portugal 2020 (POCI-01-0145-FEDER-016585, POCI-01-0145-FEDER-007274). (ii) Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) project NORTE-01-0145-FEDER- 000029. (iii) Portuguese funds through Fundacao para a Ciencia e a Tecnologia (FCT)/Ministerio da Ciencia, Tecnologia e Inovacao through the research projects PTDC/MED-QUI/29780/2017 - POCI-01-0145-FEDER-029780, PTDC/MED-QUI/2335/2021 and EXPL/BTM-ORG/1450/2021. AFC, LSF and RA were supported by FCT PhD grants (UI/BD/150829/2021, 2021.05495.BD and 2020.05483.BD), CG (2022.04678.CEECIND), FP (2022.02109.CEECIND) and HOD (2022.00943.CEECIND).### Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:Ehtics commitee/IRB of Centro Hospitalar Universitario Sao Joao gave ethical approval of this workI confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.YesAll data produced in the present study are available upon reasonable request to the authors

Gastric and gastroesophageal junction adenocarcinomas (GA/GEJA) are associated with a poor prognosis, primarily due to late disease diagnosis. Human Epidermal Growth Factor Receptor 2 (HER2) overexpression and programmed death-ligand 1 (PD-L1) expression are important biomarkers for treatment selection in locally advanced unresectable and metastatic GA/GEJA, and there is increasing interest in their role in earlier stages of disease. In this study, we aimed to evaluate HER2 and PD-L1 expression in a curative-intent GA/GEJA cohort to describe their expression patterns and analyze the association between HER2 expression and clinicopathological features. HER2 expression was evaluated in surgical and endoscopic submucosal dissection tumor samples, and PD-L1 was evaluated in HER2-positive cases. The clinical cohort included 107 patients, with 8.4% testing positive for HER2 (seven of whom also exhibited a PD-L1 combined positive score of ≥1. HER2 status was not significantly associated with survival outcomes. A pathologist-guided, region-specific analysis revealed that PD-L1 expression rarely overlaps with HER2-positive tumor areas. While the therapeutic implications of these observations remain unknown, these findings suggest that combination strategies targeting HER2 and PD-L1 might be directed toward distinct tumor subclones. The herein disclosed region-specific biomarker expression patterns may have important therapeutic and prognostic impacts, warranting further evaluation.

Mammalian glycosaminoglycans (GAGs), except hyaluronan (HA), are sulfated polysaccharides that are covalently attached to core proteins to form proteoglycans (PGs). This article summarizes key biological findings for the most widespread GAGs, namely HA, chondroitin sulfate/dermatan sulfate (CS/DS), keratan sulfate (KS), and heparan sulfate (HS). It focuses on the major processes that remain to be deciphered to get a comprehensive view of the mechanisms mediating GAG biological functions. They include the regulation of GAG biosynthesis and postsynthetic modifications in heparin (HP) and HS, the composition, heterogeneity, and function of the tetrasaccharide linkage region and its role in disease, the functional characterization of the new PGs recently identified by glycoproteomics, the selectivity of interactions mediated by GAG chains, the display of GAG chains and PGs at the cell surface and their impact on the availability and activity of soluble ligands, and on their move through the glycocalyx layer to reach their receptors, the human GAG profile in health and disease, the roles of GAGs and particular PGs (syndecans, decorin, and biglycan) involved in cancer, inflammation, and fibrosis, the possible use of GAGs and PGs as disease biomarkers, and the design of inhibitors targeting GAG biosynthetic enzymes and GAG-protein interactions to develop novel therapeutic approaches.

Gastric cancer is a dominating cause of cancer-associated mortality with limited therapeutic options. Here, we show that syndecan-4 (SDC4), a transmembrane proteoglycan, is highly expressed in intestinal subtype gastric tumors and that this signature associates with patient poor survival. Further, we mechanistically demonstrate that SDC4 is a master regulator of gastric cancer cell motility and invasion. We also find that SDC4 decorated with heparan sulfate is efficiently sorted in extracellular vesicles (EVs). Interestingly, SDC4 in EVs regulates gastric cancer cell-derived EV organ distribution, uptake, and functional effects in recipient cells. Specifically, we show that SDC4 knockout disrupts the tropism of EVs for the common gastric cancer metastatic sites. Our findings set the basis for the molecular implications of SDC4 expression in gastric cancer cells and provide broader perspectives on the development of therapeutic strategies targeting the glycan-EV axis to limit tumor progression.

Gastric cancer (GC) is the fourth leading cause of cancer-related deaths worldwide and, therefore, it is urgent to develop new and more efficient therapeutic approaches. Foretinib (FRT) is an oral multikinase inhibitor targeting MET (hepatocyte growth factor receptor) and RON (recepteur d'origine nantais) receptor tyrosine kinases (RTKs) that has been used in clinical trials for several solid tumors. Targeted uptake of therapeutic polymeric nanoparticles (NPs) represents a powerful approach in cancer cell drug delivery. Previously, a nanodelivery system composed of polymeric NPs functionalized with B72.3 antibody, which targets the tumor-associated antigen Sialyl-Tn (STn), has been developed. Herein, these NPs were loaded with FRT to evaluate its capacity in delivering the drug to multicellular tumors spheroids (MCTS) and mouse models. The data indicated that B72.3 functionalized FRT-loaded PLGA-PEG-COOH NPs (NFB72.3) specifically target gastric MCTS expressing the STn glycan (MKN45 SimpleCell (SC) cells), leading to a decrease in phospho-RTKs activation and reduced cell viability. In vivo evaluation using MKN45 SC xenograft mice revealed that NFB72.3 were able to decrease tumor growth, reduce cell proliferation and tumor necrosis. NFB72.3-treated tumors also showed inactivation of phospho-MET and phospho-RON. This study demonstrates the value of using NPs targeting STn for FRT delivery, highlighting its potential as a therapeutic application in GC. STATEMENT OF SIGNIFICANCE: Despite the advances in gastric cancer therapeutics, it remains one of the diseases with the highest incidence and mortality in the world. Combining targeted therapies with a controlled drug release is an attractive strategy to reduce drug cytotoxic effects and improve specific drug delivery efficiency to the cancer cells. Thus, we developed nanoparticles loaded with a tyrosine kinase inhibitor and targeting a specific tumor glycan exclusive of cancer cells. In in vivo gastric cancer xenograft mice models, these nanoparticles efficiently reduced tumor growth, cell proliferation and tumor necrosis area and inactivated phosphorylation of targeting receptors. This approach represents an innovative therapeutic strategy with high impact in gastric cancer.

Expression of sialyl Lewis X (SLeX) is a well-documented event during malignant transformation of cancer cells, and largely associates with their invasive and metastatic properties. Glycoproteins and glycolipids are the main carriers of SLeX, whose biosynthesis is known to be performed by different glycosyltransferases, namely by the family of β-galactoside-α2,3-sialyltransferases (ST3Gals). In this study, we sought to elucidate the role of ST3GalIV in the biosynthesis of SLeX and in malignant properties of gastrointestinal (GI) cancer cells. By immunofluorescent screening, we selected SLeX-positive GI cancer cell lines and silenced ST3GalIV expression via CRISPR/Cas9. Flow cytometry, immunofluorescence and western blot analysis showed that ST3GalIV KO efficiently impaired SLeX expression in most cancer cell lines, with the exception of the colon cancer cell line LS174T. The impact of ST3GalIV KO in the biosynthesis of SLeX isomer SLeA and non sialylated Lewis X and A were also evaluated and overall, ST3GalIV KO led to a decreased expression of SLeA and an increased expression in both LeX and LeA. In addition, the abrogation of SLeX on GI cancer cells led to a reduction in cell motility. Furthermore, ST3GalVI KO was performed in LS174T ST3GalIV KO cells, resulting in the complete abolishment of SLeX expression and consequent reduced motility capacity of those cells. Overall, these findings portray ST3GalIV as the main, but not the only, enzyme driving the biosynthesis of SLeX in GI cancer cells, with a functional impact on cancer cell motility.