José Alexandre Ferreira

Antigen-presenting-cell dysfunction drives immune exclusion, but how tumour glycans disable these cells remains incompletely defined. Immature O -glycosylation, marked by Tn and sialyl-Tn antigens, drives myeloid dysfunction in bladder cancer. Transcriptomics and human tumour profiling linked this state to reduced representation of antigen-presenting myeloid cells and attenuated lymphoid-effector programmes. In glycoengineered models, immature O -glycans promoted immunoregulatory macrophage phenotypes and contact-dependent death of monocyte-derived dendritic cells (moDCs), while surviving cells showed impaired maturation and T-cell activation. Tumour-cell CD44 standard isoform was required for DC death, which was accompanied by inflammasome-associated proteins and gasdermin D cleavage, consistent with pyroptosis. Receptor-discovery approaches converged on CD206, which bound immaturely glycosylated CD44. CD206 blockade restored DC viability. In immunocompetent mice, immature glycosylation accelerated tumour growth and depleted conventional DCs, whereas CD206 blockade restrained tumour progression and restored local and nodal myeloid and lymphoid immunity. These findings identify CD44-CD206 as a targetable glycoimmune checkpoint that promotes immune exclusion.

Immature protein O-glycosylation, characterized by the Tn antigen expression rather than complex glycans, is prevalent in advanced solid tumors and promotes immune evasion. We developed GlycoAvatars, magnetic beads coated with plasma-membrane glycoproteins from glycoengineered cancer cells reflecting these alterations, to identify cancer-immune cell interactomes. We uncovered numerous membrane and intracellular proteins involved in glycan-mediated immune signaling, providing a novel high-throughput strategy for identifying potential relevant molecular nodes foreseeing therapeutic targets.

Background: Colorectal cancer (CRC) progression is fuelled by immune evasion, yet the underlying molecular mechanisms remain to be fully characterized. CD276 (B7-H3), an immune checkpoint glycoprotein frequently overexpressed in aggressive tumors, is extensively modified by glycosylation, a process known to regulate protein stability, localization, and immune interactions. However, its glycosylation-dependent functions in CRC remain unclear. Methods: TCGA Transcriptomic data were analysed to identify glycogene alterations linked to patient prognosis. The O-glycome of advanced CRC and normal mucosa was profiled by mass spectrometry. CD276 expression and glycosylation were examined in primary tumors, lymph nodes, and metastases by immunohistochemistry, proximity ligation assays, and dual immunofluorescence. CRC proteomic datasets from PRIDE (≥ 90 cases) were reanalyzed to map CD276 immature glycosylation across differentiation states. C1GALT1 knockout CRC cell lines were generated with CRISPR-Cas9 to mimic immature glycosylation in tumors, and CD276 was silenced with siRNAs. Immunoprecipitation, lectin blotting, protein stability assays, proliferation, invasion, phosphoproteomics, and T cell co-culture experiments were used to assess functional consequences. Results: Downregulation of B3GNT6 and C1GALT1 or C1GALT1C1 defined an immature O-glycosylation phenotype associated with poor prognosis. Glycomic profiling revealed Tn- and sialyl-Tn(sTn)-enriched glycophenotypes in both epithelial- and mesenchymal-like tumors, with subtype-specific patterns. CD276 colocalized with Tn and sTn, carried immature O-glycans absent from healthy tissues, and was enriched in right-sided and metastatic CRC, correlating with worse survival. PRIDE reanalysis suggested widespread CD276 expression and revealed differentiation-linked glycosylation, which was denser in the IgV and IgC domains of epithelial-like tumors and sparser, membrane-proximal in mesenchymal-like tumors. C1GALT1 knockout in CRC cells enhanced invasion while increasing CD276 stability and transcription, driving its overexpression. Aberrantly glycosylated CD276 promoted proliferation, invasion, kinase-driven signalling, and T cell suppression while driving cytokines toward immunosuppression. TCGA confirmed that high CD276 and low C1GALT1 expression correlated with transcriptional signatures of heightened immune checkpoint activity and T cell exhaustion. Conclusions: Immature CD276 glycosylation promotes CRC aggressiveness and immune escape, representing a candidate prognostic biomarker and therapeutic target. Graphical Colorectal cancer (CRC) progression is closely linked to immune evasion, yet the molecular mechanisms underlying this process remain poorly understood. This study identifies CD276 (B7-H3) as a glycosylation-driven regulator of CRC aggressiveness and demonstrates how O -glycosylation remodelling promotes tumor immune escape. These findings establish CD276 as a potential therapeutic target and highlight the role of glycoproteoform-specific immune modulation in cancer progression.

Rationale: Gastric cancer (GC) is a leading cause of cancer-related mortality, particularly in advanced stages where prognosis and targeted treatment remain challenging. The glycocalyx, a dense network of glycans and glycoproteins, is critical for tumor progression and immune evasion, yet its molecular signatures are poorly understood. This study investigates glycan-based biomarkers of aggressiveness, focusing on paucimannosidic N-glycans, a previously underexplored glycosylation pattern in cancer.

Colorectal cancer (CRC) progression is fueled by immune evasion, yet the underlying molecular mechanisms remain to be fully characterized. CD276 (B7-H3), an immune checkpoint glycoprotein frequently overexpressed in aggressive tumours, is extensively modified by glycosylation, a process known to regulate protein stability, localization, and immune interactions. However, its glycosylation-dependent functions in CRC remain unclear. This study shows that poor-prognosis CRC tumours exhibit dysregulated glycogene expression, leading to an immature O -glycosylation phenotype that enhances malignancy. Using C1GALT1 knockout CRC cells, which recapitulate the cancer glycocalyx, it was demonstrated that this glycosylation shift promotes proliferation and invasion. We further identify CD276 as a metastasis-associated glycoprotein whose function is shaped by O -glycan modifications. Mechanistically, immature O -glycosylation of CD276 enhances invasion, suppresses T-cell activation, and induces an immunosuppressive cytokine milieu, reinforcing its role in tumour immune escape. These findings establish CD276 as a glycosylation-dependent immune checkpoint and a promising therapeutic target to overcome immune evasion in CRC. Graphical Table of Contents: Colorectal cancer (CRC) progression is closely linked to immune evasion, yet the molecular mechanisms underlying this process remain poorly understood. This study identifies CD276 (B7-H3) as a glycosylation-driven regulator of CRC aggressiveness and demonstrates how O -glycosylation remodeling promotes tumour immune escape. These findings establish CD276 as a potential therapeutic target and highlight the role of glycoproteoform-specific immune modulation in cancer progression.

Aberrant glycosylation is a hallmark of cancer, offering opportunities to enhance clinical decision-making and enable precise targeting of cancer cells. Nevertheless, alterations in the bladder urothelial carcinoma (BLCA) N-glycome remain poorly characterized. Here, we used in situ N-deglycosylation and mass spectrometry, revealing a marked enrichment of oligomannose-type N-glycans in non-invasive Ta tumors, which diminished with disease progression. A complementary analysis of The Cancer Genome Atlas (TCGA) transcriptomic data revealed downregulation of the key mannosidases in BLCA, suggesting a mechanistic basis for oligomannose accumulation, though this requires further validation. Then, targeted glycoproteomic profiling identified potential stage-specific carriers of oligomannoses. Exploratory functional annotation suggests stage-dependent differences among detected glycoproteins, ranging from metabolic regulation in Ta tumors to oxidative stress adaptation in muscle-invasive disease, highlighting glycosylation’s contribution to tumor progression. Furthermore, myeloperoxidase (MPO) was enriched in more aggressive stages. Spatial validation confirmed MPO overexpression in tumor-infiltrating immune cells and its correlation with oligomannose content. Importantly, high MPO expression combined with low mannosidase levels was linked to poor survival, suggesting biological relevance. This study suggests a dynamic, stage-specific N-glycome in BLCA and identifies oligomannose-bearing glycoproteins as exploratory leads for biomarker and therapeutic target discovery, providing a N-glycomic resource for further investigation towards glycan-based precision oncology.

Immature O -glycosylation of plasma membrane proteins, characterized by the presence of glycosites with a single GalNAc residue (the Tn antigen) rather than more extended and complex glycans, is a prominent feature of many advanced solid tumors across different origins and aetiologies. This form of glycosylation has been strongly associated with poor prognosis and is known to play a crucial role in the progression of the disease. Notably, it has been directly implicated in the crosstalk with the immune system, inducing tolerogenic phenotypes in both innate and adaptive immune cells. Despite this, the specific receptors involved in these interactions remain largely unidentified. To address this gap, we propose a novel high-throughput proteomics assisted strategy for characterizing the glycan interactome using plasma membrane-derived glycoprotein coated magnetic beads. Glycoproteins were isolated from plasma membranes of glycoengineered cell models with immature glycosylation and were then cloaked on the surface of magnetic beads, creating simplified cell “GlycoAvatars.” These GlycoAvatars were primarily employed to characterize the interactome between gastric and colorectal cancer cell lines and immune cells (dendritic cells and macrophages), providing a tool for identifying potentially relevant molecular nodes in glycan-mediated immune synapses, foreseeing their application in deciphering tumor-immune interactions.

Colorectal cancer (CRC) cells express sialylated Lewis antigens (sLe), crucial for metastasis via E‐selectin binding. However, these glycoepitopes lack cancer specificity, and E‐selectin‐targeted glycoproteins remain largely unknown. Here, we established a framework for identifying metastasis‐linked glycoproteoforms. More than 70% of CRC tumors exhibited overexpression of sLeA/X, yet without discernible associations with metastasis or survival. However, The Cancer Genome Atlas (TCGA) analysis unveiled differing expression patterns of sLeA/X‐related glycogenes correlating with disease severity, indicating context‐dependent regulation by distinct glycosyltransferases. Deeper exploration of metastatic tumor sialoglycoproteome identified nearly 600 glycoproteins, greatly expanding our understanding of the metastasis‐related glycoproteome. These glycoproteins were linked to cell adhesion, oncogenic pathways, and neuroendocrine functions. Using an in‐house algorithm, the secretin receptor (SCTR) emerged as a top‐ranked targetable glycoprotein. Tumor screening confirmed SCTR's association with poor prognosis and metastasis, with N‐glycosylation adding cancer specificity to this glycoprotein. Prognostic links were reinforced by TCGA‐based investigations. In summary, SCTR, a relatively unknown CRC glycoprotein, holds potential as a biomarker of poor prognosis and as an E‐selectin ligand, suggesting an unforeseen role in disease dissemination. Future investigations should focus on this glycoprotein's biological implications for clinical applications.