CD276 immature glycosylation drives colorectal cancer aggressiveness and T cell mediated immune escape

Abstract 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 Abstract 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.