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

Jan 20, 2026Cell communication and signaling : CCS

Immature CD276 sugar modification increases colorectal cancer aggressiveness and helps it avoid T cell immune attack

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CRISPR Gene Editing on OpenScience ↗PubMed ↗DOI ↗OA ↗

Abstract

Downregulation of B3GNT6 and C1GALT1 is associated with an immature O-glycosylation phenotype linked to poor prognosis in colorectal cancer (CRC).

Glycomic profiling identified Tn- and sialyl-Tn-enriched glycophenotypes in both epithelial and mesenchymal tumors with specific subtype patterns.
CD276, which colocalizes with Tn and sTn, exhibited immature O-glycans that were absent in healthy tissues and was more prevalent in right-sided and metastatic CRC.
Reanalysis of CRC proteomic data indicated that CD276 expression and glycosylation varied with tumor differentiation states, with distinct patterns in epithelial-like versus mesenchymal-like tumors.
Knockout of C1GALT1 in CRC cells led to increased CD276 stability and transcription, contributing to enhanced invasion and overexpression of CD276.
Aberrantly glycosylated CD276 was linked to increased tumor proliferation, invasion, and suppression of T cell activity, correlating with immune evasion.
TCGA data confirmed that high levels of CD276 and low levels of C1GALT1 expression were associated with markers of immune checkpoint activity and T cell exhaustion.

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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.

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

Abstract

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