Endothelial glycocalyx disruption to promote colorectal carcinogenesis and recurrence through formation of TLS-deficient immune microenvironment.

148

Background: The endothelial glycocalyx is a glycan-rich layer covering vascular endothelial cells and plays an essential role in vascular homeostasis by regulating permeability and immune cell trafficking. Glycocalyx disruption occurs in several pathological conditions, including diabetes, infection, and cancer. Tertiary lymphoid structures (TLS) are ectopic immune niches that contribute to antitumor immunity and have gained attention in the era of immune checkpoint inhibitors. Methods: We investigated the impact of endothelial glycocalyx disruption on colorectal tumorigenesis and tumor immune microenvironment remodeling using endothelial-specific Ext-1 knockout mice lacking heparan sulfate synthesis. In parallel, 192 human colorectal cancer cases were analyzed for recurrence. TLS-rich and TLS-poor tumors were further characterized using spatial omics analysis. Results: In the AOM/DSS-induced colorectal carcinogenesis model, Ext-1–deficient mice showed significantly increased tumor number and tumor size, indicating enhanced tumorigenesis. TLS counts were significantly lower in mutant mice compared with controls. Single-cell RNA sequencing revealed partial activation of the TGF-β pathway and enhanced Wnt transcriptional activity in mutant tumors. Foxp3 expression was increased, and immune checkpoint molecules CD274 and CTLA4 were upregulated in tumor and whole tissue samples. In human colorectal cancer, TLS-poor tumors showed increased Treg proportions and reduced cDC1 and cDC2 frequencies compared with TLS-rich tumors, suggesting impaired antigen presentation. Consistent with murine findings, CTLA4 and CD274 expression levels were higher in TLS-poor tumors. Therapeutic validation demonstrated that CTLA4 blockade significantly reduced tumor number in mutant mice compared with untreated mutant mice and control mice. Notably, all macroscopically malignant-appearing lesions were histologically adenoma or hyperplastic polyps, and no adenocarcinoma was identified. Conclusions: Endothelial glycocalyx disruption promotes colorectal tumorigenesis and recurrence through TLS suppression and remodeling of the tumor immune microenvironment. CTLA4 inhibition may represent a potential therapeutic strategy for TLS-poor colorectal cancer and warrants further investigation.