Evidence of endothelial anergy and T-cell exclusion in microsatellite stable colorectal liver metastases revealed through human hemi-liver perfusion and spatial transcriptomics.

Abstract

BACKGROUND Colorectal liver metastasis (CRLM) is the terminal stage of colorectal cancer and remains largely resistant to immunotherapeutics, yet the underlying immune escape mechanisms remain poorly understood. Through novel observational methods focused on human living and fixed tissue, we profiled the spatial and molecular features of microsatellite stable (MSS) CRLM and the surrounding liver microenvironment. METHODS First, we re-purposed normothermic hepatic machine perfusion as a preclinical model of MSS CRLM, enabling delivery of labelled T-cells through physiological vascular routes into living, tumour-bearing human livers. Second, to dissect the liver microenvironment, we spatially map the transcriptomes of MSS CRLM and adjacent liver at single-cell resolution across ~ 1.9 million cells, and perform multiplexed immunofluorescence applying a19-marker panel to a validation cohort. RESULTS Whilst T-cells readily extravasated within the peri-tumoural liver, they failed to extravasate into MSS CRLM. Spatial analyses revealed a progressive shift in endothelial phenotypes across regions: from a non-inflamed state in normal liver, to a highly inflamed, peri-tumoural endothelium, and finally to an anergic, angiogenic endothelial population within CRLM characterised by reduced expression of adhesion molecules required for T-cell extravasation. CD4 T-cells that extravasate in the peri-tumoural liver are TCR-reactive yet exhausted, whereas CD4 T-cells within CRLM show stress responses, impaired cytokine gene expression and reduced TCR reactivity, residing within hypoxic niches. These divergent CD4 phenotypes are associated with distinct fibroblast-myeloid niches across peri-tumoural and tumour regions. CONCLUSION Based on these observations, MSS CRLM impair T-cell extravasation and simultaneously drive intra-tumoural endothelial anergy, peri-tumoural T-cell exhaustion and an intra-tumoural T-cell stress response. Further mechanistic work is needed to validate these findings and guide the development of novel immunotherapeutics.