Colorectal Air-Liquid Interface Organoids Preserve Tumour-Immune Architecture and Reveal Local Treg Expansion After PD-1 Blockade.

BACKGROUND/OBJECTIVES: Interactions between colorectal tumours and their immune microenvironment critically influence disease progression and response to immunotherapy. However, most organoid systems fail to preserve the complex architecture and immune composition of the original tissue. Here, we applied the air-liquid interface (ALI) organoid model to paired tumour and perilesional colon tissues from colorectal cancer patients to evaluate its ability to retain immune and genetic features and to reproduce responses to chemotherapy and immune checkpoint blockade. METHODS: Fresh human tumour and matched healthy colon tissues were processed to generate ALI organoids. Their histological organization, immune cell composition (including CD45(+) subsets), and genomic profiles were compared with those of the parental tissues and with conventional Matrigel organoids, either alone or co-cultured with peripheral blood mononuclear cells (PBMCs). Organoids were exposed to 5-FU and nivolumab (anti-PD-1) to assess local immune modulation. RESULTS: ALI organoids faithfully preserved the three-dimensional architecture, native immune infiltrates, and somatic mutational landscape of the source tissues. Importantly, upon PD-1 blockade with nivolumab, ALI organoids consistently exhibited a local expansion of regulatory T cells (Tregs), a phenomenon that could contribute to adaptive immune resistance. This response was not reproduced in PBMC-Matrigel co-culture systems, highlighting the importance of preserving endogenous tumour-immune interactions. CONCLUSIONS: Patient-derived ALI organoids represent a physiologically relevant platform that conserves key structural, immunological, and genomic hallmarks of colorectal cancer. By capturing clinically relevant immune remodeling events, such as Treg expansion following PD-1 blockade, this model provides a powerful tool for dissecting tumour-immune interactions.