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Colorectal Air–Liquid Interface Organoids Preserve Tumour-Immune Architecture and Reveal Local Treg Expansion After PD-1 Blockade
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Laura Córdoba
1,2,3,*,†
,
Francisco J. Cueto
2,3,4,†,
Ramón Cantero-Cid
5,6,7,†,
Rebeca Abad-Moret
5,
Esteban Díaz
5,
Jaime Álvarez-Benayas
8,
Jesús Fernández-Felipe
2,3,9,
Jesús Jiménez-Rodríguez
1,2,3,
Daniel Arvelo-Rosario
2,3,
Pablo Mata-Martínez
2,10,
Marina Arranz-Álvarez
1,
Yaiza Pedroche-Just
1,
Sandra Nieto-Torrero
1,
Jaime Valentín-Quiroga
2,3,
Verónica Terrón-Arcos
2,3,
Jaime Fernández-Pascual
2,10,
Paloma Gómez-Campelo
1,
Nieves Cubo-Mateo
8
,
Olivia Fernández-Medina
2,10,
Laura Hurtado-Navarro
2,3,9,
Gonzalo Sáenz de Santa María
2,3,
Julia del Prado-Montero
2,3,
Agustín L. Santos
1,2,3,
Roberto Lozano-Rodríguez
2,3,
Carlos del Fresno
2,10 and
Eduardo López-Collazo
1,2,3,9,11,*add
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1
Biobank Platform, IdiPAZ, La Paz University Hospital, 28046 Madrid, Spain
2
The Innate Immune Response Group, IdiPAZ, La Paz University Hospital, 28046 Madrid, Spain
3
Tumour Immunology Lab, IdiPAZ, La Paz University Hospital, 28046 Madrid, Spain
4
Department of Medical Oncology, La Paz University Hospital, 28046 Madrid, Spain
5
Digestive Surgery Service, La Paz University Hospital, 28046 Madrid, Spain
6
Digestive Surgery Department, Faculty of Medicine, Universidad Autónoma de Madrid, 28046 Madrid, Spain
7
Translational Research and Innovation in Surgery Group, La Paz University Hospital, 28046 Madrid, Spain
8
ARIES Research Group, Universidad Antonio de Nebrija, 28248 Madrid, Spain
9
Faculty of Health Sciences, Universidad UNIE, 28015 Madrid, Spain
10
Immunomodulation Laboratory, IdiPAZ, La Paz University Hospital, 28046 Madrid, Spain
11
Biomedical Research Networking Center (CIBER), Respiratory Diseases (CibeRes), 28029 Madrid, Spain
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Cancers 2026, 18(1), 132; https://doi.org/10.3390/cancers18010132 (registering DOI)
Submission received: 2 December 2025
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Revised: 24 December 2025
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Accepted: 26 December 2025
/
Published: 30 December 2025
(This article belongs to the Special Issue Dendritic Cells and Immune Checkpoints Within the Tumor Microenvironment: Mechanisms, Models, and Therapy)
Simple Summary
Understanding how colorectal tumours interact with immune cells is critical for improving immunotherapy responses. In this study, we used air–liquid interface organoids derived from patient tumours and adjacent healthy colon tissues to reproduce the original tumour microenvironment in the laboratory. These organoids preserved the three-dimensional tissue structure, the native immune infiltrates, and the genetic mutations of the original samples more faithfully than conventional Matrigel-based cultures. When compared with Matrigel organoids co-cultured with blood immune cells, the air–liquid interface model more accurately reflected the immune and molecular features of each patient’s tissue. Notably, treatment with the immune checkpoint inhibitor nivolumab (anti–PD-1) led to a consistent expansion of regulatory T cells, recapitulating a mechanism of adaptive resistance. This approach offers a physiologically relevant platform to study colorectal cancer–immune interactions and to predict patient-specific responses to immunotherapy.
Abstract
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.
