Researchers at VIB and VUB have developed a powerful new way to study how the immune system behaves inside lung tumours. By combining a patient-relevant mouse model with single-cell technologies, the team provides one of the most comprehensive immune maps to date of lung adenocarcinoma, which is the most common subtype of lung cancer. Their work appears in Nature Communications.
“We created a lung cancer model that closely mimics how tumours grow in patients,” said Damya Laoui (VIB-VUB Center for Inflammation Research).
“Combined with a new tracking method for cells, this allow us to tell the difference between immune cells inside the tumour tissue and those that are just passing by in the bloodstream. That distinction makes a big difference. It allows us to see much more clearly how immune cells behave and change once they are inside the tumour.”
Lung cancer remains the leading cause of cancer-related deaths worldwide, leading to almost 20% of all cancer mortality. Developing effective therapies begins with preclinical studies that mostly rely on subcutaneous tumour models, where cancer cells are implanted under the skin. While practical, these models fail to capture the lung’s unique immune landscape.
To address this gap, the researchers developed a lung adenocarcinoma model, in which tumours grow directly inside the lung. The model allows researchers to dissect tumour nodules separately from adjacent healthy lung tissue, mirroring how patient samples are handled in the clinic. When the team compared their model to human lung adenocarcinoma datasets, they found it closely reproduced key immune features observed in patients, such as dysfunctional natural killer (NK) cells inside tumours and increased regulatory and exhausted T cells.
“Our goal was to build a model that reflects what we actually see in patients. By placing the tumour in its natural environment, the lung, we capture immune dynamics that are simply absent in subcutaneous models,” said Pauline Bardet (VIB-VUB), PhD student and co-first author of the study.
A central innovation of the study is SEPARATE-Seq (streptavidin enabled partitioning and tag evaluation for RNA-sequencing). In organs like the lung, immune cells are distributed across different compartments: within blood vessels, in the tissue itself, or in the airways. Standard single-cell RNA sequencing cannot easily distinguish between immune cells that are truly infiltrating a tumour and those merely passing through its blood supply. SEPARATE-Seq overcomes this limitation by ‘labelling’ immune cells in the blood.
“Location matters enormously,” said Laoui, senior author of the study.
“An immune cell inside a blood vessel is not experiencing the same signals as one embedded in tumour tissue. With SEPARATE-Seq, we can finally resolve that difference at single-cell resolution.”
The method is broadly applicable beyond lung cancer and can be used in other diseases where different immune cell populations need to be distinguished.
By combining SEPARATE-Seq with spatial transcriptomics, the researchers mapped not only which immune cells are present, but also where they are precisely located inside tumours.
The analyses revealed several spatial patterns. The team identified a ring of lipid-associated tumour-associated macrophages lining the tumour edge, alongside defined hubs of interferon-stimulated immune and non-immune cells within the tumour, that were enriched in specific dendritic cell states. They observed increased infiltration of hypoxic, tumour-associated neutrophils, enrichment of plasma cells in the tumour, and a shift of NK cells toward an immature, dysfunctional state upon tumour entry.
Many of these features were also observed in human lung adenocarcinoma samples, underscoring the translational relevance of the model.
“This level of spatial and molecular resolution allows us to see how immune cells specialise within defined tumour niches,” said Lize Allonsius, PhD student and co-first author of the study.
“It highlights how strongly the tumour microenvironment reshapes immune function.”
Beyond establishing the model, the team provides a comprehensive multiomics dataset and makes it accessible through an interactive online tool, offering a valuable resource for researchers studying tumour immunology and lung cancer biology.
“Therapies succeed or fail based on how immune cells behave inside real tumours,” Laoui said.
“If our models do not faithfully reflect patient biology, we risk drawing misleading conclusions. With this work, we provide a framework that brings preclinical research one step closer to the clinic.”