Researchers affiliated with VIB and VUB have pioneered a sophisticated methodology for examining the immune system’s activity within the microenvironment of lung tumors. Through the integration of a patient-aligned murine model with advanced single-cell analytical techniques, this research collective has produced one of the most exhaustive immunological atlases documented for lung adenocarcinoma, the predominant form of lung malignancy. The findings of their investigation are published in the esteemed journal, Nature Communications.

We engineered a preclinical model of lung cancer that closely emulates the growth patterns observed in human patients. When coupled with an innovative cellular tracking mechanism, this approach empowers us to differentiate between immune cells residing within the tumorous tissue and those merely circulating in the bloodstream. This crucial distinction markedly enhances our comprehension, enabling a clearer visualization of how immune cells function and transform once they have infiltrated the tumor mass.

Prof. Damya Laoui, VIB-VUB Center for Inflammation Research

A Preclinical Model Yielding Enhanced Patient Relevance

Globally, lung cancer continues to be the foremost determinant of cancer-associated mortality, accounting for nearly 20% of all cancer-related deaths. The development of efficacious therapeutic interventions is predicated on preclinical investigations, which predominantly employ subcutaneous tumor models. These models involve the implantation of cancerous cells beneath the cutaneous layer. Although practical, these models are insufficient in fully encapsulating the distinct immunological milieu characteristic of the pulmonary system.

To address this deficiency, the scientific team devised a lung adenocarcinoma model wherein neoplastic growths develop directly within the lung parenchyma. A critical feature of this model is its capacity to allow for the meticulous separation of tumor nodules from the surrounding healthy pulmonary tissue, mirroring the methodologies employed in clinical analysis of patient-derived samples. Upon comparing their model with existing human lung adenocarcinoma datasets, the researchers ascertained that it accurately recapitulated principal immunological characteristics identified in patients, including impaired natural killer (NK) cell functionality within tumors and an escalation in regulatory and exhausted T cells.

“Our principal objective was to construct a model that truly reflects the conditions encountered in patients,” stated Pauline Bardet (VIB-VUB), a doctoral candidate and a lead author of the publication. “By situating the tumor within its native physiological environment, the lung, we are able to capture intricate immune dynamics that are inherently absent in subcutaneous models.”

Introduction of SEPARATE-Seq Technology

A pivotal advancement underpinning this research is the development of SEPARATE-Seq, an acronym for Streptavidin Enabled PARtitioning And Tag Evaluation for RNA-Sequencing. In organs such as the lungs, immune cells are dispersed across various compartments: within vascular structures, embedded within the tissue matrix, or present in the respiratory passages. Conventional single-cell RNA sequencing methodologies struggle to definitively differentiate between immune cells that have genuinely invaded a tumor and those merely transiting through its vascular network. SEPARATE-Seq effectively surmounts this challenge by imparting a distinctive ‘label’ to immune cells circulating within the blood.

“Localization exerts a profound influence,” elucidated Prof. Damya Laoui, the senior author of the study. “An immune cell situated within a blood vessel experiences an entirely different set of signaling cues compared to one integrated into the tumorous tissue. SEPARATE-Seq provides us with the unprecedented ability to resolve this critical distinction at the resolution of individual cells.”

This innovative technique possesses broad applicability extending beyond the confines of lung cancer research, rendering it valuable for investigations into other pathologies where discerning between disparate immune cell populations is paramount.

Immune Cell Reorganization Within Tumors

By integrating SEPARATE-Seq with spatial transcriptomics, the research team was able to meticulously map not only the types of immune cells present but also their exact spatial positioning within the tumor architecture.

Their comprehensive analyses revealed several noteworthy spatial organizational patterns. The investigators identified a peripheral ring of lipid-associated tumor-associated macrophages encasing the tumor’s periphery. Furthermore, distinct clusters of interferon-stimulated immune and non-immune cells were observed within the tumor mass, exhibiting enrichment in specific dendritic cell phenotypes. The study also documented heightened infiltration of hypoxic, tumor-associated neutrophils, a concentration of plasma cells within the tumor milieu, and a discernible shift in NK cells towards an immature, compromised state upon their entry into the tumor.

Significantly, many of these observed characteristics were also present in human lung adenocarcinoma specimens, thereby underscoring the clinical translational value of the developed model.

“This degree of spatial and molecular granularity permits us to observe how immune cells differentiate and specialize within delineated tumor niches,” commented Lize Allonsius, a doctoral candidate and a co-first author of the study. “It powerfully illustrates the extent to which the tumor microenvironment actively reshapes immune functionality.”

A Valuable Resource for the Scientific Community

In addition to the successful establishment of the preclinical model, the research group has curated and made publicly accessible an extensive multiomics dataset. This valuable resource is presented through an interactive online platform, offering a significant boon to researchers engaged in the study of tumor immunology and lung cancer pathogenesis.

“The success or failure of therapeutic interventions is intrinsically linked to the behavior of immune cells within actual tumors,” emphasized Laoui. “If our preclinical models do not accurately mirror patient biology, we risk deriving flawed conclusions. This current research provides a foundational framework that significantly advances preclinical studies closer to clinical relevance.”