Abstract P02: Vascularised Lymphoid Organoids Recreate Tertiary Lymphoid Structures for Quantitative Tumour-Immune Studies

Abstract

Tertiary lymphoid structures (TLS) are temporary immune cell clusters that form in non-lymphoid tissues during chronic inflammation and cancer. Their presence has been associated with better patient outcomes, especially within tumors, and a stronger response to immunotherapies. However, the in vivo study of TLS is complicated by their heterogeneous formation and the complex interplay between immune, stromal, and vascular components. Current in vitro models lack the architecture and microenvironmental cues necessary to capture these dynamics. To address these limitations, we developed a novel human in vitro model by integrating tonsil-derived lymphoid organoids with a vascularised tumor microenvironment using the OrganiXTM microfluidic platform. Tonsil tissue was dissociated and processed to yield single stromal and immune cells, which were assembled via hanging drop culture into dense, three-dimensional organoids. These structures recapitulated hallmark features of secondary lymphoid organs, including CD19+ B cells, CD3+, CD4+, and CD8+ T cells, CD11c+ dendritic cells, and functional antigen-presenting cells, validated with flow cytometry and advanced imaging. Embedding these organoids into a fibrin-collagen hydrogel, co-cultured with endothelial and fibroblast cells, enabled the development of perfusable vascular networks, which are crucial for lymphocyte recruitment and immune cell trafficking. In proximity to tumor spheroids, organoids supported immune-tumor cross-talk, as evidenced by the migration and reactivation of lymphocyte subsets, germinal centers formation, and the spatial organization of T and B cell zones. Functional relevance was demonstrated by the effective antigen-specific activation of T cells and the robust, targeted migration of CD133-directed CAR-T cells within the vascularized system. This advanced in vitro platform provides a physiologically relevant tool for dissecting TLS biology, immune-tumor interactions, and immunotherapy performance, thereby paving the way for mechanistic investigations and personalized assessment of anti-cancer immunotherapies in a controlled and tunable setting.