P607: A NOVEL LYMPH NODE-MIMICKING 3D CULTURE SYSTEM DISPLAYS LONG-TERM T CELL-DEPENDENT CLL PROLIFERATION AND SURVIVAL

Abstract

Background: Primary chronic lymphocytic leukemia (CLL) cells, despite originating from a proliferative disease, rapidly undergo apoptosis in vitro in the absence of microenvironmental survival signals1. No current system permits long-term expansion of CLL cells in vitro due to difficulties of mimicking a physiological microenvironment. The lymph node (LN) is the critical site of in vivo CLL proliferation and is also thought to play a key role in the development of resistance to targeted agents2,3. Developing an in vitro culture system for CLL with pathophysiological relevance will greatly benefit studies of CLL proliferation, microenvironment, clonal outgrowth and relevant drug screening. Aims: To design an in vitro CLL culture system that incorporates key aspects of the CLL LN; contribution of non-CLL cells and cytokines, and induction of drug resistance and prolonged proliferation of CLL cells. Methods: Primary CLL cells were cultured in ultra-low attachment (ULA) plates in parallel to standard 2D cultures. PBMCs were cultured with or without T cells (in a specific CLL:T cell ratio to mimic the LN composition), or PBMCs were co-cultured with primary lymph node fibroblasts. CLL cells were either stimulated directly with a B cell cocktail (BCC) consisting of IL-2, IL-15, IL-21 and CpG and/or indirectly via a T cell stimulation of anti-CD3/CD28. Results: Compared to 2D cultures, 3D cultures showed several important CLL LN features. First, significantly increased CLL proliferation independent of IGHV mutation status, which was abrogated in the absence of T cells, or by JAK inhibitors. Notably, treatment with BTK inhibitors significantly inhibited CLL proliferation and resulted in disintegration of the 3D spheroid architecture. Second, co-culture with LN-derived stromal cells further increased CLL proliferation, reaching a maximum of 8 generations. Third, 3D cultures could be expanded approximately 3-4-fold over a course of 6 weeks using the 3D model. Fourth, when PBMCs were stimulated with BCC, spheroids developed proliferation center-like structures after 4 weeks of culture where T cells localized together with enrichment of Ki-67+ CLL cells. Finally, either B or T cell stimulation resulted in an induction of venetoclax resistance, showing that T cells are able to confer drug resistance to CLL cells in this model. Summary/Conclusion: We present a 3D culture system that underlines the role of T cells in sustained CLL proliferation, permitting investigation of CLL cells in the context of a protective niche consisting of multiple cell types, thereby opening up new avenues for clinically useful applications.