Scaffold‐free three‐dimensional culture of chronic lymphocytic leukaemia preserves B–T‐cell cross‐talk and enables drug response profiling

Chronic lymphocytic leukaemia (CLL) presents a complex biological and clinical landscape, strongly influenced by the tumour microenvironment (TME). To better capture this heterogeneity, we have developed patient-derived lymphoma spheroids from CLL samples (CLL-PDLS), which recapitulate the cross-talk between malignant cells and the autologous immune microenvironment enabling efficient drug screening for further studies. CLL spheroids incorporate tumour B cells together with autologous T cells, which self-organize into a three-dimensional (3D) disc-shaped structure. Cultures can be maintained for up to 6 days under optimal conditions, preserving cell viability and high proliferative activity. T cells exhibit a range of activation and exhaustion phenotypes across both CD4+ and CD8+ subsets, including expression of Programmed Cell Death Protein 1 (PD-1), CD25, T cell immunoreceptor with Ig and ITIM domains (TIGIT) and CD69. The cellular composition remains stable, with tumour B cells representing approximately 90% of the population and T cells comprising 10%. The model was further validated by assessing responses to conventional therapies, including ibrutinib and venetoclax. In conclusion, this 3D spheroid model captures key functional aspects of B-T interactions in CLL, maintaining the dynamic interactions between malignant B cells and their autologous immune components, partially reproducing microenvironmental features, and offering a promising platform for preclinical therapeutic evaluation.