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

Summary

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.