Single-cell characterization of 3D CLL spheroid model that recapitulates lymph node microenvironment-driven phenotypes and in vivo drug responses to ibrutinib

Haselager, M. V.; Lashgari, D.; Jongejan, A.; Roos, J. L.; Dijkema, M.; Dubois, J.; Peters, F. S.; Kater, A. P.; Moerland, P. D.; Eldering, E.

doi:10.64898/2026.10.31.763314

bioRxiv · 10.64898/2026.10.31.763314

Single-cell characterization of 3D CLL spheroid model that recapitulates lymph node microenvironment-driven phenotypes and in vivo drug responses to ibrutinib

Haselager, M. V.·Lashgari, D.·Jongejan, A.·Roos, J. L.·Dijkema, M.·Dubois, J.·Peters, F. S.·Kater, A. P.·Moerland, P. D.·Eldering, E.

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Abstract

Lymph node (LN) microenvironmental signals promote chronic lymphocytic leukemia (CLL) cell survival, proliferation, and treatment resistance. We previously developed a 3D culture model that recapitulates key features of this protective LN niche. Using single-cell RNA sequencing of ex vivo paired peripheral blood and LN samples, together with matched patient-derived CLL spheroids, we investigated how microenvironmental cues shape CLL phenotypes and their response to ibrutinib. A total of 3 functional CLL states (resting, activated, proliferating) were identified, with expanded populations of activated and proliferating subsets in 3D cultures. After 5 days of in vitro ibrutinib treatment, CLL cells shifted toward a resting phenotype, reflecting published responses in patients at 6 months. Activated CLL cells showed relative resistance to ibrutinib, associated with TLR9-MALT1-MYC pathway activation. The 3D model recapitulated tissue-specific T cell phenotypes observed in patients, and computational analyses predicted several T-to-CLL cell-mediated interactions supporting the LN niche. Collectively, these data reveal distinct ibrutinib responses among CLL subsets and establish the 3D model as a physiologically relevant platform.

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Haselager, M. V., Lashgari, D., Jongejan, A., Roos, J. L., Dijkema, M., Dubois, J., Peters, F. S., Kater, A. P., Moerland, P. D., Eldering, E.. 2026-11-03. Single-cell characterization of 3D CLL spheroid model that recapitulates lymph node microenvironment-driven phenotypes and in vivo drug responses to ibrutinib. https://doi.org/10.64898/2026.10.31.763314

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