DLBCL-microenvironment interactions: cytokine profiling and ECM-mediated ibrutinib resistance in a 3D bone-based model.

Diffuse large B-cell lymphoma (DLBCL) frequently relapses following therapy, partly due to microenvironment-mediated drug resistance. Bone marrow involvement is associated with poor prognosis, yet preclinical models that faithfully recapitulate its extracellular matrix (ECM) remain limited. We used a decellularized human bone-derived 3D scaffold to evaluate the impact of extracellular matrix (ECM) interactions on proliferation, cytokine secretion, and ibrutinib sensitivity in four DLBCL cell lines (OCI-LY1, OCI-LY18, RIVA, NU-DUL-1). Functional assays and cytokine profiling were performed under 2D and 3D culture conditions. Compared with 2D cultures, the 3D ECM model induced a profound remodeling of the DLBCL secretome, with upregulation of a coordinated network of pro-migratory chemokines, particularly CXCL9, CCL22, CCL17, CCL4 and CXCL1/2/3. ECM engagement enhanced DLBCL migration and promoted scaffold colonization, indicating a positive feedback loop between tumor cells and the microenvironment. While all DLBCL cell lines were sensitive to ibrutinib in 2D, ECM-adherent OCI-LY18 and RIVA cells showed reduced drug-induced apoptosis in 3D. This effect was dependent on direct ECM contact, was not reproduced by inert 3D scaffold, and was fully reversible upon disruption of cell-ECM interaction. Mechanistically, ECM adhesion was associated with activation of the AKT/mTOR pathway. The human bone-derived 3D ECM model reveals that direct tumor-matrix interactions induce reversible ibrutinib resistance and reshape the cytokine milieu in DLBCL. These findings highlight the role of ECM as a dynamic regulator of drug response and support the use of physiologically relevant 3D models to investigate microenvironment-driven resistance and guide therapeutic strategies.