Impact of 3D culture on drug response in B-NHL. (A-C) Comparison of B-cell death induced by doxorubicin in SUDHL4 (A), HLY1 (B), and DOHH2 (C) cultured in 2D or in 3D alone (upper panel) or with TSC and Matrigel (2D+TSC or 3D+Mg+TSC) (lower panel). For 2D culture, B cells were seeded alone or in the presence of TSC. For 3D culture, 100 confluent capsules, containing B cells alone or B cells with TSC in the presence of Matrigel, were seeded. 2D and 3D cultures were exposed to doxorubicin at a concentration corresponding to its IC50, and GFP^neg B-cell death was evaluated after 24 hours by flow cytometry. (D) Comparison of cell death induced by ABT-199 (1 µM) in DOHH2 cells cultured in 2D alone (upper panel) or in 2D+TSC or 3D+TSC+Matrigel (lower panel) at confluency, for 24 hours. The data represent mean ± standard error of, at least, 3 independent experiments (Wilcoxon test, *P < .05, ****P < .0001). (E) Doxorubicin diffusion in spheroids. Flow cytometry analyses of doxorubicin fluorescence in cells cultured in suspension (2D) or in 3D alone (3D) or in 3D with Matrigel and TSC (3D+ Mg+TSC) before confluence (D4) or at confluence (D9-D10) and treated with 1µg/ml doxorubicin for 4 hours. Numbers represent the median fluorescence intensity of cells.