Figure 5.

CLL spheroids enable the study of drug resistance and T cell cytotoxicity. (A) Paired baseline and refractory samples of an ibrutinib-treated CLL patient were stimulated with IL-2/15/21/CpG and cultured in ULA plates (3D) for 4 d in the presence of 1 µM ibrutinib. Culture plates were placed in an IncuCyte live-cell imager, which imaged and quantified the spheroid area every 12 h. The data represents 9 spheroids from 2 patients cultured in each condition. Error bars represent the mean ± SEM. (B–E) CLL samples were stimulated as indicated and/or cocultured with 3T40 fibroblast in ULA plates (3D) for 1 wk. Afterwards, cells were incubated with a titration of 0.001–10 µM venetoclax for an additional 24 h, during which the culture plates were placed in an IncuCyte live-cell imager, which imaged and quantified spheroid area every hour. The data represents 7 spheroids from 4 patients cultured in each condition (E). Viability was measured by flow cytometry using DiOC6 and TO-PRO-3 viability dyes (B), including expression of Bcl-XL (C) and Mcl-1 (D). Error bars represent the mean ± SEM (n = 3), *P < 0.05, **P < 0.01 (paired t test). Each group was compared with the unstimulated control group. (F) CTV-labeled CLL cells were cocultured with healthy donor T cells in a 4:1 effector to target ratio in the presence of 1 ng/mL blinatumomab for 24 h. Viability was measured by flow cytometry using MitoTracker Orange and TO-PRO-3 viability dyes. Error bars represent the mean ± SEM (n = 4). CLL = chronic lymphocytic leukemia; CTV = CellTrace^™ Violet; IL = interleukin; SEM = standard error of mean; ULA = ultra-low attachment; 2D = two-dimensional; 3D = 3-dimensional.