Fig. 5: Evaluation of short-term AML progenitors in vitro.

A Schematic description of colony-forming unit (CFU) assay. AML PDX cells, isolated from NSG donor mice, or primary AML cells were treated with the different ADCs for 96 h in vitro/ex vivo. Subsequently, cells were washed and plated in methylcellulose and colonies were counted after 14 d. Figure was created with BioRender.com and Adobe Illustrator, in parts modified from STEMCELL Technologies. B–D AML-388, AML-393 and AML-669 PDX cells (3 × 10⁵ cells per condition, respectively) were treated with indicated concentrations of ADCs for 96 h. On day 4, all remaining cells were plated in methylcellulose for CFU assay in technical duplicates without further treatment and incubated for 14 d. On day 14, cell clusters ( > 20 cells) and colonies ( > 50 cells) were counted and normalized to untreated control. mean±s.d.; AML-393 and AML-669 (B): n = 3 biological replicates; AML-388 (B–D): n = 2 biological replicates, each including two technical replicates. Quantification of colony numbers (B, C) and representative pictures of the whole well (scale bar = 5000 µm; PlanApo 2 × 0.10/8.50 mm objective) and zoom-ins (scale bar = 500 µm; Plan-ACHROMAT 4x/0.10 Ph0 objective) (D) are displayed. E, F Primary cells corresponding to AML-393 PDX (3 × 10⁵ cells per condition) were treated with 0.3 or 1 µg/ml 20D9h3-DUBA or 20D9h3-MMAF (E) or 0.3 µg/ml IgG1-DUBA or 20D9h3-LALA-DUBA (F) for 96 h. On day 4, all remaining cells were plated in methylcellulose in technical duplicates without further treatment, incubated for 14 d and colonies were counted as described before. mean±s.d.; n = 2 technical replicates.