Fig. 6: Evaluation of long-term AML progenitors in vitro and in vivo.

A Schematic description of long-term culture initiating cell (LTC-IC) and leukemia-initiating cell (LIC) assay. AML PDX cells, isolated from NSG donor mice were treated with the different ADCs for 48-96 h in vitro/ex vivo (as individually stated). For LTC-IC assay, cells were then cultivated on a feeder layer of murine fibroblasts for 5 weeks and afterwards plated in methylcellulose for colony analysis on day 14. For LIC assay, cells were transplanted into NSG mice and engraftment was monitored by bioluminescence imaging (BLI). Figure was created with BioRender.com and Adobe Illustrator, in parts modified from STEMCELL Technologies. B AML PDX cells of AML-388 and AML-393 (5 × 10⁶ cells per condition, respectively) were treated with 0.025 µg/ml or 0.3 µg/ml 20D9h3-DUBA or 20D9h3-MMAF for 48 h. All remaining cells were harvested and co-cultured with irradiated SLSL-J-IL3-neo murine fibroblasts for 5 weeks without further treatment, then they were plated in methylcellulose in technical duplicates and incubated for 14 d. In analogy to the CFU assay, cell clusters ( > 20 cells) and colonies ( > 50 cells) were counted and normalized to untreated control. mean±s.d.; n = 2 technical replicates. Luc-positive mCherry-positive AML PDX cells of AML-388 (1 × 10⁵ cells per condition and mouse, (C) and AML-393 (5 × 10⁴ cells per condition and mouse, (D) were treated ex vivo with indicated concentrations of 20D9h3-DUBA or 20D9h3-MMAF for 96 h. On day 4, remaining cells were injected into ten-weeks old (AML-393) or 26-weeks-old (AML-388) male NSG recipient mice and engraftment was repeatedly monitored by BLI for up to 181 d. Right side: BLI image of one representative mouse per treatment group for AML-393 PDX cells. mean±s.d.; n = 5 mice per group. Dashed line = detection limit.