Fig. 3

ETO2::GLIS2 expression induces leukemic transformation in FL HSPCs in vivo. A Experimental design of in vivo experiments. EG-expressing FL, FBM and CB HSPCs (via lentiviral vectors delivery or CRISPR/Cas9-editing) were injected into NSG mouse strains following 2–11 days of culture. Mice were monitored daily and sacrificed when exhibiting disease symptoms. Recovered cells from primary (1ry) recipients were serially transplanted into secondary (2ry) and tertiary (3ry) recipients. Phenotypic and transcriptomic analysis were performed on the recovered cells and murine organs were assessed by histological analysis. B Kaplan–Meier survival plot of 1ry recipients injected with FL, FBM and CB CTL vs EG cells. Median survival of mice with FL EG-transduced and CRISPR-edited cells are 141 days and 302 days respectively, 275.5 and 371 days for FBM EG-transduced and CRISPR-edited cells respectively. C Percent of hCD45⁺ (left panel) and GFP⁺ cells (gated in hCD45⁺ cells) (right panel) measured in the BM of 1ry mice injected with CTL and EG FL and CB cells. Data from mice with CRISPR/Cas9-edited (GFP⁻) cells are presented in light red (left panel). D Representative flow cytometry analysis of cells recovered from the vertebral column (VC) of 1ry recipients injected with FL and CB EG cells (lentiviral delivery). E Percent of CD33⁺KIT⁺, KIT⁺CD56⁺ and KIT⁺CD41⁺ cells in the BM of 1ry mice, gated in hCD45⁺(GFP⁺) cells. CRISPR/Cas9-edited (GFP⁻) cells are shown in light red. F Fluorescent in-situ hybridization (FISH) analysis of human cells recovered from a 1ry sick NSG recipient injected with FL EG-CRISPR/Cas9-edited cells. G Kaplan–Meier survival plot of mice serially transplanted with 1 × 10⁶ FL EG cells and FBM EG cells. Median survival of 2ry recipients was 85 and 84 days for FL and FBM groups respectively. Median survival of 3ry recipients was 50 and 96 days for FL and FBM groups respectively. H Percent of CD33⁺KIT⁺, KIT⁺CD56⁺ and KIT⁺CD41⁺ cells in BM of 2ry and 3ry mice injected with FL EG cells, gated in hCD45⁺(GFP⁺ or GFP⁻ (Inv(16) engineered)) cells. Data of mice with CRISPR/Cas9-edited (GFP⁻) cells are presented in light red. Statistical significance is indicated as p values (B and G: Log-rank test, C and E: Kruskal–Wallis test, H: Mann–Whitney test). *: p < 0.05, **: p < 0.01, ***: p < 0.001. ns, non significant