Fig. 5

Treatment-induced CD31⁺Sca-1^high re-vascularization mediates LSC resistance. a Schematic design of the experiments. BM MNCs from diseased Mll^PTD/wt/Flt3^ITD/ITD mouse (CD45.1/CD45.2) were transplanted into normal wt recipient mice (CD45.1, 6 Gy) to generate a cohort of leukemic mice with a similar disease onset time. At day 14 post transplantation, the mice were treated with AC220 (20 mg/kg/day, oral gavage) or vehicle for 3 weeks. After completion of treatment, a cohort of treated mice (n = 10 mice per group) were monitored for survival and another cohort of mice (n = 9 mice per group) were euthanized and assessed for BM vascular changes and LSC burden by 2nd transplantation. b Frequency and number of AML cells in BM and spleen from AC220-treated versus vehicle-treated AML mice. c TNFα mRNA expression in BM MNCs by Q-RT-PCR (left) and TNFα protein concentrations in BM plasma by Luminex assay (right) in AC220-treated versus vehicle-treated AML mice. d and e Long bones (femurs and tibias) from AML mice treated with vehicle or AC220 for 3 weeks were evaluated for: BM EC Sca-1^high and Sca-1^low subfractions (d left, representative plots; right, aggregate results) by flow cytometry analysis (n = 4 mice per group), and CD31⁺Sca-1^high EC-lined vessels (i.e., arterioles) by CD31 (FITC) and Sca-1 (PE) IF staining (e left) and quantification (e right) (n = 3 mice per group). For e: Yellow arrows indicate CD31⁺Sca-1^high EC-lined vessels; white arrows indicate CD31⁺Sca-1^low EC-lined vessels; scale bars represent a size of 50 µm. f Gata2 levels in BM ECs from AML mice treated with AC220 or vehicle for 3 weeks, analyzed by Q-RT-PCR (n = 4 mice per group). g miR-126 levels in BM ECs (left) and LSKs (right) from AML mice treated with vehicle or AC220 for 3 weeks, analyzed by Q-RT-PCR. h Representative plots of cell cycling of BM LSKs (left) and frequency of quiescent BM LSKs (i.e., LSKs in G0 phase, LSC^G0, right) from AML mice treated with vehicle or AC220 for 3 weeks, analyzed by Ki-67 and DAPi staining and flow cytometry analysis. i and j Survival of AML mice treated with vehicle or AC220 for 3 weeks (primary, i) and survival of 2nd recipient mice (2nd survival, j) receiving BM cells from AC220-treated or vehicle-treated AML donors. Two of three independent experiments with similar results were shown. Results represent mean ± SEM. Significance values: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001