Hypoxia increases clonogenicity and engraftment of CML stem cells. The relative frequencies of primitive CML (A) or cord blood progenitors (B) were determined by LTC-IC limiting dilution assays for 2 independent samples. Cells were treated under 21% or 0.5% O2 for 96 hours and were harvested for long-term, drug-free culture. The progenitor frequency was calculated on the basis of colony formation and was scaled against 100% for the 0 µM imatinib group incubated at 21% O2. The cord blood results (B) show the average of 2 samples with similar trends (n = 2). For the CML samples, colonies from each treatment condition were pooled, and the percentage of BCR-ABL1-positive cells was determined by FISH, with percentages indicated on the top of each column. (C) BM engraftment of CD34+ CML cells in NSG mice. CD34+ CML cells (2 × 106 cells/mouse) from 2 CP CML patients (CML#3 and CML#4) were treated with 21% or 0.5% O2 and DMSO or imatinib for 96 hours and were injected into sublethally irradiated mice. The percentage and the total number of engrafted human CD45+ cells in the BM of recipient mice are shown in the left and right panels for each patient sample, respectively. Open symbols (CML#3) and filled symbols (both CML#3 and CML#4) represent experiments using 5 μM or 1 μM imatinib, respectively. Data were available for only 3 groups with open symbols (untreated at 21% O2, untreated at 0.5% O2, and imatinib-treated at 21% O2). Each point represents data from a single mouse. Horizontal bars indicate mean with numbers shown for each condition, and error bars indicate standard error of the mean. (D) The average percentage of BCR-ABL1 positive cells in each group of mice was determined by FISH. *P < .05.
