BMP4^high BCP-ALL blasts exhibit an increased leukemogenic activity. (A) Outline of the in vivo experimental design for panels B-E and H. BMP4^high and BMP4^low leukemic blasts were IV-transplanted into NSG mice at day 0. Recipient mice were monitored for disease symptoms and the presence of blasts in blood and were euthanized upon the appearance of clinical signs. (B) Nine primary samples of pediatric BCP-ALL patients were chosen according to BMP4 mRNA expression (4 BMP4^high primary samples [patients #1, #2, #3, #8] and 5 BMP4^low primary samples [patients #4, #5, #6, #7, #9] as shown in supplemental Table 1) and xenografted into 3 to 5 mice. Mice were followed for 4 months or were euthanized when they showed clinical signs. Kaplan-Meier curves show reduced survival in mice xenografted with BMP4^high primary samples (P = .0001; log-rank test). (C) Nalm6 or Nalm6-BMP4 (0.5 x 10⁵ cells per mouse) were xenografted into NSG mice. Reduced survival rate was observed in mice injected with Nalm6-BMP4 ALL blasts (P = .007; log-rank test). (D) IL-6 serum levels in NSG mice transplanted with Nalm6 or Nalm6-BMP4 cells and in control healthy mice (n = 5-6 mice per group). Each dot represents a single transplanted mouse, and lines represent the mean level of IL-6 in each group (**P ≤ .01 and #P ≤ .05 represent statistical significances relative to control healthy mice [*] or Nalm6 mice [#]). (E) Percentages (mean ± standard deviation [SD]) and absolute numbers of leukemic blasts recovered from the spleen of mice xenografted with either Nalm6 (n = 7) or Nalm6-BMP4 (n = 10) cells. Each dot represents a transplanted mouse, and bars and lines represent the mean percentages and absolute numbers (**P ≤ .01 and ***P ≤ .001). (F) Proliferation rate of Nalm6 and Nalm6-BMP4 leukemic cells recovered from the spleen of xenografted mice (n = 6), determined by 7-AAD staining and analyzed by flow cytometry. Results represent the mean ± SD of cells in G₀/G₁ phases (**P ≤ .01). Representative flow cytometry histograms are shown in the right panel. (G) Relative viability of Nalm6-BMP4 leukemic cells with respect to Nalm6 cells recovered from spleens of xenografted mice (n = 4) and cultured during 48 hours with different concentrations of methotrexate (MTX) and cytarabine (Ara-C) (*P ≤ .05). (H) Percentage of CD19⁺ leukemic blasts infiltrated in CNS in either Nalm6 (n = 7) or Nalm6-BMP4 (n = 10) mice. Lines represent average percentages of CNS-infiltrating CD19⁺ cells for each cohort (P = .058). Data in (D-H) panels were compared using 2-tailed Mann-Whitney U tests.