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P.S. Godavarthy et al.
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Figure 1. Inhibition of E-selectin affects survival in mice with chronic myeloid leukemia. (A) Representative two-photon in vivo microscopy image of the bone marrow (BM) calvarium of an unirradiated Rag-2-/-CD47-/-IL-2 receptor γ-/- mouse injected with 200,000-500,000 unsorted human chronic myeloid leukemia (CML) cells [from peripheral blood (PB) or BM], labeled with CMTMR (orange; white arrows), 2 h prior to in vivo microscopy. Vessels were visualized via the injection of dextran-FITC (1 mg per injection), while bones were visualized in blue due to second harmonic generation. The scale bar represents 50 mm. (B) Time of contact (seconds), determined by in vivo microscopy, between the calvarial endothelium and human unsorted CML cells from the PB of one patient labeled with CMTMR and injected into vehicle- or GMI-1271 (20 mg/kg/dose)- treated unirradiated Rag-2-/-CD47-/-IL-2 receptor γ-/- mice (P<0.0001, t-test). The mice had been treated with vehicle or GMI-1271 2 h before transplantation. (C) Adhesion (presented as fold change) of unsorted human CML cells from PB or BM to E-selectin-coated wells (50,000 CML cells/well) in the presence of vehicle vs. GMI-1271 (P=0.001, t-test) (n=5) after incubation for 6 h. Different symbols signify individual patients. The experiment was performed in duplicate. (D) Kaplan-Meier-style survival curve for Balb/c recipients of BCR-ABL1-transduced BM treated with vehicle (black solid line), 20 mg/kg/dose GMI-1271 intraperitoneally (long gray dashes), 100 mg/kg imatinib orally (black dots) and the combination of both imatinib and GMI-1271 (short gray dashes) beginning on day 9 after transplantation. Vehicle and imatinib were administered daily, while GMI-1271 was given twice daily. The difference in survival between mice treated with imatinib or imatinib plus GMI-1271 was statistically signi- ficant (P=0.028, log-rank test, n=15). (E) Distance (in mm) from endothelium of CMTMR-labeled GFP+ (BCR-ABL1+) Lin- cells (200,000) from FVB mice with established CML- like disease transplanted by intravenous injection into Tie2-GFP mice (FVB background) and imaged by in vivo microscopy 19 h after injection [P=0.001, analysis of variance /(ANOVA); Tukey test]. The imaging analysis was performed by ImageJ. (F) Percentage of GFP+ (BCR-ABL1+) Lin- c-Kit+ Sca-1+ cells of total leukocytes which homed to the BM (P=0.03, t-test) or spleen (P=0.048, t-test) of vehicle (black)- or GMI-1271 (white)-treated mice 18 h after transplantation (n=4). A total of 2.5 × 106 unsorted, BCR- ABL1-transduced cells had been injected. (G, H) Southern blot showing distinct proviral integration sites and, consequently, disease clonality in spleens (taken at the time of death, as shown in Figure 1D) of Balb/c recipients of BCR-ABL1-transduced BM treated with vehicle (lanes 1-4), imatinib (lanes 5-9), GMI-1271 (lanes 10-14) or imatinib plus GMI-1271 (lanes 15-19) (G) and disease clonality (H). The difference in disease clonality between imatinib- and imatinib plus GMI-1271-treated recipients as a mea- sure of engraftment of leukemia-initiating clones is statistically significant (P=0.007, ANOVA; Tukey test). Data are expressed as mean ± standard deviation.
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