Synergistic effects of CPX and imatinib on CD34⁺ cells from CML patients at diagnosis. Mobilized CD34⁺ cells from 2 CML patients (A) and 3 healthy donors (B) were expanded in SFM supplemented with growth factors starting with 1 × 10³ cells/well with increasing doses of CPX (0-40 μM). The average cell number per well was determined every third day for a total of 12 days in culture. Mean ±SE of replicate experiments is shown. (C) Isobolograms for the effects of imatinib and CPX (C) or GC7 (D) on cellular proliferation. Cells were incubated with a combination of imatinib and CPX (C) or GC7 (D) for 48 hours, and cytotoxicity was assessed using an MTT-based assay. Results were analyzed using Calcusyn software. (E) High-resolution cell-cycle analysis and tracking of CML CD34⁺ cell division. Flow cytometry dot plot showing high-resolution cell-cycle analysis with Ki-67 (FL1) and 7-AAD (FL3) staining. (left) No drug control. (right) Imatinib 5 μM/CPX 1 μM combination. G₁ arrest with combination therapy can be clearly seen in the right plot. (F) Representative flow cytometry dot plots of CFSE (FL1) versus CD34PE (FL2) for viable (PI-negative) primary CML cells remaining after treatment with no drug control, CPX 1 μM, imatinib 5 μM, or combination imatinib 5 μM/CPX 1 μM. Tracking of cell division demonstrates the antiproliferative effect of imatinib 5 μM, which is enhanced by the addition of CPX 1 μM. (G) Total cell numbers (expressed as percentage of no drug control ± SD) remaining at the end of treatment for each condition studied using CML (⊡) and normal (▪) cells. (H) Number of quiescent (CFSE^max) CML (⊡) and normal (▪) CD34⁺ cells remaining viable at the end of 72-hour culture in the presence of imatinib, CPX, and the combination.