Cell division tracking and response to tyrosine kinase inhibitors in vitro. (A) CFSE experiments to track cell division and apoptosis following 72 hours in vitro with or without nilotinib and with or without IM. CML CD34⁺ cells were stained with CFSE and then treated for 72 hours with 5 μM nilotinib or IM before analysis. All plots shown are from the same representative patient, and a minimum of 3 patient samples were assayed in all experiments. CFSE fluorescence in cells remaining after treatment with (i) 5GF alone, (ii) 5 μM nilotinib, (iii) 5 μM IM, and (iv) 5 μM nilotinib + 5 μM IM. All treatments are shown as open peaks overlayed on the 5GF-alone control (dark fill). CD34 expression versus CFSE fluorescence of cells remaining after treatment with PBS (v), 5 μM nilotinib (vi), 5 μM IM (vii), and 5 μM nilotinib + 5 μM IM (viii). The box indicates undivided cells (CD34⁺ CFSE^max) and the number is the percent of total viable cells in this gate. Caspase expression versus CFSE fluorescence of cells remaining after treatment with PBS (ix), 5 μM nilotinib (x), 5 μM IM (xi), and 5 μM nilotinib + 5 μM IM (xii). The small black box and numbers indicate undivided cells (CD34⁺ CFSE^max) and the larger blue box and numbers indicate caspase-3–positive cells. It can be clearly seen that the undivided cells were exclusively caspase-3–negative cells. (B) Fold-change in recovery of CD34⁺ cells remaining undivided and in division 1 after drug treatment. The percentage recovery of input for CD34⁺ cells remaining undivided and in division 1 after 72 hours of culture with nilotinib with or without IM was calculated as previously described.^5–7  The percentage recovery in the no drug control was normalized to 1 arbitrary unit and each drug treatment scaled accordingly to give a fold-change in recovery relative to the no drug control. Nilotinib treatment caused a concentration-dependent accumulation of undivided cells. Results are the mean of at least 3 replicate experiments. *P < .05 with respect to no drug control, labeled 0.