Abstract
Background/Objectives: Multidrug resistance (MDR) remains a major pathophysiological barrier to effective chemotherapy based on anthracyclines, including daunorubicin (DNR), in the treatment of leukemia. However, conventional population-level measurements of drug uptake do not resolve variability in uptake kinetics among individual leukemia cells, which may influence intracellular drug accumulation and therapeutic response. Methods: In this study, real-time DNR uptake was quantified at the single-cell level using a microfluidic biochip that enabled long-term cellular retention and continuous monitoring. Both wild-type drug-sensitive leukemia cells and a multidrug-resistant mutant overexpressing the P-glycoprotein (P-gp) efflux pump were examined. Results: Kinetic analysis revealed that DNR uptake in drug-sensitive cells was well described by a single dominant uptake process, whereas uptake in MDR cells required a model incorporating two kinetically distinct processes. In both cell populations, pronounced cell-to-cell variation was observed in uptake rates and intracellular drug retention, indicating substantial functional heterogeneity within phenotypically similar cells. This variability persisted following the treatment with an MDR inhibitor and obscured the differences between inhibitor-treated and untreated cells when the uptake was compared across different single cells. To overcome this limitation, a same-single-cell analysis (SASCA) approach was employed, enabling direct comparison of DNR uptake in the same individual cell before and after inhibitor exposure, thereby revealing enhanced intracellular DNR retention and accelerated uptake kinetics following inhibition. Conclusions: Together, these results demonstrate that real-time single-cell kinetic analysis reveals functionally relevant heterogeneity in multidrug-resistant leukemia cells and provides insight into the pathophysiology of MDR that cannot be obtained from population-averaged measurements.