Measurement of imatinib uptake by flow cytometry

Abstract

One of the essential parameters of targeted therapy efficiency in cancer treatment is the amount of drug reaching the therapeutic target area. Imatinib (IM) was the first specifically targeted drug to be developed and has revolutionized the treatment of patients with chronic myeloid leukemia (CML). To evaluate cellular uptake of IM, we developed a method based on the chemical structure of the molecule and using the natural UV fluorescence that we quantified by flow cytometry. In two CML cell lines, we obtained a satisfactory relationship between intracellular IM (ICIM) levels and media concentrations, and we found a strong correlation between ICIM at 1 h and IM efficacy at 24 h, demonstrating that ICIM at 1 h might be a relevant predictive parameter of cell sensitivity. Our method was more sensitive than the standard physicochemical method. We applied our method to primary cells and found cell morphology-dependant IM accumulation. Moreover, in CML cells from patients at diagnosis, IM accumulation was heterogeneous. In all cases, ICIM at the single-cell level was much higher than in culture media arguing in favor of a predominantly active uptake process. We developed a simple method directly applicable to primary cells that has shown two major advantages: only a small number of cells are required, and cell subsets can be identified according to morphological criteria and/or the presence of particular antigenic sites. This method provides a new tool to assess CML cell sensitivity to IM, and ICIM levels in native CML cells could be used to monitor therapeutic response. ' 2012 International Society for Advancement of Cytometry

Lysosomal Sequestration Determines Intracellular Imatinib Levels

“…Similar observations were made in other cell lines: intracellular imatinib concentrations were found to be 35-to .100-fold higher than those in the incubation medium. Our findings support reports in the literature that describe high intracellular imatinib levels in peripheral blood mononuclear cells from CML patients (Bouchet et al, 2013), in leukemic cell lines K562 and KCL22 (le Coutre et al, 2004), and in HL-60 cells (Bourgne et al, 2012). To explain these findings, most studies infer the existence of an active uptake mechanism involving a saturable drug transporter (Bourgne et al, 2012;Bouchet et al, 2013).…”

Adhesion to stromal cells mediates imatinib resistance in chronic myeloid leukemia through ERK and BMP signaling pathways

“…The intracellular IM was measured using a fluorimeter as described earlier 34 . Standard curve was prepared using IM stock solution diluted in the lysis buffer and intracellular IM in different cell types was measured.…”

Beyond the message: advantages of snapshot proteomics with single‐cell mass cytometry in solid tumors

“…See protocol [46] for conjugating antibodies with these heavy-metals. Beyond this, users can employ quantum dot labeled antibodies to gain additional channels [47], iodine for cell cycle tracking [124], RNA transcripts [131][132][133], platinum drug uptake [128,129], cell cycle status and DNA synthesis [130], cell size [125], apoptosis [48], and viability [27] Proteins, phospho-proteins, chromatin modifications [145], RNA transcripts [146], fluorescent drug uptake [147], metabolism and redox state [148,149], cell cycle status and DNA synthesis [150], cell size and granularity, apoptosis [151] and viability [152] Poly-adenylated RNA transcripts, CITE-seq probes [144] Minimum cells per sample needed at start of protocol Max is an example maximum range of the instrument scale; error is the typical error for that scale. c Includes capabilities of novel instruments such as Cytek TM Aurora (Cytek Biosciences), ZE5 TM (Bio-Rad), and FACSymphony TM (BD).…”