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Updated: Oct 17, 2026
Automated Imaging and Analysis for the Quantification of Fluorescently Labeled Macropinosomes
Published on: August 24, 2021
Determination of intracellular organelles implicated in daunorubicin cytoplasmic sequestration in multidrug-resistant
C Bour-Dill1, M P Gramain, J L Merlin
1Laboratoire de Recherche en Oncologie, Centre Alexis Vautrin, Vandoeuvre-les-Nancy cedex, France.
Background:
Anthracycline resistance is known to be mediated by P-glycoprotein (P-gp) or multidrug-resistance related protein (MRP) as well as intracellular sequestration of drugs.
Methods:
The resistance phenotype of doxorubicin-selected MCF-7(DXR) human breast adenocarcinoma cell line was characterized by cellular and nuclear daunorubicin efflux, P-gp and MRP expression and apoptosis induction. Daunorubicin sequestration was investigated through organelle markers (lysosomes, endoplasmic reticulum and Golgi apparatus) and daunorubicin co-localization by dual-color image analysis fluorescence microscopy using high numerical aperture objective lenses to achieve the smallest field depth and the best lateral resolution. Signal to noise and specificity ratios were optimized for daunorubicin and organelle fluorescent probes labeling.
Results:
An original image analysis procedure was developed to investigate daunorubicin and organelles co-localization. The reliability of the image analysis was controlled through chromatic shift and intensity linearity measurement using calibrated microbeads. The main contribution (65%) of Golgi vesicles in daunorubicin sequestration was demonstrated. Although no rational relationship could be established between daunorubicin sequestration and apoptosis induction, no apoptosis was observed in MCF-7(DXR) cells.
Conclusions:
In addition to P-glycoprotein mediated drug efflux and without MRP overexpression, MCF-7(DXR) daunorubicin resistance phenotype involves drug sequestration within intracellular vesicles identified as Golgi vesicles and resistance to apoptosis induction.
