Quantitative Assessment of P-Glycoprotein Expression and Function Using Confocal Image Analysis

Abstract

P-glycoprotein is implicated in clinical drug resistance; thus, rapid quantitative analysis of its expression and activity is of paramout importance to the design and success of novel therapeutics. The scope for the application of quantitative imaging and image analysis tools in this field is reported here at "proof of concept" level. P-glycoprotein expression was utilized as a model for quantitative immunofluorescence and subsequent spatial intensity distribution analysis (SpIDA). Following expression studies, p-glycoprotein inhibition as a function of verapamil concentration was assessed in two cell lines using live cell imaging of intracellular Calcein retention and a routine monolayer fluorescence assay. Intercellular and sub-cellular distributions in the expression of the p-glycoprotein transporter between parent and MDR1-transfected Madin-Derby Canine Kidney cell lines were examined. We have demonstrated that quantitative imaging can provide dose-response parameters while permitting direct microscopic analysis of intracellular fluorophore distributions in live and fixed samples. Analysis with SpIDA offers the ability to detect heterogeniety in the distribution of labeled species, and in conjunction with live cell imaging and immunofluorescence staining may be applied to the determination of pharmacological parameters or analysis of biopsies providing a rapid prognostic tool.

LightSpot®-FL-1 Fluorescent Probe: An Innovative Tool for Cancer Drug Resistance Analysis by Direct Detection and Quantification of the P-glycoprotein (P-gp) on Monolayer Culture and Spheroid Triple Negative Breast Cancer Models

“…For this approach, flow cytometry is mainly used, expressly requiring living cells in suspension. Secondly, direct P-gp quantification can be made with enzyme-linked immunosorbent assays, specific antibody immunostaining using Western blotting, immunocytochemistry or immunohistochemistry methodologies [5,45]. These approaches can be applied to cells in suspension, in monolayers, or on tissue sections, and allowed us to specifically detect and localize the P-gp.…”

“…However, these approaches present difficulties regarding antibody penetration inside cells and/or cell masses and imply expensive and time-consuming experimentation. Thus, they only target extracellularmembrane P-gp, and not the intracellular part [45]. Indeed, the P-gp located on organelle membranes, such as the endoplasmic reticulum and lysosomes, also plays an important role in cellular drug resistance that must not be underestimated [46,47].…”

Chemical biology fluorescent tools for in vitro investigation of the multidrug resistant P-glycoprotein (P-gp) expression in tumor cells

“…The list includes western blot, immunohistochemistry, immunouorescence staining, reverse transcription quantitative real-time PCR (RT-qPCR) and more recently mass spectrometry-based proteomics techniques. 8,9 Despite clear advantages, each approach also comes with its own set of drawbacks and limitations. Western blot, for example, is a multistep low throughput technique which provides only relative quantication.…”

“…Sulfocyanine 3 labeled conjugates (5, 10, 24, 29, 34) showed a particular intracellular staining pattern. Other conjugates bearing a boron dipyrromethene (BODIPY) core (3,8,13,22, 27 (BODIPY FL), 12 (BODIPY 564/570) and 4, 9 (BODIPY 650/665)) or a 7-nitrobenz-2-oxa-1,3-diazole (NBD) core (11,30) showed potential for global P-gp direct detection and quantification. These fluorescent conjugates holds key advantages over existing methods for drug resistance evaluation with regards to P-gp expression and could be used as innovative tools in preclinical assays and clinical diagnosis.…”

Spatial Intensity Distribution Analysis Reveals Abnormal Oligomerization of Proteins in Single Cells

“…More recently, SpIDA was compared with alternative techniques to examine the intracellular accumulation of CellTrace calcein red-orange (Life Technologies, Carlsbad, CA) in a colorectal adenocarcinoma cell line and bovine aortic endothelial cells (12), demonstrating that SpIDA is a user-friendly tool compatible with conventional confocal microscopy that can be rapidly applied to obtain quantitative information on intracellular concentration and kinetics of fluorophore uptake. It was also proposed to use SpIDA as a rapid prognostic tool that can be used on biopsy tissue (13), given its full compatibility with immunofluorescence. SpIDA also revealed new insights, to our knowledge, into the receptor quaternary structure of the serotonin 5-HT2C receptor (11).…”