Evaluation of a method for detection of cells with reduced drug retention in solid tumours

Abstract

A method for detection of cells with reduced drug retention was evaluated in solid tumours. After a 1 h incubation with daunorubicin (DNR), the right angle scatter (RAS), forward angle scatter (FAS), and specific fluorescence (Fluo) were measured in sensitive and resistant cells; only Fluo was related qualitatively, but not quantitatively, to resistance. Various incubation conditions were examined. When the pH of the incubation medium increased, the DNR retention increased in sensitive and resistant cells. In contrast, when the cell concentration increased, the DNR retention decreased. Using sensitive and resistant cell lines, a proportion of resistant cells lower than 10% can be detected in a mixture. To analyse cells from solid tumours, the cells were dissociated by repeated fine needle aspirations. Tumours from 22 patients have been processed with this technique; 8 samples were classified as S (sensitive); 2 as R (resistant); and 12 as I (intermediate). Further experiments were run to study and improve the method. Another method of detection of dead cells was tested. The intra‐assay variability of the technique was found to be less than 10%. When the study was performed with different fragments of the same tumour, the variation, corresponding to the tumour heterogeneity, rose to 21 to 36%. The inter‐assay reproducibility was too high, so a variant of this technique has been adapted, using verapamil or cyclosporin A, which is able to block DNR efflux; this new method allows tumour cells to be used as their own controls. © 1992 Wiley‐Liss, Inc.

Influence of S9788, a new modulator of multidrug resistance, on the cellular accumulation and subcellular distribution of daunorubicin in p‐glycoprotein‐expressing MCF7 human breast adenocarcinoma cells

“…The flow cytometric procedure reported in this paper is interesting, because both parameters can be determined simultaneously; therefore, the evaluation of multidrug-resistance modulators is applicable to clinical specimens of either hematological malignancies (33) or solid tumors (3). The additional labeling by propidium iodide is useful for avoiding experimental artefacts encountered with conventional functional analyses of multidrug resistance, which cannot distinguish membrane-altered cells Daunorubicin was selected among other anthracyclines, because its fluorescence intensity did not overlap the fluorescence signal of propidium iodide in the sensitive cell line (data not shown).…”

Validation of a single point flow cytometric assay for determining P-glycoprotein activity in multidrug resistant cell lines

“…As each tumour cell sample has its own internal control (sample without inhibitor) the need for an external reference cell line for comparison is not required. Furthermore, factors such as pH, temperature, cell number, cell size, known to modify DNR retention and measured fluorescence in cells (McGown et al 1983) are readily controlled in a split single sample assay which is not the case in other flow cytometric assays (Herweijer et al 1989;Carpentier et al 1992) of this assay has also been verified by demonstrating a high correlation with the amount of P-glycoprotein present as determined by Western blot. Herweijer et al described an online flow cytometric assay measuring uptake of DNR by sensitive and resistant cell lines with the enhancement of intracellular drug accumulation by addition of inhibitors of P-glycoprotein.…”

“…Carpentier (Carpentier et al 1992) has also described two methods for detection of drug resistant cells in solid tumours. Their initial method involved measuring specific fluorescence as reflected by DNR retention by tumour cells run in parallel with known drug resistant cell lines used as a reference.…”