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

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 bad, 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.