Sensitive immunofluorescence detection of the expression of P-glycoprotein in malignant cells

Because reversal of multidrug resistance increases chemotoxicity, early detection of low P-glycoprotein expression is clinically relevant for justifying early treatment of those patients that might benefit most from reversal therapy. We elected to score P-glycoprotein in single tumor cells, because the gene is rarely amplified, mRNA levels do not necessarily correlate with protein levels, and many normal hematopoietic or stroma cells within tumors and leukemic marrows also express P-glycoprotein. We enhanced the "signal-to-noise" ratio for detecting low P-glycoprotein levels by a novel complex made by pre-incubating mouse peroxidase-antiperoxidase, used solely to provide a stable framework for attaching multiple DTAF-labeled F(ab')2 fragments of rabbit antimouse IgG. We improved specificity by using both C219 and C494, which are directed against separate internal P-glycoprotein epitopes. We standardized staining with two series of negative and positive controls, in which P-glycoprotein was quantified by immunoblot, and confirmed sensitivity by staining a low-expression cell line and "mixed" samples containing small numbers of positive cells. We measured P-glycoprotein by flow cytometry, examining aliquots by differential interference contrast microscopy to identify malignant cells, in which we confirmed P-glycoprotein staining by fluorescence microscopy. We detected low P-glycoprotein expression in clinical samples of leukemic blasts, distinguishing them from normal P-glycoprotein-expressing hematopoietic cells. This assay may be valuable for early diagnosis of low, but potentially important expression of P-glycoprotein, thereby allowing early application of reversal therapy.