ATP-binding Cassette Transporters Substantially Reduce Estimates of ALDH-positive Cancer Cells based on Aldefluor and AldeRed588 Assays

Jin Won Park,Kyung-Han Lee,Kyung-Ho Jung,Jin Hee Lee,Youngjoo Byun,Seung Hwan Moon,Young Seok Cho

doi:10.1038/s41598-019-42954-9

Abstract

Aldehyde dehydrogenase (ALDH) assays measure the accumulated fluorescence of enzyme products. However, cancer cells frequently co-express ALDH and ATP-binding cassette (ABC) transporters, which might mediate efflux of ALDH assay reagents. We demonstrate expression of active multidrug resistance protein1 (MDR1), multidrug resistance-associated protein (MRP), and breast cancer resistance protein (BCRP) in CT26 cancer cells as well as expression of MRP and BCRP in HT29 cancer cells. Without transporter inhibition, only small portions of both cell types were estimated to be ALDH-positive based on Aldefluor and AldeRed588 assays. However, MK-571 (MRP inhibitor) and novobiocin (BCRP inhibitor) substantially increased the rate of ALDH-positive CT26 cells based on either Aldefluor or AldeRed588 assays. Verapamil (MDR inhibitor) did not influence assay results. MK-571 also substantially increased the rate of ALDH-positive HT29 cells. Limiting dilution assays demonstrated greater numbers of tumor-spheres formed by Aldefluor-positive compared to -negative CT26 cells selected in the presence of MK-571 or novobiocin but not in their absence. These results reveal that Aldefluor and AldeRed588 products are efficient substrates for MRP- and BCRP-mediated efflux and substantially reduce estimated ALDH positivity rates in cancer cells. These findings demonstrate that complete blockade of these transporters is important to ensure accurate ALDH assay results and to develop newer assay techniques.

Highlights

Given the crucial role of CSCs in tumor resistance and recurrence, targeting specific markers to recognize and isolate these cells has become an important tool for cancer research[9,10]
Verapamil, MK-571, and novobiocin were used as multidrug resistance protein1 (MDR1), MRP1/2, and breast cancer resistance protein (BCRP) inhibitors, respectively
Histograms of CT26 mouse colon cancer cells incubated with Efluxx-ID Green dye and analyzed by Fluorescence-activated cell sorting (FACS) showed a substantial right shift in the presence of verapamil and MK-571 and a significant but mild right shift in the presence of novobiocin (Fig. 1A)

Summary

Introduction

Given the crucial role of CSCs in tumor resistance and recurrence, targeting specific markers to recognize and isolate these cells has become an important tool for cancer research[9,10]. High ALDH activity is widely used as a detection and isolation marker that is often expressed in CSCs from various organs[9,10] This underlines the importance of identifying factors that influence the results of Aldefluor assays. Fluorescence-activated cell sorting (FACS) using fluorescent reagents is currently the standard method to identify cells that have increased ALDH activity In these assays, ALDH mediates conversion of reagent substrates into products that can no longer diffuse out of cells. ALDH mediates conversion of reagent substrates into products that can no longer diffuse out of cells This leads to increased intracellular accumulation of fluorescent signals for detection. A clear understanding of how Aldefluor and AldeRed[588] reagents undergo efflux through ABC transporters is important to ensure accurate assays of cancer cell ALDH activity. We further assessed how activities of these transporters reduce reagent retention and lead to underestimation of ALDH positivity based on assays using Aldefluor and AldeRed[588]

Methods

Results

Conclusion