Correlating NAD(P)H lifetime shifts to tamoxifen resistance in breast cancer cells: A metabolic screening study with time-resolved flow cytometry

Abstract

Time-resolved °ow cytometry (TRFC) was used to measure metabolic di®erences in estrogen receptor-positive breast cancer cells. This specialty cytometry technique measures °uorescence lifetimes as a single-cell parameter thereby providing a unique approach for high-throughput cell counting and screening. Di®erences in °uorescence lifetime were detected and this was associated with sensitivity to the commonly prescribed therapeutic tamoxifen. Di®erences in °uorescence lifetime are attributed to the binding states of the auto°uorescent metabolite NAD(P)H. The function of NAD(P)H is well described and in general involves cycling from a reduced to oxidized state to facilitate electron transport for the conversion of pyruvate to lactate. NAD(P)H °uorescence lifetimes depend on the bound or unbound state of the metabolite, which also relates to metabolic transitions between oxidative phosphorylation and glycolysis. To determine if fundamental metabolic pro¯les di®er for cells that are sensitive to tamoxifen compared to those that are resistant, large populations of MCF-7 breast cancer cells were screened and °uorescence lifetimes were quanti¯ed. Additionally, metabolic di®erences associated with tamoxifen sensitivity were measured with a Seahorse HS mini metabolic analyzer (Agilent Technologies Inc. Santa Clara, CA) and confocal imaging. Results show that tamoxifen-resistant breast cancer cells have increased utilization of glycolysis for energy production compared to tamoxifen-sensitive breast cancer cells. This work is impacting because it establishes an early step toward developing a reliable screening technology in which large cell censuses can be di®erentiated for drug sensitivity in a label-free fashion.