Correlating NAD(P)H lifetime shifts to treatment of breast cancer cells: a metabolic screening study with time-resolved flow cytometry

Abstract

Here we present a simple, high-throughput, time-resolved flow cytometer to detect changes in NAD(P)H autofluorescence lifetime. The lifetime of this metabolite is characterized by its binding state, when bound to its coenzyme the metabolite exhibits a longer lifetime (1-7 ns) indicating a preferential energy generation state of oxidative phosphorylation. A shorter lifetime, 0.1-1 ns, however, indicates an unbound coenzyme which has been correlated to the ATP energy pathway of glycolysis. The work here in is to demonstrate the capabilities of simple, time-resolved cytometry to show subtle changes in autofluorescence lifetime detection. Flow cytometry results are validated by the use of the Agilent Seahorse HS Mini. Results include the method of ATP energy generation measured by the Seahorse as a comparison to flow cytometry lifetime shifts. Measurements were made using an estrogen receptor positive breast cancer cell line and the same cell line under developed Tamoxifen resistance. The outcome of this work is a map of the metabolic profile of tamoxifen resistance in breast cancer.

Metabolic profiling of tamoxifen-sensitive and tamoxifen-resistant breast cancer cells measuring the optical redox ratio of NAD(P)H/FAD utilizing a high-throughput time-resolved flow cytometer

“…As we saw here, intensity results were not as clear as the optical redox ratio results for our experiment. With the intensity measurements the second experiment produced conflicting results with the first and we were not able to confidently say that glycolysis was the primary metabolic cycle for tamoxifen resistant MCF-7 cells as we have previously reported 12 .…”

“…In the context of this study our goal was to determine if the redox ratio could be used to explore the metabolic differences between tamoxifen resistant and tamoxifen sensitive MCF-7 breast cancer cells. Our group has already been able to demonstrate a clear shift in the metabolism of resistant cell populations towards glycolysis as a means of metabolism through fluorescence lifetime analysis with flow cytometry, as well as explore direct metabolic changes due to environmental conditions with time-resolved flow cytometry 12,13 . Thus, the goal of this work was to explore resistance phenomena in terms of the metabolic optical redox ratio.…”