Autofluorescent metabolic measurements of an optical redox ratio, NAD(P)H/FAD, have been utilized as a means of measuring cancer progression, treatment impact, subtype determination, and more. This optical redox ratio is traditionally measured through intensity with microscopy, but there is potential to adapt this technique for high throughput analysis using time-resolved flow cytometry with autofluorescence lifetime measurements. A fluorescence lifetime approach to these measurements allows for fluorophore concentration independent measurements that can provide new information to the field. The two fluorescent metabolites of interest that allow for redox analysis are NAD(P)H/NAD(P)+ and FADH2/FAD. Variations in the redox state and binding of these metabolites to their respective coenzymes, have been correlated to the cycles in which cells metabolize glucose into ATP, either oxidative phosphorylation (OXPHOS) or glycolysis. This technique will be used herein to study the metabolism of MCF-7 tamoxifen resistant and sensitive breast cancer cells using flow cytometry, first on a fluorescence intensity basis of the two metabolites. Our results show there is a clear shift towards an increased redox ratio for tamoxifen resistant cells, indicating a greater reliance on glycolysis as a means of metabolism. Future work will focus on adapting the intensity based redox ratio approach through high-throughput flow cytometry used here to a fluorescence lifetime based measurement.
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 to 7 ns) indicating a preferential energy generation state of oxidative phosphorylation. A shorter lifetime, 0.1 to 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.
We present a cytometric and metabolic screening tool that measures shifts in NAD(P)H autofluorescence and autofluorescence lifetimes from single cells based on metabolite-enzyme interactions. Short autofluorescence lifetimes of NAD(P)H (~0.1-1ns) indicate the metabolite is unbound from metabolic enzymes and the cell is favoring glycolysis for energy production. In contrast, longer autofluorescence lifetimes of NAD(P)H (~1-7ns) are an inference that the metabolite is bound to metabolic enzymes and the cell is respiring under oxidative phosphorylation. Using a simple time-resolved flow cytometer we are able to measure autofluorescence lifetimes of MCF-7 and T47D breast cancer cells, which we relate to metabolic changes within each cell line. In order to determine the resolution limits of our time-resolved instrument, we first treated cells under different conditions that directly alter the metabolic pathway that drives their energy production. We deprived cells of serum in their growth media, which drives the cell to utilize glycolysis as a metabolic pathway. By comparing normal to deprived cells, we were able to determine if our cytometry system is able to measure differences in the autofluorescence lifetimes. Results show a decrease in lifetime and autofluorescence intensity for both T47D and MCF7 following serum deprivation. Initial cytometric analysis illustrates consistent lifetime data with respect to fluorescence lifetimes and fluorescence intensities decreasing as expected. This study is a preliminary confirmation that our timeresolved cytometer can effectively detect autofluorescence signals, albeit with some limitations in lifetime resolution. Future work will include refinement of lifetime analysis, frequency domain approaches, and improving sensitivity.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.