Though much of the interest in fluorescence in the past has been on measuring spectral qualities such as wavelength and intensity, there are two other highly useful intrinsic properties of fluorescence: lifetime (or decay) and anisotropy (or polarization). Each has its own set of unique advantages, limitations, and challenges in detection when it comes to use in biological studies. This review will focus on the property of fluorescence lifetime, providing a brief background on instrumentation and theory, and examine the recent advancements and applications of measuring lifetime in the fields of high-throughput fluorescence lifetime imaging microscopy (HT-FLIM) and time-resolved flow cytometry (TRFC). In addition, the crossover of these two methods and their outlooks will be discussed.
The active metabolite of tamoxifen, 4‐hydroxytamoxifen, functions as an anti‐estrogen in breast cancer cells and thus inhibits proliferation. While tamoxifen continues to be successfully used to treat estrogen‐dependent breast cancer, most patients receiving treatment will develop chemoresistance over time. Two commonly reported biomarkers of tamoxifen resistance are decreased expression of insulin‐like growth factor 1 receptor (IGF‐1R) and increased expression of epidermal growth factor receptor (EGFR). In prior work we have shown that these receptors facilitate chemoresistance and have unique regulatory functions measurable in resistant cell lines compared with nonresistant. Thus, we hypothesized that these receptors and a newly identified biomarker, integrin β1, may be used to search for the presence of resistant breast cancer cells within a population of cells that are sensitive to tamoxifen therapy. We tested this by designing a straightforward cell‐labeling approach to measure differences in the receptor expression of resistant vs. sensitive cells cytometrically. Our results show that separation is possible when observing the expression of IGF‐1R as well as integrin β1. Interestingly, we found no detectable difference in EGFR expression between tamoxifen resistant and ‐sensitive cells when measured with cytometry despite the fact that EGFR is upregulated in resistant cells. Our long‐term goal is to utilize sorting to isolate tamoxifen resistant subpopulations of cells by receptor expression level. Isolating rare resistant cells that reside within a population of drug‐sensitive cells will offer new insights into why chemoresistance occurs.
A multi-color spectral flow cytometry panel targeting receptors on MCF-7 breast cancer cells is used to potentially identify a tamoxifen resistant subpopulation. Results using separate resistant and normal cells indicate detection and sorting is plausible.
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