Abstract P1-01-08: Optical metabolic imaging of the effects of acute and intermittent hypoxia in murine breast cancer cells

Abstract

Abstract About 90% of the patients diagnosed with breast cancer succumb due to metastases and not from primary tumor. There are currently few methods that can distinguish indolent and aggressive breast tumors. Recent studies have shown differences in the metabolic profiles of primary, circulating, and metastatic breast cancer cells. One of the key regulators of metastases in breast cancer is the transcription factor Twist. The goal of our study was to determine the relationship between Twist expression and cellular metabolism, and the effect of this relationship on metastatic potential. Multiphoton microscopy can provide non-invasive, label-free and reproducible measurements of cellular metabolism. An optical redox ratio of endogenous fluorescence from nicotinamide adenine dicnucleotide (NADH) and flavin adenine dinucleotide (FAD) (calculated as FAD/NADH+FAD) can reflect the balance between mitochondrial oxidative phosphorylation and glucose catabolism in the cell. Because hypoxia is known to promote metastasis, we studied the effect of both intermittent and acute hypoxia on cellular metabolism in 4T1 murine breast cancer cells. We generated a 4T1-Twist− cell line using CRISPR-Cas9 technology. Western blots of Twist protein expression confirmed that Twist expression was significantly lower in the selected cell population compared with the parental 4T1 line. Both cell lines were exposed to 0.5% oxygen for 3 hours and one hour of reoxygenation (acute hypoxia), and 3 cycles of one-hour hypoxia followed by one-hour reoxygenation (intermittent hypoxia). Multiphoton imaging of endogenous fluorescence as well as metabolic flux analysis (Seahorse Biosciences) was performed for both hypoxic exposures in both cell lines. Our results indicate a significant increase in the optical redox ratio in 4T1 cells when exposed to acute hypoxia, indicating a possible increase in oxidative metabolism. There were no signfiicant differences in the 4T1-Twist− line. On the other hand, we observed a decrease in the optical redox ratio in response to intermittent hypoxia in the 4T1 cells and the opposite effect in the 4T1-Twist− line. In addition to these cell lines, we will also present data from sibling cell lines of the 4T1 with varying Twist levels and hence metastatic potential. These data indicate indicate potentially measurable metabolic differences between indolent and aggressive breast cancer cells when subject to a hypoxia-reoxygenation regimen. Citation Format: Lisa Rebello, Narasimhan Rajaram. Optical metabolic imaging of the effects of acute and intermittent hypoxia in murine breast cancer cells [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-01-08.