About 90% of the patients diagnosed with breast cancer succumb due to metastases and not from pri... more 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.
Muscle derived stem cells (MDSCs) and myoblast play an important role in myotube regeneration whe... more Muscle derived stem cells (MDSCs) and myoblast play an important role in myotube regeneration when muscle tissue is injured. However, these cells can be induced to differentiate into adipocytes once exposed to PPARγ activator like EPA and DHA that are highly suggested during pregnancy. The objective of this study aims at determining the identity of trans-differentiated cells by exploring the effect of EPA and DHA on C2C12 undergoing differentiation into brown and white adipocytes. DHA but not EPA committed C2C12 cells reprograming into white like adipocyte phenotype. Also, DHA promoted the expression of lipolysis regulating genes but had no effect on genes regulating β-oxidation referring to its implication in lipid re-esterification. Furthermore, DHA impaired C2C12 cells differentiation into brown adipocytes through reducing the thermogenic capacity and mitochondrial biogenesis of derived cells independent of UCP1. Accordingly, DHA treated groups showed an increased accumulation of...
Metabolic changes in breast cancer cells of varying metastatic potential on exposure to chronic a... more Metabolic changes in breast cancer cells of varying metastatic potential on exposure to chronic and intermittent hypoxia are determined using optical redox ratio obtained via two-photon microscopy.
Accurate methods for determining metastatic risk from the primary tumor are crucial for patient s... more Accurate methods for determining metastatic risk from the primary tumor are crucial for patient survival. Cell metabolism could potentially be used as a marker of metastatic risk. Optical imaging of the endogenous fluorescent molecules nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) provides a non-destructive and label-free method for determining cell metabolism. The optical redox ratio (FAD/FAD+NADH) is sensitive to the balance between glycolysis and oxidative phosphorylation (OXPHOS). We have previously established that hypoxia-reoxygenation stress leads to metastatic potential-dependent changes in optical redox ratio. The objective of this study was to monitor the changes in optical redox ratio in breast cancer cells in response to different periods of hypoxic stress as well various levels of hypoxia to establish an optimal protocol. We measured the optical redox ratio of highly metastatic 4T1 murine breast cancer cells under normoxic conditions and...
Results from imaging the optical redox ratio in murine breast cancer cells at normoxic and hypoxi... more Results from imaging the optical redox ratio in murine breast cancer cells at normoxic and hypoxic conditions are presented. These data compare well with measurements of glycolysis and oxidative phosphorylation using a metabolic flux analyzer.
The development of prognostic indicators of breast cancer metastatic risk could reduce the number... more The development of prognostic indicators of breast cancer metastatic risk could reduce the number of patients receiving chemotherapy for tumors with low metastatic potential. Recent evidence points to a critical role for cell metabolism in driving breast cancer metastasis. Endogenous fluorescence intensity of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) can provide a label-free method for assessing cell metabolism. We report the optical redox ratio of FAD/(FAD + NADH) of four isogenic triple-negative breast cancer cell lines with varying metastatic potential. Under normoxic conditions, the redox ratio increases with increasing metastatic potential (168FARN>4T07>4T1), indicating a shift to more oxidative metabolism in cells capable of metastasis. Reoxygenation following acute hypoxia increased the redox ratio by 43 ± 9% and 33 ± 4% in the 4T1 and 4T07 cells, respectively; in contrast, the redox ratio decreased 14 ± 7% in the non-metastatic 67NR cell line. These results demonstrate that the optical redox ratio is sensitive to the metabolic adaptability of breast cancer cells with high metastatic potential and could potentially be used to measure dynamic functional changes that are indicative of invasive or metastatic potential.
About 90% of the patients diagnosed with breast cancer succumb due to metastases and not from pri... more 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.
Muscle derived stem cells (MDSCs) and myoblast play an important role in myotube regeneration whe... more Muscle derived stem cells (MDSCs) and myoblast play an important role in myotube regeneration when muscle tissue is injured. However, these cells can be induced to differentiate into adipocytes once exposed to PPARγ activator like EPA and DHA that are highly suggested during pregnancy. The objective of this study aims at determining the identity of trans-differentiated cells by exploring the effect of EPA and DHA on C2C12 undergoing differentiation into brown and white adipocytes. DHA but not EPA committed C2C12 cells reprograming into white like adipocyte phenotype. Also, DHA promoted the expression of lipolysis regulating genes but had no effect on genes regulating β-oxidation referring to its implication in lipid re-esterification. Furthermore, DHA impaired C2C12 cells differentiation into brown adipocytes through reducing the thermogenic capacity and mitochondrial biogenesis of derived cells independent of UCP1. Accordingly, DHA treated groups showed an increased accumulation of...
Metabolic changes in breast cancer cells of varying metastatic potential on exposure to chronic a... more Metabolic changes in breast cancer cells of varying metastatic potential on exposure to chronic and intermittent hypoxia are determined using optical redox ratio obtained via two-photon microscopy.
Accurate methods for determining metastatic risk from the primary tumor are crucial for patient s... more Accurate methods for determining metastatic risk from the primary tumor are crucial for patient survival. Cell metabolism could potentially be used as a marker of metastatic risk. Optical imaging of the endogenous fluorescent molecules nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) provides a non-destructive and label-free method for determining cell metabolism. The optical redox ratio (FAD/FAD+NADH) is sensitive to the balance between glycolysis and oxidative phosphorylation (OXPHOS). We have previously established that hypoxia-reoxygenation stress leads to metastatic potential-dependent changes in optical redox ratio. The objective of this study was to monitor the changes in optical redox ratio in breast cancer cells in response to different periods of hypoxic stress as well various levels of hypoxia to establish an optimal protocol. We measured the optical redox ratio of highly metastatic 4T1 murine breast cancer cells under normoxic conditions and...
Results from imaging the optical redox ratio in murine breast cancer cells at normoxic and hypoxi... more Results from imaging the optical redox ratio in murine breast cancer cells at normoxic and hypoxic conditions are presented. These data compare well with measurements of glycolysis and oxidative phosphorylation using a metabolic flux analyzer.
The development of prognostic indicators of breast cancer metastatic risk could reduce the number... more The development of prognostic indicators of breast cancer metastatic risk could reduce the number of patients receiving chemotherapy for tumors with low metastatic potential. Recent evidence points to a critical role for cell metabolism in driving breast cancer metastasis. Endogenous fluorescence intensity of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) can provide a label-free method for assessing cell metabolism. We report the optical redox ratio of FAD/(FAD + NADH) of four isogenic triple-negative breast cancer cell lines with varying metastatic potential. Under normoxic conditions, the redox ratio increases with increasing metastatic potential (168FARN>4T07>4T1), indicating a shift to more oxidative metabolism in cells capable of metastasis. Reoxygenation following acute hypoxia increased the redox ratio by 43 ± 9% and 33 ± 4% in the 4T1 and 4T07 cells, respectively; in contrast, the redox ratio decreased 14 ± 7% in the non-metastatic 67NR cell line. These results demonstrate that the optical redox ratio is sensitive to the metabolic adaptability of breast cancer cells with high metastatic potential and could potentially be used to measure dynamic functional changes that are indicative of invasive or metastatic potential.
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Papers by Lisa Rebello