FAD Fluorescence Lifetime Research Articles

Abstract B31: Autofluorescence imaging of macrophage metabolism during tumor-mediated 3D migration

Abstract Macrophages are key regulators of tumor progression, and can be targeted for cancer treatment. However, reprogramming macrophages for anti-tumor activity is challenging due to their plasticity. Standard assessment of macrophage function (e.g., ELISA) lacks the sensitivity to capture cell-level heterogeneity in living macrophages. Therefore, single cell imaging and analysis tools are needed to study heterogeneous macrophage behavior within the tumor microenvironment. Macrophages adapt their metabolism to alter both phenotype and function. Thus, we propose the combination of optical metabolic imaging (OMI) and microdevice co-culture to observe cellular level macrophage heterogeneity in response to tumor stimuli. OMI enables noninvasive, dynamic imaging of intact, living samples by measuring autofluorescence from the metabolic co-enzymes NAD(P)H and FAD. Cellular level redox balance is evaluated with the optical redox ratio, defined as the ratio of NAD(P)H to FAD intensity. Additionally, NAD(P)H and FAD fluorescence lifetimes report on metabolic enzyme binding activities. Three-layer microdevices consisting of murine PyVMT breast carcinoma cells, collagen (200 micron thickness), and RAW 264.7 macrophages, respectively, were designed to monitor tumor-mediated macrophage polarization and migration. Metabolic changes with macrophage migration were measured with OMI at 24, 48, and 72 hours after culture. Redox ratio and NAD(P)H mean lifetime increased over time in co-cultured macrophages (p <0.05). Macrophage mono-cultures exhibited lower redox ratio and NAD(P)H lifetime (p<0.05) than in co-culture, consistent with the metabolism of naïve and cytokine-stimulated M2-like macrophages, respectively. Phenotype and function of co-cultured macrophages were confirmed with polymerase chain reaction (PCR) gene expression analysis and immunostaining. Furthermore, volumetric imaging visualized macrophage migration distance-dependent changes in NAD(P)H autofluorescence intensity. These results demonstrate that OMI and microdevice co-culture can capture dynamic changes in macrophage metabolism throughout 3D tumor-stimulated polarization and migration. Overall, this approach could enhance our understanding of macrophage metabolism and function, thus revealing novel therapeutic targets within the tumor microenvironment. Citation Format: Tiffany M. Heaster, Jiaquan Yu, David J. Beebe, Melissa C. Skala. Autofluorescence imaging of macrophage metabolism during tumor-mediated 3D migration [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B31.

Abstract 4100: Personalized medicine in pancreatic cancer using cellular-resolution optical metabolic imaging

Abstract Pancreatic cancer has the worst prognosis of all cancers (5-year survival rate of 7%). There is a critical need for a tool to match individual patients with optimal drugs for their cancer. The goal of this work is to validate Optical Metabolic Imaging (OMI) of patient-derived pancreatic tumor organoids as a high-throughput predictive drug screen for patients. Drug induced changes in cell metabolism precede changes in tumor volume and thus present an earlier marker of treatment response. OMI is sensitive to cellular metabolism because it measures the intrinsic fluorescent properties of NAD(P)H and FAD, coenzymes of metabolic reactions. OMI endpoints include the optical redox ratio (the fluorescence intensity of NAD(P)H divided by the fluorescence intensity of FAD), and the fluorescence lifetimes of NAD(P)H and FAD. The redox ratio reflects the cellular redox balance, and the fluorescence lifetimes report on the binding activity of these coenzymes. OMI, performed using high-resolution multiphoton microscopy, has the unique ability to non-invasively monitor metabolism in living, intact 3D samples on the single-cell level, and can thus quantify inter-cellular heterogeneity in drug response. OMI also allows for high-throughput screening of potential cancer drugs and drug combinations on patient biopsy samples cultured ex vivo. These patient-specific organoids are grown in a 3D matrix that mimics the natural tumor environment. Drug response can be quantified separately in tumor cells and tumor-associated fibroblasts. Organoids were successfully generated from 9 of 11 surgically resected pancreatic tumors and 2 of 2 pancreatic intraepithelial neoplasias. These organoids were treated with a panel of standard and experimental therapies, and early metabolic changes were measured using OMI. Changes at the single-cell level were quantified using the OMI Index, a linear combination of the optical redox ratio and the mean NAD(P)H and FAD fluorescence lifetimes. Patient survival data after surgery and drug treatment was compared to drug response in organoids to that same treatment. Early results have focused on the standard gemcitabine + 5-fluorouracil (G+F) adjuvant treatment. Organoids generated from a patient whose CA19-9 biomarker levels increased while on G+F adjuvant therapy contained a subpopulation of 68% of the cells which were resistant to G+F therapy. Moreover, no G+F resistant cell subpopulations were found in organoids generated from a patient with no signs of disease recurrence over 14 months of G+F adjuvant therapy. Follow-up with the remaining patients is on-going. The ability of OMI to capture heterogeneous cell-level drug responses shows promise for predicting treatment response of individual pancreatic cancer patients. Citation Format: Joe T. Sharick, Tongcheng Qian, Jillian K. Johnson, Lingjun Li, Cheri A. Pasch, Dustin A. Deming, Alexander A. Parikh, Melissa C. Skala. Personalized medicine in pancreatic cancer using cellular-resolution optical metabolic imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4100.

Abstract P5-01-01: Personalized neoadjuvant treatment planning using optical metabolic imaging

Abstract Currently, there are no reliable methods to optimize treatment regimens for individual breast cancer patients. Oncologists choose drug treatments based on expression levels of tumor cell signaling receptors (i.e. HER2, ER, PR) and other factors, and assess whether the treatment is effective after significant time has passed. Unfortunately, over one third of patients exhibit resistance to their initial treatment, increasing their risk of future metastasis and death. Morbidities from sub-optimal drug regimens could be reduced with a personalized drug screen for breast cancer at the time of diagnosis. With the vast number of therapeutic options available to patients (>50 drugs approved with more on the way), a high-throughput screening technology is needed to accurately evaluate how a patient will respond to these options. Here we present Optical Metabolic Imaging (OMI) of tumor-derived organoids as a predictive drug screening platform for individual breast cancer patients. Changes in cell metabolism precede changes in tumor volume and thus present an earlier marker of treatment response. OMI is sensitive to these early changes by exploiting the intrinsic fluorescent properties of NAD(P)H and FAD, coenzymes of metabolic reactions. OMI endpoints include the optical redox ratio (the fluorescence intensity of NAD(P)H divided by the fluorescence intensity of FAD), as well as the fluorescence lifetimes of NAD(P)H and FAD. The redox ratio reflects the cellular redox balance, and the fluorescence lifetimes report on the binding activity of these coenzymes. OMI has the unique ability to non-invasively monitor metabolism in living, intact samples on the single-cell level, and can thus quantify heterogeneity in drug response. Changes were quantified at the single-cell level using the OMI Index, a linear combination of the optical redox ratio and the mean NAD(P)H and FAD fluorescence lifetimes. This index was derived using a multivariate analysis of variance and has been shown previously to correlate with treatment response in human cancer cells. OMI also allows for high-throughput screening of potential cancer drugs and drug combinations on patient biopsy samples cultured ex vivo. These samples are grown as organoids in a 3D matrix that mimics the natural tumor environment. Organoids were successfully generated from core needle biopsies of untreated breast tumors. These organoids were treated with the patient's prescribed neoadjuvant therapy, and early metabolic changes were quantified using OMI. Organoids grew from a variety of untreated breast tumor subtypes, and early metabolic changes could be resolved at the single-cell level after only 24 hours of treatment in vitro. In parallel, each patient's Residual Cancer Burden (RCB) score was quantified by a surgical pathologist after neoadjuvant treatment and served as gold standard validation of tumor drug response. Results from an early cohort of patients suggest that OMI could be used to predict patient clinical response to therapy. A linear combination of OMI variables measured in vitro in only 48 hours correlated strongly with patient RCB score (Pearson correlation coefficient=0.97, n=5). This methodology could allow oncologists to determine the ideal treatment regimen for their patients at the time of diagnosis. Citation Format: Sharick JT, Walsh AJ, Sanders ME, Kelley MC, Meszoely IM, Hooks MA, Burkard ME, Esbona K, Choudhary A, Skala MC. Personalized neoadjuvant treatment planning using optical metabolic imaging [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-01-01.

Abstract 3702: Quantitative imaging of metabolic changes in macrophage subsets

Abstract Macrophages play an important role in tumor therapeutic response. However, current methods cannot image heterogeneities in macrophage activity on a single-cell level over time within intact, living samples. Macrophage metabolism is closely linked to macrophage function, as M1-like macrophages rely on aerobic glycolysis and M2-like macrophages rely on fatty acid oxidation and oxidative phosphorylation. Thus, we propose to develop optical metabolic imaging (OMI) using two-photon microscopy to monitor cell-level changes in macrophage polarization and functional activity. OMI exploits the auto-fluorescence intensities and lifetimes of the metabolic co-enzymes NAD(P)H and FAD, and has been previously used to dynamically image in vivo tumors with cell-level resolution. The optical redox ratio (fluorescence intensity of NAD(P)H divided by FAD) provides a global measure of redox balance within individual cells. The fluorescence lifetimes of NAD(P)H and FAD provide information in protein-binding activities in metabolic reactions. OMI was used to monitor metabolic changes in macrophages on a single-cell level during polarization and after metabolic perturbations. RAW 264.7 macrophages were stimulated towards an M1- or M2-like phenotype, and imaged with OMI at 24, 48, and 72 hours post-polarization. M1- and M2-like macrophages exhibited significant differences (p<0.05) in redox ratio and FAD fluorescence lifetime values at 48 and 72 hours post-polarization. The M1-like and M2-like character of the macrophages were validated with quantitative polymerase chain reaction (qPCR) analysis. Population density modeling of single-cell metabolism established differences in intra-sample heterogeneity across subsets over time. M1- and M2-like macrophages were also treated with 2-deoxy-glucose (glycolysis inhibitor), etomoxir (fatty acid oxidation inhibitor), and cyanide (oxidative phosphorylation inhibitor. Treatment with etomoxir caused a significant decrease in redox ratio in M2-like macrophages (p<0.05) with no significant effect on M1-like macrophages. In contrast, the redox ratio decreased in M1-like macrophages upon inhibition of glycolysis (p<0.05). Comparable to previously published data, inhibition of oxidative phosphorylation in both groups yielded a decreased NAD(P)H mean lifetimes (p<0.05) and increased FAD mean lifetimes (p<0.05). These results establish OMI as a method to image heterogeneity in macrophage populations within intact, living samples. This method could be used to understand macrophage interactions in the microenvironment of intact in vivo tumors. Citation Format: Tiffany M. Heaster, David L. Elion, Rebecca S. Cook, Melissa C. Skala. Quantitative imaging of metabolic changes in macrophage subsets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3702. doi:10.1158/1538-7445.AM2017-3702

Autofluorescence imaging identifies tumor cell-cycle status on a single-cell level.

The goal of this study is to validate fluorescence intensity and lifetime imaging of metabolic co-enzymes NAD(P)H and FAD (optical metabolic imaging, or OMI) as a method to quantify cell-cycle status of tumor cells. Heterogeneity in tumor cell-cycle status (e. g. proliferation, quiescence, apoptosis) increases drug resistance and tumor recurrence. Cell-cycle status is closely linked to cellular metabolism. Thus, this study applies cell-level metabolic imaging to distinguish proliferating, quiescent, and apoptotic populations. Two-photon microscopy and time-correlated single photon counting are used to measure optical redox ratio (NAD(P)H fluorescence intensity divided by FAD intensity), NAD(P)H and FAD fluorescence lifetime parameters. Redox ratio, NAD(P)H and FAD lifetime parameters alone exhibit significant differences (p<0.05) between population means. To improve separation between populations, linear combination models derived from partial least squares - discriminant analysis (PLS-DA) are used to exploit all measurements together. Leave-one-out cross validation of the model yielded high classification accuracies (92.4 and 90.1 % for two and three populations, respectively). OMI and PLS-DA also identifies each sub-population within heterogeneous samples. These results establish single-cell analysis with OMI and PLS-DA as a label-free method to distinguish cell-cycle status within intact samples. This approach could be used to incorporate cell-level tumor heterogeneity in cancer drug development.

In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia

Metabolic imaging of the relative amounts of reduced NADH and FAD and the microenvironment of these metabolic electron carriers can be used to noninvasively monitor changes in metabolism, which is one of the hallmarks of carcinogenesis. This study combines cellular redox ratio, NADH and FAD lifetime, and subcellular morphology imaging in three dimensions to identify intrinsic sources of metabolic and structural contrast in vivo at the earliest stages of cancer development. There was a significant (P < 0.05) increase in the nuclear to cytoplasmic ratio (NCR) with depth within the epithelium in normal tissues; however, there was no significant change in NCR with depth in precancerous tissues. The redox ratio significantly decreased in the less differentiated basal epithelial cells compared with the more mature cells in the superficial layer of the normal stratified squamous epithelium, indicating an increase in metabolic activity in cells with increased NCR. However, the redox ratio was not significantly different between the superficial and basal cells in precancerous tissues. A significant decrease was observed in the contribution and lifetime of protein-bound NADH (averaged over the entire epithelium) in both low- and high-grade epithelial precancers compared with normal epithelial tissues. In addition, a significant increase in the protein-bound FAD lifetime and a decrease in the contribution of protein-bound FAD are observed in high-grade precancers only. Increased intracellular variability in the redox ratio, NADH, and FAD fluorescence lifetimes were observed in precancerous cells compared with normal cells.

Conformational transitions accompanying oligomerization of yeast alcohol oxidase, a peroxisomal flavoenzyme.

Alcohol oxidase (AO) is a homo-octameric flavoenzyme which catalyzes methanol oxidation in methylotrophic yeasts. AO protein is synthesized in the cytosol and subsequently sorted to peroxisomes where the active enzyme is formed. To gain further insight in the molecular mechanisms involved in AO activation, we studied spectroscopically native AO from Hansenula polymorpha and Pichia pastoris and three putative assembly intermediates. Fluorescence studies revealed that both Trp and FAD are suitable intramolecular markers of the conformation and oligomeric state of AO. A direct relationship between dissociation of AO octamers and increase in Trp fluorescence quantum yield and average fluorescence lifetime was found. The time-resolved fluorescence of the FAD cofactor showed a rapid decay component which reflects dynamic quenching due to the presence of aromatic amino acids in the FAD-binding pocket. The analysis of FAD fluorescence lifetime profiles showed a remarkable resemblance of pattern for purified AO and AO present in intact yeast cells. Native AO contains a high content of ordered secondary structure which was reduced upon FAD-removal. Dissociation of octamers into monomers resulted in a conversion of beta-sheets into alpha-helices. Our results are explained in relation to a 3D model of AO, which was built based on the crystallographic data of the homologous enzyme glucose oxidase from Aspergillus niger. The implications of our results for the current model of the in vivo AO assembly pathway are discussed.