Fluorescence Lifetimes Of NAD Research Articles

Metabolic activity and intracellular pH in induced pluripotent stem cells differentiating in dermal and epidermal directions

Induced pluripotent stem cells (iPSC) are a promising tool for personalized cell therapy, in particular, in the field of dermatology. Metabolic plasticity of iPSC are not completely understood due to the fact that iPSC have a mixed mitochondrial phenotype, which still resembles that of somatic cells. In this study we investigated the metabolic changes in iPSC undergoing differentiation in two directions, dermal and epidermal, using two-photon fluorescence microscopy combined with FLIM. Directed differentiation of iPSC into dermal fibroblasts and keratinocyte progenitor cells was induced. Cellular metabolism was examined on the basis of the fluorescence of the metabolic cofactors NAD(P)H and FAD. The optical redox ratio (FAD/NAD(P)H) and the fluorescence lifetimes of NAD(P)H and FAD were traced using two-photon fluorescence microscopy combined with FLIM. Evaluation of the intracellular pH was carried out with the fluorescent pH sensor SypHer-2 and fluorescence microscopy. In this study, evaluation of the metabolic status of iPSC during dermal and epidermal differentiation was accomplished for the first time with the use of optical metabolic imaging. Based on the data on the FAD/NAD(P)H redox ratio and on the fluorescence lifetimes of protein-bound form of NAD(P)H and closed form of FAD, we registered a metabolic shift toward a more oxidative status in the process of iPSC differentiation into dermal fibroblasts and keratinocyte progenitor cells. Biosynthetic processes occurring in dermal fibroblasts associated with the synthesis of fibronectin and versican, that stimulate increased energy metabolism and lower the intracellular pH. No intracellular pH shift is observed in the culture of keratinocyte progenitor cells, which reflects the incomplete process of differentiation in this type of cells. Presented results provide the basis for further understanding the metabolic features of iPSC during differentiation process, which is essential for developing new treatment strategies in cell therapy and tissue engineering.

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.

Metabolic cofactors NAD(P)H and FAD as potential indicators of cancer cell response to chemotherapy with paclitaxel

Paclitaxel, a widely used antimicrotubular agent, predominantly eliminates rapidly proliferating cancer cells, while slowly proliferating and quiescent cells can survive the treatment, which is one of the main reasons for tumor recurrence and non-responsiveness to the drug. To improve the efficacy of chemotherapy, biomarkers need to be developed to enable monitoring of tumor responses. In this study we considered the auto-fluorescent metabolic cofactors NAD(P)H and FAD as possible indicators of cancer cell response to therapy with paclitaxel. It was found that, among the tested parameters (the fluorescence intensity-based redox ratio FAD/NAD(P)H, and the fluorescence lifetimes of NAD(P)H and FAD), the fluorescence lifetime of NAD(P)H is the most sensitive in tracking the drug response, and is capable of indicating heterogeneous cellular responses both in cell monolayers and in multicellular tumor spheroids. We observed that metabolic reorganization to a more oxidative state preceded the morphological manifestation of cell death and developed faster in cells that were more responsive to the drug. Our results suggest that noninvasive, label-free monitoring of the drug-induced metabolic changes by noting the NAD(P)H fluorescence lifetime is a valuable approach to characterize the responses of cancer cells to anti-cancer treatments and, therefore, to predict the effectiveness of chemotherapy.

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

Method to Detect the Cellular Source of Over-Activated NADPH Oxidases Using NAD(P)H Fluorescence Lifetime Imaging.

Fluorescence-lifetime imaging microscopy (FLIM) is a technique to generate images, in which the contrast is obtained by the excited-state lifetime of fluorescent molecules instead of their intensity and emission spectrum. The ubiquitous coenzymes NADH and NADPH, hereafter NAD(P)H, in cells show a short fluorescence lifetime ≈400 psec in the free-state and a longer fluorescence lifetime when bound to enzymes. The fluorescence lifetime of NAD(P)H in this state depends on the binding-site on the specific enzyme. In the case of NADPH bound to members of the NADPH oxidases family we measured a fluorescence lifetime of 3650 psec as compared to enzymes typically active in cells, in which case fluorescence lifetimes of ∼2000 psec are measured. Here we present a robust protocol based on NAD(P)H fluorescence lifetime imaging in isolated cells to distinguish between normally active enzymes and NADPH oxidases, mainly responsible for oxidative stress. © 2017 by John Wiley & Sons, Inc.

Two-photon FLIM of NAD(P)H and FAD in mesenchymal stem cells undergoing either osteogenic or chondrogenic differentiation

BackgroundMetabolic plasticity and the versatility of different lineages of stem cells as they satisfy their energy demands are not completely understood. In this study we investigated the metabolic changes in mesenchymal stem cells (MSCs) undergoing differentiation in two directions, osteogenic and chondrogenic, using two-photon fluorescence microscopy combined with FLIM.MethodsDifferentiation was induced by incubating the human bone marrow MSCs in osteogenic or chondrogenic mediums. Cellular metabolism was examined on the basis of the fluorescence of the metabolic cofactors NAD(P)H and FAD. The optical redox ratio (FAD/NAD(P)H) and the fluorescence lifetimes of NAD(P)H and FAD were traced using two-photon fluorescence microscopy combined with FLIM. The cells were imaged before the induction of differentiation (day 0) and on days 7, 14, and 21 of osteogenic and chondrogenic differentiation.ResultsBased on the data for the FAD/NAD(P)H redox ratio and on the fluorescence lifetimes of protein-bound NAD(P)H, we registered a metabolic shift toward a more glycolytic status in the process of MSC differentiation. The difference was that, in osteogenic differentiation, an increase in oxidative phosphorylation preceded the shift to the glycolytic status in the process of such MSC differentiation. The fluorescence lifetime characteristics of FAD indicated the stimulation of an unknown metabolic pathway, where protein-bound FAD participates.ConclusionsIn this study, probing of the metabolic status of MSCs during osteogenic and chondrogenic differentiation was implemented for the first time with the use of optical metabolic imaging of the two cofactors - NAD(P)H and FAD. Our data suggest that biosynthetic processes, associated, presumably, with the synthesis of collagen, drive energy metabolism in differentiating cells, and promote a metabolic shift from a more oxidative to a more glycolytic state.

Abstract 4241: Predicting clinical response in breast cancer using cellular-resolution optical metabolic imaging

Abstract While over 50 drugs have been approved by the FDA to treat breast cancer, there are no reliable methods for optimizing treatment regimens for individual patients. Currently, oncologists choose drug treatments based on expression levels of tumor cell signaling receptors (i.e. HER2, ER) and assess whether the treatment is effective after weeks or months of precious time have passed. Unfortunately, over one third of patients exhibit resistance to their initial treatment. The toxic side effects and morbidities resulting from suboptimal drug regimens could be eradicated by applying a personalized medicine approach to breast cancer treatment. This approach would allow clinicians to determine the optimal treatment plan for individual patients early on, at the time of diagnosis. While current methods track therapy response via changes in tumor size (i.e. MRI, mammography, ultrasound), changes in cell metabolism precede changes in tumor size and thus present an earlier marker of treatment response. Optical metabolic imaging (OMI) is sensitive to these early changes in metabolism 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 state, and the fluorescence lifetimes of NAD(P)H and FAD report on the binding activity of these coenzymes. Additionally, OMI has the unique ability to measure these endpoints in individual cells, which allows for the detection of heterogeneous subpopulations of responsive or resistant cells within a tumor. 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. We have demonstrated that OMI accurately predicts treatment response in organoids derived from breast cancer xenografts compared with gold standard tumor growth curves in vivo. We have also shown that OMI can measure drug response and detect heterogeneous cell populations in organoids derived from triple negative, ER+, and HER2+ human breast tumors. The ability of OMI to predict treatment response has also been demonstrated in the polyoma middle-T mouse model of breast cancer, which exhibits more cellular heterogeneity than cell line xenografts and also incorporates the influence of the immune system on cancer drug response. Preliminary data shows that OMI of organoids generated from biopsies of newly diagnosed breast cancer patients can accurately predict how the patient clinically responds to neoadjuvant treatment. This methodology could allow oncologists to determine the ideal treatment regimen for their patients at the time of diagnosis. Citation Format: Joe T. Sharick, Alex J. Walsh, Melinda E. Sanders, Ingrid Meszoely, Mary A. Hooks, Mark C. Kelley, Melissa C. Skala. Predicting clinical response in breast cancer using cellular-resolution optical metabolic imaging. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4241.

In Vivo Autofluorescence Imaging of Tumor Heterogeneity in Response to Treatment

Subpopulations of cells that escape anti-cancer treatment can cause relapse in cancer patients. Therefore, measurements of cellular-level tumor heterogeneity could enable improved anti-cancer treatment regimens. Cancer exhibits altered cellular metabolism, which affects the autofluorescence of metabolic cofactors NAD(P)H and FAD. The optical redox ratio (fluorescence intensity of NAD(P)H divided by FAD) reflects global cellular metabolism. The fluorescence lifetime (amount of time a fluorophore is in the excited state) is sensitive to microenvironment, particularly protein-binding. High-resolution imaging of the optical redox ratio and fluorescence lifetimes of NAD(P)H and FAD (optical metabolic imaging) enables single-cell analyses. In this study, mice with FaDu tumors were treated with the antibody therapy cetuximab or the chemotherapy cisplatin and imaged in vivo two days after treatment. Results indicate that fluorescence lifetimes of NAD(P)H and FAD are sensitive to early response (two days post-treatment, P<.05), compared with decreases in tumor size (nine days post-treatment, P<.05). Frequency histogram analysis of individual optical metabolic imaging parameters identifies subpopulations of cells, and a new heterogeneity index enables quantitative comparisons of cellular heterogeneity across treatment groups for individual variables. Additionally, a dimensionality reduction technique (viSNE) enables holistic visualization of multivariate optical measures of cellular heterogeneity. These analyses indicate increased heterogeneity in the cetuximab and cisplatin treatment groups compared with the control group. Overall, the combination of optical metabolic imaging and cellular-level analyses provide novel, quantitative insights into tumor heterogeneity.

Two-photon fluorescence lifetime imaging monitors metabolic changes during wound healing of corneal epithelial cells in vitro

Early and correct diagnosis of delayed or absent corneal epithelial wound healing is a key factor in the prevention of infection and consecutive destruction of the corneal stroma with impending irreversible visual loss. Two-photon microscopy (TPM) is a novel technology that has potential to depict epithelial cells and to evaluate cellular function by measuring autofluorescence properties such as fluorescence intensity and fluorescence lifetimes of metabolic co-factors such as NAD(P)H. Using non-invasive TPM in a tissue-culture scratch model and an organ-culture erosion model, fluorescence intensity and fluorescence lifetimes of NAD(P)H were measured before and during closure of the epithelial wounds. Influence of temperature and selective inhibition of metabolism on intensity and lifetimes were tested additionally. Decrease of temperature resulted in significant increase of fluorescence lifetimes and decrease of the relative amount of free NAD(P)H due to decreased global metabolism. Increase in temperature and upregulation of glycolysis through blocking the mitochondrial electron transport chain by rotenone resulted in increased intensity, decreased lifetimes and increase in the relative amount of free NAD(P)H. Changes of lifetimes and free:protein-bound NAD(P)H ratios were similar to changes measured during wound healing in both scratch and erosion models. Fluorescence lifetime measurements (FLIM) detected enhancement of cellular metabolism following epithelial damage in both models. The prospective detection of cellular autofluorescence in vivo, in particular FLIM of metabolic cofactor NAD(P)H, has the potential to become an indispensible tool in clinical use to differentiate healing from non-healing epithelial cells and to evaluate effects of newly developed substances on cellular metabolism in preclinical and clinical trials.

Enzyme Selective Flim in Microglia: Studying the Role of NADPH Oxidase in Chronic Neuroinflammation

Understanding the pathological mechanisms in the brain is the decisive step in developing novel specific therapeutic strategies and in curing chronic neuroinflammatory or neurodegenerative diseases like multiple sclerosis (MS). For this purpose, techniques which confer high molecular specificity and spatial resolution, which are non-invasive and allow for dynamics visualization in vivo are necessary. We already showed that NAD(P)H-based fluorescence lifetime imaging (FLIM) is the appropriate technique to detect the activation of NADPH oxidase in murine polymorphonuclear cells and in cells of Nicotiana tabacum during phagocytosis. The fluorescence lifetime of NAD(P)H if bound to NADPH oxidase increases to approx. 3600 ps as compared to the case of NAD(P)H-dependent metabolic enzymes, i.e. approx. 2000 ps. A central yet still not fully understood role in neuronal injury is assigned to microglial NADPH oxidase: this might be a neurotoxic effect due to direct production of reactive oxygen species or, indirectly, due to the induction of NO synthase or a protective role if NADPH oxidase is involved in the phagocytosis of noxious supra-molecular structures like beta-amyloids. By means of NAD(P)H-based FLIM we investigate the activation of NADPH oxidase in murine and human peripheral macropahges as compared to microglia, i.e. macrophages of the central nervous system. The same response to specific chemical activation and to phagocytosis of Stafiloccocus aureus functionalized beads is demonstrated for macrophages and microglia of new-born mice. However, this response is different for microglia of adult, healthy mice as also reflected by their reduced phagocytotic capacity. In pathological context, we investigate the activation of NADPH oxidase in microglia of adult, healthy mice as compared to microglia of mice affected by a murine model of MS. Thus, we were able to identify a new mechanism in chronic neuroinflammation.