Metabolic Cofactors NAD Research Articles

Label-Free Assessment of Key Biological Autofluorophores: Material Characteristics and Opportunities for Clinical Applications.

Autofluorophores are endogenous fluorescent compounds that naturally occur in the intra and extracellular spaces of all tissues and organs. Most have vital biological functions - like the metabolic cofactors NAD(P)H and FAD+, as well as the structural protein collagen. Others are considered to be waste products - like lipofuscin and advanced glycation end products - which accumulate with age and are associated with cellular dysfunction. Due to their natural fluorescence, these materials have great utility for enabling non-invasive, label-free assays with direct ties to biological function. Numerous technologies, with different advantages and drawbacks, are applied to their assessment, including fluorescence lifetime imaging microscopy, hyperspectral microscopy, and flow cytometry. Here, the applications of label-free autofluorophore assessment are reviewed for clinical and health-research applications, with specific attention to biomaterials, disease detection, surgical guidance, treatment monitoring, and tissue assessment - fields that greatly benefit from non-invasive methodologies capable of continuous, in vivo characterization.

Metabolism in the large intestine wall after injury of the thoracic spinal cord (experimental study)

Ailm: to assess metabolic processes in the serous layer of the large intestine in the acute period of spinal injury.Materials and Methods: the experiment was carried out on laboratory animals — Wistar rats (n = 20). Spinal injury was simulated by complete transection of the spinal cord at the level of Th5-Th6 vertebrae. Metabolic changes in the intestinal wall were assessed before injury, 3 and 24 hours after spinal cord transection. Metabolism was assessed in vivo using fluorescence time-resolved macroimaging technology (macroFLIM) by autofluorescence in the spectral channel of the metabolic cofactor NAD(P)H.Results: a significant (p = 0.041) increase in the mean fluorescence lifetime (τm) by 12% and the lifetime of the long component (τ2) of the NAD(P)H cofactor by 13% (p = 0.008) was detected 24 hours after injury. MacroFLIM showed an increase in the intensity of metabolism in the large intestine wall.Conclusion: for the first time in an in vivo experiment, it has been shown that the acute period of spinal injury is accompanied by an increase in metabolic activity in the tissues of the large intestine. The revealed phenomenon confirms the role of the large intestine in compensatory reactions to spinal injury and open up new possibilities for therapy in the acute period of spinal cord injury.

Monitoring the Intracellular pH and Metabolic State of Cancer Cells in Response to Chemotherapy Using a Combination of Phosphorescence Lifetime Imaging Microscopy and Fluorescence Lifetime Imaging Microscopy.

The extracellular matrix (ECM), in which collagen is the most abundant protein, impacts many aspects of tumor physiology, including cellular metabolism and intracellular pH (pHi), as well as the efficacy of chemotherapy. Meanwhile, the role of collagen in differential cell responses to treatment within heterogeneous tumor environments remains poorly investigated. In the present study, we simultaneously monitored the changes in pHi and metabolism in living colorectal cancer cells in vitro upon treatment with a chemotherapeutic combination, FOLFOX (5-fluorouracil, oxaliplatin and leucovorin). The pHi was followed using the new pH-sensitive probe BC-Ga-Ir, working in the mode of phosphorescence lifetime imaging (PLIM), and metabolism was assessed from the autofluorescence of the metabolic cofactor NAD(P)H using fluorescence lifetime imaging (FLIM) with a two-photon laser scanning microscope. To model the ECM, 3D collagen-based hydrogels were used, and comparisons with conventional monolayer cells were made. It was found that FOLFOX treatment caused an early temporal intracellular acidification (reduction in pHi), followed by a shift to more alkaline values, and changed cellular metabolism to a more oxidative state. The presence of unstructured collagen markedly reduced the cytotoxic effects of FOLFOX, and delayed and diminished the pHi and metabolic responses. These results support the observation that collagen is a factor in the heterogeneous response of cancer cells to chemotherapy and a powerful regulator of their metabolic behavior.

Abstract 2386: Label free imaging for rapid assessment of tumor viability in live tumor fragments

Abstract Live tumor fragments (LTF) that maintain a patient's relevant tumor microenvironment provides an opportunity to study treatment response. To verify viability of the fragments over time, we have devised and applied a label-free quantitative microscopy-based approach to monitor LTF health. This method directly assesses intrinsic fluorescence from metabolic co-factors and allows the spatial and temporal visualization of cell status. Using the label-free method of multiphoton fluorescence lifetime microscopy (MP-FLIM), we can assess excised tumor samples over 48 hours to determine the health of individual cells. Specifically, with MP-FLIM, we can analyze signals generated by the intrinsically fluorescent metabolic co-factor NAD(P)H which correlates with mitochondrial outer membrane permeabilization and the irreversible cascades leading to cell death. Our findings using MP-FLIM were confirmed using a standard caspase 3/7 live apoptosis assay. These data demonstrate MP-FLIM can detect and quantify cell viability without the use of potentially toxic dyes, thus enabling longitudinal multi-day studies assessing the effects of therapeutic agents on LTF. Citation Format: Jonathan Ouellette, Ellen Wargowski, Eric Wait, Chris Zahm, Scott Johnson, Jon Oliner. Label free imaging for rapid assessment of tumor viability in live tumor fragments [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2386.

Autofluorescence imaging permits label free cell type assignment in unmodified airway and reveals the dynamic formation of airway secretory cell associated antigen passages (SAPs).

The specific functional properties of a tissue are distributed amongst its component cell types. The various cells act coherently, as an ensemble, in order to execute a physiologic response. Modern approaches for identifying and dissecting novel physiologic mechanisms would benefit from an ability to identify specific cell types in live tissues that could then be imaged in real-time. Current techniques require the use of fluorescent genetic reporters that are not only cumbersome, but which only allow the simultaneous study of 3 or 4 cell types at a time. We report a non-invasive imaging modality that capitalizes on the endogenous autofluorescence signatures of the metabolic cofactors NAD(P)H and FAD. By marrying morphological characteristics with autofluorescence signatures, all seven of the airway epithelial cell types can be distinguished simultaneously in mouse tracheal explant in real-time. Furthermore, we find that this methodology for direct cell type specific identification avoids pitfalls associated with the use of ostensibly cell type-specific markers that are, in fact, altered by clinically relevant physiologic stimuli. Finally, we utilize this methodology to interrogate real-time physiology and identify dynamic secretory cell associated antigen passages (SAPs) that form in response to cholinergic stimulus. The identical process has been well documented in the intestine where the dynamic formation of secretory and goblet cell associated antigen passages (SAPs and GAPs) enable luminal antigen sampling. Given that airway secretory cells can be stimulated to make mucous within hours, we suspect that both SAPs and GAPs are also used for luminal antigen sampling in the airway. This hypothesis is supported by our observation that secretory cells with airway SAPs are frequently juxtaposed to antigen presenting cells.

Fluorescence Lifetime Imaging Microscopy (FLIM) reveals spatial-metabolic changes in 3D breast cancer spheroids

Cancer cells are mechanically sensitive to physical properties of the microenvironment, which can affect downstream signaling to promote malignancy, in part through the modulation of metabolic pathways. Fluorescence Lifetime Imaging Microscopy (FLIM) can be used to measure the fluorescence lifetime of endogenous fluorophores, such as the metabolic co-factors NAD(P)H and FAD, in live samples. We used multiphoton FLIM to investigate the changes in cellular metabolism of 3D breast spheroids derived from MCF-10A and MD-MB-231 cell lines embedded in collagen with varying densities (1 vs. 4 mg/ml) over time (Day 0 vs. Day 3). MCF-10A spheroids demonstrated spatial gradients, with the cells closest to the spheroid edge exhibiting FLIM changes consistent with a shift towards oxidative phosphorylation (OXPHOS) while the spheroid core had changes consistent with a shift towards glycolysis. The MDA-MB-231 spheroids had a large shift consistent with increased OXPHOS with a more pronounced change at the higher collagen concentration. The MDA-MB-231 spheroids invaded into the collagen gel over time and cells that traveled the farthest had the largest changes consistent with a shift towards OXPHOS. Overall, these results suggest that the cells in contact with the extracellular matrix (ECM) and those that migrated the farthest had changes consistent with a metabolic shift towards OXPHOS. More generally, these results demonstrate the ability of multiphoton FLIM to characterize how spheroids metabolism and spatial metabolic gradients are modified by physical properties of the 3D ECM.

Effect of Collagen Matrix on Doxorubicin Distribution and Cancer Cells' Response to Treatment in 3D Tumor Model.

The extracellular matrix (ECM) plays an important role in regulation of many aspects of tumor growth and response to therapies. However, the specifics of the interaction of chemotherapeutic agents with cancer cells in the presence of collagen, the major component of ECM, is still poorly investigated. In this study, we explored distribution of doxorubicin (DOX) and its effects on cancer cells' metabolism in the presence of collagen with different structures in 3D models. For this, a combination of second harmonic generation imaging of collagen and multiphoton fluorescence microscopy of DOX, and metabolic cofactor NAD(P)H was used. It was found that collagen slowed down the diffusion of DOX and thus decreased the cellular drug uptake. Besides nuclei, DOX also targeted mitochondria leading to inhibition of oxidative phosphorylation, which was more pronounced in the cells growing in the absence of collagen. As a result, the cells in collagen displayed better viability upon treatment with DOX. Taken together, our data illustrate that tumor collagen contributes to heterogeneous and sub-optimal response to DOX and highlight the challenges in improving drug delivery and efficacy.

Simultaneous Probing of Metabolism and Oxygenation of Tumors In Vivo Using FLIM of NAD(P)H and PLIM of a New Polymeric Ir(III) Oxygen Sensor.

Tumor cells are well adapted to grow in conditions of variable oxygen supply and hypoxia by switching between different metabolic pathways. However, the regulatory effect of oxygen on metabolism and its contribution to the metabolic heterogeneity of tumors have not been fully explored. In this study, we develop a methodology for the simultaneous analysis of cellular metabolic status, using the fluorescence lifetime imaging microscopy (FLIM) of metabolic cofactor NAD(P)H, and oxygen level, using the phosphorescence lifetime imaging (PLIM) of a new polymeric Ir(III)-based sensor (PIr3) in tumors in vivo. The sensor, derived from a polynorbornene and cyclometalated iridium(III) complex, exhibits the oxygen-dependent quenching of phosphorescence with a 40% longer lifetime in degassed compared to aerated solutions. In vitro, hypoxia resulted in a correlative increase in PIr3 phosphorescence lifetime and free (glycolytic) NAD(P)H fraction in cells. In vivo, mouse tumors demonstrated a high degree of cellular-level heterogeneity of both metabolic and oxygen states, and a lower dependence of metabolism on oxygen than cells in vitro. The small tumors were hypoxic, while the advanced tumors contained areas of normoxia and hypoxia, which was consistent with the pimonidazole assay and angiographic imaging. Dual FLIM/PLIM metabolic/oxygen imaging will be valuable in preclinical investigations into the effects of hypoxia on metabolic aspects of tumor progression and treatment response.

A Label-Free Segmentation Approach for Intravital Imaging of Mammary Tumor Microenvironment.

The ability to visualize complex and dynamic physiological interactions between numerous cell types and the extracellular matrix (ECM) within a live tumor microenvironment is an important step toward understanding mechanisms that regulate tumor progression. While this can be accomplished through current intravital imaging techniques, it remains challenging due to the heterogeneous nature of tissues and the need for spatial context within the experimental observation. To this end, we have developed an intravital imaging workflow that pairs collagen second harmonic generation imaging, endogenous fluorescence from the metabolic co-factor NAD(P)H, and fluorescence lifetime imaging microscopy (FLIM) as a means to non-invasively compartmentalize the tumor microenvironment into basic domains of the tumor nest, the surrounding stroma or ECM, and the vasculature. This non-invasive protocol details the step-by-step process ranging from the acquisition of time-lapse images of mammary tumor models to post-processing analysis and image segmentation. The primary advantage of this workflow is that it exploits metabolic signatures to contextualize the dynamically changing live tumor microenvironment without the use of exogenous fluorescent labels, making it advantageous for human patient-derived xenograft (PDX) models and future clinical use where extrinsic fluorophores are not readily applicable.

Circadian transcription factor NPAS2 and the NAD+ -dependent deacetylase SIRT1 interact in the mouse nucleus accumbens and regulate reward.

Substance use disorders are associated with disruptions to both circadian rhythms and cellular metabolic state. At the molecular level, the circadian molecular clock and cellular metabolic state may be interconnected through interactions with the nicotinamide adenine dinucleotide (NAD+ )-dependent deacetylase, sirtuin 1 (SIRT1). In the nucleus accumbens (NAc), a region important for reward, both SIRT1 and the circadian transcription factor neuronal PAS domain protein 2 (NPAS2) are highly enriched, and both are regulated by the metabolic cofactor NAD+ . Substances of abuse, like cocaine, greatly disrupt cellular metabolism and promote oxidative stress; however, their effects on NAD+ in the brain remain unclear. Interestingly, cocaine also induces NAc expression of both NPAS2 and SIRT1, and both have independently been shown to regulate cocaine reward in mice. However, whether NPAS2 and SIRT1 interact in the NAc and/or whether together they regulate reward is unknown. Here, we demonstrate diurnal expression of Npas2, Sirt1 and NAD+ in the NAc, which is altered by cocaine-induced upregulation. Additionally, co-immunoprecipitation reveals NPAS2 and SIRT1 interact in the NAc, and cross-analysis of NPAS2 and SIRT1 chromatin immunoprecipitation sequencing reveals several reward-relevant and metabolic-related pathways enriched among shared gene targets. Notably, NAc-specific Npas2 knock-down or a functional Npas2 mutation in mice attenuates SIRT1-mediated increases in cocaine preference. Together, our data reveal an interaction between NPAS2 and SIRT1 in the NAc, which may serve to integrate cocaine's effects on circadian and metabolic factors, leading to regulation of drug reward.

Autofluorescence Imaging of Treatment Response in Neuroendocrine Tumor Organoids.

Simple SummaryGastroenteropancreatic neuroendocrine tumors (GEP-NET) account for roughly 60% of all neuroendocrine tumors, and low/intermediate grade human GEP-NETs have relatively slow growth rates that many laboratory culture methods fail to capture. Patient-derived cancer organoids (PDCOs) are an attractive model to address this need for relevant 3D cultures of GEP-NETs for laboratory drug testing. However, traditional measurements of drug response are not effective in GEP-NET PDCOs due to the small volume of tissue and slow growth rates that are characteristic of the disease. Here, we test a label-free, non-destructive optical metabolic imaging (OMI) method to measure drug response in live GEP-NET PDCOs. OMI measured a response to the novel treatment combination of ABT-263 and everolimus in five out of seven PDCO lines, at 72 h post-treatment. Overall, this work shows that OMI provides single-cell metabolic measurements of drug response in PDCOs to guide drug development for GEP-NET patients.Gastroenteropancreatic neuroendocrine tumors (GEP-NET) account for roughly 60% of all neuroendocrine tumors. Low/intermediate grade human GEP-NETs have relatively low proliferation rates that animal models and cell lines fail to recapitulate. Short-term patient-derived cancer organoids (PDCOs) are a 3D model system that holds great promise for recapitulating well-differentiated human GEP-NETs. However, traditional measurements of drug response (i.e., growth, proliferation) are not effective in GEP-NET PDCOs due to the small volume of tissue and low proliferation rates that are characteristic of the disease. Here, we test a label-free, non-destructive optical metabolic imaging (OMI) method to measure drug response in live GEP-NET PDCOs. OMI captures the fluorescence lifetime and intensity of endogenous metabolic cofactors NAD(P)H and FAD. OMI has previously provided accurate predictions of drug response on a single cell level in other cancer types, but this is the first study to apply OMI to GEP-NETs. OMI tested the response to novel drug combination on GEP-NET PDCOs, specifically ABT263 (navitoclax), a Bcl-2 family inhibitor, and everolimus, a standard GEP-NET treatment that inhibits mTOR. Treatment response to ABT263, everolimus, and the combination were tested in GEP-NET PDCO lines derived from seven patients, using two-photon OMI. OMI measured a response to the combination treatment in 5 PDCO lines, at 72 h post-treatment. In one of the non-responsive PDCO lines, heterogeneous response was identified with two distinct subpopulations of cell metabolism. Overall, this work shows that OMI provides single-cell metabolic measurements of drug response in PDCOs to guide drug development for GEP-NET patients.

Poly(ADP-ribose) polymerase 1 regulates mitochondrial DNA repair in an NAD-dependent manner

Mitochondrial DNA is located in organelle that house essential metabolic reactions and contains high reactive oxygen species. Therefore, mitochondrial DNA suffers more oxidative damage than its nuclear counterpart. Formation of a repair enzyme complex is beneficial to DNA repair. Recent studies have shown that mitochondrial DNA polymerase (Pol γ) and poly(ADP-ribose) polymerase 1 (PARP1) were found in the same complex along with other mitochondrial DNA repair enzymes, and mitochondrial PARP1 level is correlated with mtDNA integrity. However, the molecular basis for the functional connection between Pol γ and PARP1 has not yet been elucidated because cellular functions of PARP1 in DNA repair are intertwined with metabolism via NAD+ (nicotinamide adenosine dinucleotide), the substrate of PARP1, and a metabolic cofactor. To dissect the direct effect of PARP1 on mtDNA from the secondary perturbation of metabolism, we report here biochemical studies that recapitulated Pol γ PARylation observed in cells and showed that PARP1 regulates Pol γ activity during DNA repair in a metabolic cofactor NAD+ (nicotinamide adenosine dinucleotide)-dependent manner. In the absence of NAD+, PARP1 completely inhibits Pol γ, while increasing NAD+ levels to a physiological concentration that enables Pol γ to resume maximum repair activity. Because cellular NAD+ levels are linked to metabolism and to ATP production via oxidative phosphorylation, our results suggest that mtDNA damage repair is coupled to cellular metabolic state and the integrity of the respiratory chain.

Abstract 4993: Label-free metabolic imaging of T cell response to the tumor microenvironment

Abstract Numerous factors including low pH, low glucose, and high lactic acid levels in tumors create an immunosuppressive tumor microenvironment. This immunosuppression is partially due to the metabolic requirements of T cell activation, which are difficult to meet in the tumor microenvironment. The goal of this study is to use label-free optical metabolic imaging (OMI) of the metabolic co-factors NAD(P)H and FAD in CD3-conjugated (activated) and control T cells to monitor metabolic changes of T cells within tumor micro environmental conditions. Single-cell measurements include the optical redox ratio (fluorescence intensity of NAD(P)H divided by FAD), which measures the oxidation-reduction state of the cell, and the mean fluorescence lifetimes (τm) of NAD(P)H and FAD, which measure enzyme-binding activity in the cell. Jurkat T cells were conjugated with CD3 antibody over a two-day incubation period to achieve activation. Control and CD3-conjugated cells were exposed to environments with increasing concentrations of lactic acid (0-20 mM), hydrochloric acid (HCl; pH 6.4- 7.4), and glucose deprivation to mimic the tumor microenvironment. OMI monitored metabolic changes on a single-cell level without confounding factors associated with cell surface marker labeling. Treatment with lactic acid resulted in a significant dose-dependent decrease in the FAD τm and optical redox ratio (p<0.005). Along with changes in metabolism, OMI also resolved impaired mobility of the T cells with increasing lactic acid concentrations. Lactic acid affects both pH and metabolism, so cells were separately exposed to different concentrations of HCl (pH 6.4 - pH 7.4) to monitor pH effects alone. In control cells, the optical redox ratio decreased with decreasing pH (p<0.005). Conversely, in the presence of CD3, the optical redox ratio increased with decreasing pH (p<0.005). CD3 conjugation in glucose deprived environments also resulted in increases in both optical redox ratio and NAD(P)H τm. Cellular viability assays indicate apoptosis only in the lowest pH environment (pH 6.4, p<0.005). These studies indicate that OMI is a promising label-free approach to investigate the effects of the tumor microenvironment on T cell metabolism, activation, and drug response. Citation Format: Ava VanDommelen, Amani Gillette, Manish S. Patankar, Melissa Skala. Label-free metabolic imaging of T cell response to the tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4993.

Abstract 2782: Neuroendocrine tumor spheroid growth and drug response assessment using optical metabolic imaging

Abstract Gastroenteropancreatic neuroendocrine tumors (GEP-NET) are the second most common form of gastrointestinal cancer, and account for roughly 60% of all neuroendocrine tumors. Unfortunately, no animal models or cell lines exist that reflect the in vivo characteristics of human GEP-NETs. Specifically, human GEP-NETs have low proliferation rates that animal models and cell lines fail to capture. Therefore, drug testing for GEP-NETs often begins in patients without rigorous pre-clinical validation. There is a need to predict the most effective treatments prior to clinical trials for GEP-NET patients, to increase treatment efficacy and reduce toxicities. Here we show that a three-dimensional spheroid model of primary GEP-NET patient tumors is representative of the original patient tumors including histological features and markers of proliferation (Ki67). Furthermore, this study uses the spheroid model to test a novel drug combination on GEP-NET spheroids, specifically ABT263, a Bcl-2 family inhibitor, and everolimus, a standard GEP-NET treatment that inhibits mTOR. Previous studies have shown that ABT263 induces apoptosis in cancer cells when used in combination with everolimus, making this a promising drug combination for GEP-NET patients. Treatment response to ABT263, everolimus, and the combination, was tested in three-dimensional spheroids derived from seven primary GEP-NET patients, using two-photon optical metabolic imaging (OMI). OMI captures the fluorescence lifetime (τm) and intensity of metabolic cofactors NAD(P)H and FAD. These metrics can be combined into a single number, the OMI index, which was developed to separate responsive and non-responsive cells. The OMI index is the linear combination of the norm-centered redox ratio (NAD(P)H intensity / FAD intensity) + NAD(P)H τm - FAD τm. The OMI index also provides single cell response information from intact spheroids, so that heterogeneity in response can be quantified. Altogether, two-photon OMI in primary patient GEP-NET spheroids provides a novel tool to measure metabolic response to drug treatment. At 72 hours post-treatment, all patients except two showed a decrease in OMI index, corresponding with a response, to the combination treatment. Heterogeneous response was resolved with two distinct subpopulations of cell metabolism in one of the nonresponsive patients. No changes in spheroid diameter were observed with any treatment, due to the slow growth rate of the GEP-NET tumors and spheroids, further highlighting a need for molecular measures of drug response in these samples. We have shown that representative GEP-NET models can be generated in patient-derived tumor spheroids. Additionally, ABT263 plus everolimus is a promising combination treatment for GEP-NETs. Overall, this work shows that patient-derived tumor spheroids can be established from GEP-NETs, and that OMI provides single-cell metabolic measurements of drug response to guide drug development for patients. Citation Format: Amani Gillette, Christopher Babiarz, Cheri Pasch, Dustin Deming, Melissa Skala. Neuroendocrine tumor spheroid growth and drug response assessment using optical metabolic imaging [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2782.

Interrogation of tumor metabolism in tissue samples ex vivo using fluorescence lifetime imaging of NAD(P)H

Exploring metabolism in human tumors at the cellular level remains a challenge. The reduced form of metabolic cofactor NAD(P)H is one of the major intrinsic fluorescent components in tissues and a valuable indicator of cellular metabolic activity. Fluorescence lifetime imaging (FLIM) enables resolution of both the free and protein-bound fractions of this cofactor, and thus, high sensitivity detection of relative changes in the NAD(P)H-dependent metabolic pathways in real time. However, the clinical use of this technique is still very limited. The applications of metabolic FLIM could be usefully expanded by probing cellular metabolism in tissues ex vivo. For this, however, the development of appropriate tissue preservation protocols is required in order to maintain the optical metabolic characteristics in the ex vivo sample in a state similar to those of the tumor in vivo. Using mouse tumor models of different histological types—colorectal cancer, lung carcinoma and melanoma—we tested eight different methods of tissue handling by comparing NAD(P)H fluorescence decay parameters ex vivo and in vivo as measured with two-photon excited FLIM microscopy. It was found that the samples placed in 10% BSA on ice immediately after excision maintained the same fluorescence lifetimes and free/bound ratios as measured in vivo for at least 3 hours. This protocol was subsequently used for metabolic assessments in fresh postoperative samples from colorectal cancer patients. A high degree of inter- and intra-tumor heterogeneity with a trend to a more oxidative metabolism was detected in T3 colorectal tumors in comparison with normal tumor-distant colon samples. These results suggest that the methodology developed on the basis of FLIM of NAD(P)H in tissues ex vivo show promise for interrogating the metabolic state of patients’ tumors.

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 1941: Fluorescence lifetime imaging of treatment response in neuroendocrine tumor organoids

Abstract Gastroenteropancreatic neuroendocrine tumors (GEP-NET) are now the second most common form of gastrointestinal cancer, and account for roughly 60% of all neuroendocrine tumors. Unfortunately, no animal models or cell lines exist that reflect the in vivo characteristics of human GEP-NET’s for use as predictive models. Therefore, pre-clinical work is often not representative of the clinical outcome, so clinical trials begin in patients without rigorous pre-clinical validation of the treatment regimen. There is an established need to predict the most effective treatments prior to clinical trials for GEP-NET patients, to increase treatment efficacy and reduce toxicities. Here we show that a three-dimensional organoid model of primary patient tumors is representative of the original patient tumors. Furthermore, this study aims to use the organoid model to test a novel pre-clinical combination of ABT263, an experimental cancer drug that has been shown to enhance the effects of other chemotherapeutic agents, and the standard drug everolimus, on GEP-NET organoids. Previous studies have shown that ABT263, which targets Bcl-2, Bcl-XL and Bcl-w, induces apoptosis when used in combination with everolimus, making this a promising drug combination for GEP-NET patients. Treatment response to ABT263, everolimus, and the combination, was tested in three-dimensional organoids derived from six primary GEP-NET patients, using two-photon fluorescence lifetime imaging microscopy (FLIM). FLIM captures the fluorescence lifetime of metabolic cofactors NAD(P)H and FAD. Further, the fluorescence intensity of NAD(P)H divided by that of FAD (the redox ratio) was measured in all samples. Altogether, two-photon imaging of NAD(P)H and FAD fluorescence intensities and lifetimes (optical metabolic imaging, or OMI) in primary patient GEP-NET organoids provides a novel tool to measure metabolic changes with drug treatment. OMI measured metabolic differences induced by treatment in primary patient GEP-NETs when compared to control, at 72 hours post-treatment. All patients except one showed a decrease in redox ratio, which corresponds with a response, to the combination treatment. As metabolic imaging is performed to collect treatment response from more patients, this OMI data will be integrated into a multivariate analysis of variance to predict treatment response. Independent measures of organoid response, including changes in organoid diameter and immunohistochemistry, have been used to validate OMI predictions. We have shown that ABT263 plus everolimus is a promising combination treatment for GEP-NET’s. Overall, this work establishes the feasibility of personalized drug screens for GEP-NET’s using OMI in primary tumor organoids. Additionally, this platform can be used to identify new treatment regimens for improved GEP-NET patient outcomes, such as the ABT263 plus everolimus combination tested here. Citation Format: Amani A. Gillette, Christopher P. Babiarz, Cheri A. Pasch, Dustin A. Deming, Melissa C. Skala. Fluorescence lifetime imaging of treatment response in neuroendocrine tumor organoids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1941.

An NAD+-dependent novel transcription factor controls stage conversion in Entamoeba.

Developmental switching between life-cycle stages is a common feature among parasitic pathogens to facilitate disease transmission and pathogenesis. The protozoan parasite Entamoeba switches between invasive trophozoites and dormant cysts, but the encystation process remains poorly understood despite being central to amoebic biology. We identify a transcription factor, Encystation Regulatory Motif-Binding Protein (ERM-BP), that regulates encystation. Down-regulation of ERM-BP decreases encystation efficiency resulting in abnormal cysts with defective cyst walls. We demonstrate that direct binding of NAD+ to ERM-BP affects ERM-BP conformation and facilitates its binding to promoter DNA. Additionally, cellular NAD+ levels increase during encystation and exogenous NAD+ enhances encystation consistent with the role of carbon source depletion in triggering Entamoeba encystation. Furthermore, ERM-BP catalyzes conversion of nicotinamide to nicotinic acid, which might have second messenger effects on stage conversion. Our findings link the metabolic cofactors nicotinamide and NAD+ to transcriptional regulation via ERM-BP and provide the first mechanistic insights into Entamoeba encystation.

Po-323 In vivo monitoring of tumour responses to chemotherapy using multiparameter fluorescence imaging

IntroductionAn increasing number of recent studies suggest that tumour response to chemotherapy cannot be fully described in terms of only interaction of the drug with the main cellular target (e.g. DNA or microtubules) but can include multiple physiological and physicochemical changes. Insight into drug-induced functional alterations in cancer cells and tissues is crucially important for understanding of mechanisms of a drug action and for development new approaches to enable monitoring of the early-treatment response.Material and methodsIn our studies, we focus on multiparameteric analysis of tumour responses to chemotherapy using advanced fluorescence imaging techniques. Previously, we developed methodologies for in vivo probing several parameters, including cytosolic pH, metabolic status, and viscosity of plasma membrane, in mouse tumour models. Mapping of cytosolic pH in tumours is performed using ratiometric genetically encoded sensor SypHer2 and fluorescence whole-body imaging [Shirmanova et al. BBA-GS 2015]. Imaging of cellular metabolism is based on the visualisation of fluorescence intensities and lifetimes of intrinsic metabolic cofactors NAD(P)H and FAD [Shirmanova et al. Sci.Rep. 2017]. Viscosity is measured using molecular BODIPY-based rotor and fluorescence lifetime imaging microscopy (FLIM) [Shimolina et al. Sci.Rep. 2017].Results and discussionsWe showed that acidification of cytosolic pH occurs after therapy with cisplatin in vivo, and this, likely, favours metabolic reorganisation of cells. Treated tumours exhibited a decreased relative contribution from free (cytosolic, protein-unbound) NAD(P)H, indicating a metabolic shift from glycolysis towards oxidative metabolism. It is interesting that optical metabolic imaging could allow early detection of tumour response to chemotherapy, before there are changes in tumour size, and metabolic changes were the same for therapeutic agents with different mechanisms of action (cisplatin, paclitaxel). Moreover, tumours treated with cisplatin displayed decreased plasma membrane viscosity. Presumably, this alteration of viscosity participates in cisplatin-induced apoptosis. Similar changes of pH, energy metabolism and viscosity were previously reported for cisplatin-treated cultured cancer cells.ConclusionThese results, therefore, suggest that all investigated parameters play a role in the cytotoxicity of the drugs and may provide a useful approach for monitoring tumour responses to chemotherapy. The study was supported by the Russian Science Foundation (#14-25-00129).

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.